SERINE RECOMBINASES
20240417754 ยท 2024-12-19
Inventors
- Ami S. Bhatt (Stanford, CA, US)
- Matthew G. Durrant (Oakland, CA, US)
- Joshua C. Tycko (Stanford, CA, US)
- Patrick D. Hsu (Oakland, CA, US)
- Alison Fanton (Oakland, CA, US)
- Michael C. Bassik (Stanford, CA, US)
- Lacramioara Bintu (Stanford, CA, US)
Cpc classification
C12Y301/22
CHEMISTRY; METALLURGY
C12N9/22
CHEMISTRY; METALLURGY
International classification
Abstract
Provided herein are recombinases and compositions, methods of identification and methods of using thereof.
Claims
1. A system for DNA modification comprising: a polypeptide comprising a recombinase having an amino acid sequence having at least 70% identity to any of SEQ ID NOs: 1-74, or a nucleic acid encoding thereof; and a first polynucleotide comprising a donor recognition sequence for the recombinase.
2. The system of claim 1, wherein the recombinase has an amino acid sequence having at least 70% identity to any of SEQ ID NOs: 2, 6, 10, 12, 18, 19, 26, 29, 61, 65, or 66.
3. A system for DNA modification comprising: a polypeptide comprising a recombinase having an amino acid sequence with at least 70% identity to one or more of the following: 1) X.sub.1aX.sub.2aX.sub.3aX.sub.4aX.sub.5aX.sub.6aX.sub.7aX.sub.8aX.sub.9aX.sub.10aX.sub.11aX.sub.12aX.sub.13aX.sub.14aX.sub.15aX.sub.16aX.sub.17aX.sub.18aX.sub.19aX.sub.20aX.sub.21aX.sub.22aX.sub.23aX.sub.24aX.sub.25aX.sub.26aX.sub.27aX.sub.28aX.sub.29aX.sub.30aX.sub.31aX.sub.32aX.sub.33aX.sub.34a, wherein: X.sub.1a is A, E, I, L, S, T, V, or Y; X.sub.2a is A, D, E, G, K, Q, R, S, or T; X.sub.6a is E or G; X.sub.8a is A, C, F, L, M, or V; X.sub.10a is A, F, I, L, M, T, or V; X.sub.13a is F, H, I, L, M, N, or V; X.sub.14a is A, G, S, or V; X.sub.15a is A, D, I, L, S, T, or V; X.sub.17a is A, G, or S; X.sub.21a is K, R, S, or V; X.sub.22a is A, D, E, G, K, N, S, or T; X.sub.23a is A, E, I, K, M, N, Q, S, or T; X.sub.24a is F, I, L, M, S, or T; X.sub.26a is D, E, L, Q, S, or V; X.sub.27a is E, N, Q, or R; X.sub.32a is A, F, H, I, K, L, M, N, Q, R, S, or V X.sub.34a is A, E, G, H, K, L, M, N, Q, R, S, or V; and X.sub.3a, X.sub.4a, X.sub.5a, X.sub.7a, X.sub.9a, X.sub.11a, X.sub.12a, X.sub.16a, X.sub.18a, X.sub.19a, X.sub.20a, X.sub.25a, X.sub.28a, X.sub.29a, X.sub.30a, X.sub.31a, and X.sub.33a are each individually selected from any amino acid; 2) X.sub.1bX.sub.2bX.sub.3bX.sub.4bX.sub.5bX.sub.6bX.sub.7bX.sub.8bX.sub.9bX.sub.10bX.sub.11bX.sub.12bX.sub.13bX.sub.14bX.sub.15bX.sub.16bX.sub.17bX.sub.18b, wherein X.sub.1b is A, G, or I; X.sub.2b is D, E, G, N, P, S, T, or V; X.sub.3b is D, G, N, Q, or S; X.sub.4b is A, H, N, Q, R, T, V, or Y; X.sub.6b is A, D, E, H, I, L, P, Q, R, T, or Y; X.sub.7b is A, D, E, Q, or R; X.sub.8b is F, I, K, or L; X.sub.10b is D, E, F, G, N, Q, R, S, T, or V; X.sub.11b is A, I, L, S, T, or V; X.sub.12b is D, E, I, K, L, N, Q, R, S, T, or V; X.sub.13b is A, D, E, K, M, N, R, S, T, or V; X.sub.14b is A, G, Q, R, S, or T; X.sub.16b is A, D, E, K, L, Q, R, or T; and X.sub.18b is A, L, M, or V; and X.sub.5b, X.sub.9b, X.sub.15b, and X.sub.17b are each individually selected from any amino acid; 3) X.sub.1cX.sub.2cX.sub.3cX.sub.4cX.sub.5cX.sub.6cX.sub.7cX.sub.8cX.sub.9cX.sub.10cX.sub.11cX.sub.12cX.sub.13cESX.sub.16cX.sub.17cKX.sub.19cX.sub.20cX.sub.21cX.sub.22cX.sub.23cX.sub.24cX.sub.25cX.sub.26c, wherein X.sub.1c is A, D, F, I, L, M, N, S, or Y; X.sub.4c is A, I, K, M, S, or V; X.sub.6c is A, F, G, I, L, M, or V; X.sub.10c is Q, R, or T; X.sub.11c is A, G, or S; X.sub.13c is D, E, G, N, Q, or S; X.sub.17c is A, H, K, N, R, S, T, or V; X.sub.21c is L, M, R, or Y; X.sub.22c is A, I, N, Q, S, T, or V; X.sub.23c is A, E, F, I, K, L, N, R, T, or V; X.sub.25c is A, F, H, L, N, Q, S, T, or Y; X.sub.26c is A, I, L, M, N, R, S, T, V, or Y; and X.sub.2c, X.sub.3c, X.sub.5c, X.sub.7c, X.sub.8c, X.sub.9c, X.sub.12c, X.sub.16c, X.sub.19c, X.sub.20c, and X.sub.24c are each individually selected from any amino acid; 4) X.sub.1dX.sub.2dX.sub.3dX.sub.4dX.sub.5dX.sub.6dX.sub.7dX.sub.8dX.sub.9dX.sub.10dX.sub.11dX.sub.12dX.sub.13dX.sub.14dX.sub.15dX.sub.16dX.sub.17dX.sub.18dX.sub.19dX.sub.20dX.sub.21dX.sub.22dX.sub.23dX.sub.24dX.sub.25dX.sub.26dX.sub.27dX.sub.28d, wherein: X.sub.1d is E, K, N, T, G, S, L, D, V, A, R, or P; X.sub.2d is E, H, I, T, G, S, L, D, V, A, or P; X.sub.4d is M, I, T, S, L, V, A, R or P; X.sub.5d is E, K, N, I, T, G, S, D, Q, V, A, R, or P; X.sub.6d is E, G, S, D, A, R, or P; X.sub.7d is I, L, D, A, or R; X.sub.8d is M, H, K, T, L, V, Q, D, A, or R; X.sub.9d is E, K, I, T, G, S, L, D, Q, V, or A; X.sub.10d is E, K, H, D, Q, V, A, or R; X.sub.11d is M, H, I, S, L, V, Q, A, or R; X.sub.12d is Q, E, K, N, M, S, L, D, V, A, or R; X.sub.13d is E, K, H, G, S, L, D, Q, A, or R; X.sub.14d is E, Y, K, N, I, H, L, V, or A; X.sub.16d is E, K, I, T, G, S, L, D, Q, A, or R; X.sub.17d is E, K, H, T, G, D, Q, A, or R: X.sub.19d is Q, E, K, N, T, G, S, D, V, A, or R; X.sub.20d is Q, E, K, N, T, G, S, V, D, A, or R; X.sub.21d is I, S, W, L, V, F, A, or R; X.sub.22d is Q, E, M, T, G, S, L, V, D, or A; X.sub.23d is E, K, N, I, T, G, S, D, A, R, or P; X.sub.24d is E, M, I, L, D, Q, or A: X.sub.25d is E, Y, I, L, V, F, A, or R; X.sub.26d is E, M, T, G, S, L, D, V, A, or R; X.sub.27d is E, K, N, G, S, L, D, Q, A, or R; X.sub.28d is Q, E, G, V, D, A, R, or P; and X.sub.3d, X.sub.15d, and X.sub.18d are each individually selected from any amino acid; 5) X.sub.1eX.sub.2eX.sub.3eX.sub.4eX.sub.5eX.sub.6eX.sub.7eX.sub.8eX.sub.9eX.sub.10eX.sub.11eX.sub.12eX.sub.13eX.sub.14eX.sub.15eX.sub.16eX.sub.17eX.sub.18e, wherein: X.sub.1e is A, D, E, H, K, N, Q, R, or S; X.sub.2e is A, D, E, F, G, H, K, M, N, Q, R, S, W, or Y; X.sub.3e is E, F, or Y; X.sub.4e is F, H, L, W, or Y; X.sub.6e is A, D, E, F, I, K, L, M, N, Q, R, S, T, or Y; X.sub.7e is F, I, Q, S, T, or V; X.sub.8e is A, G, K, L, N, R, S, T, or V; X.sub.9e is A, D, E, H, K, N, Q, R, T, or Y; X.sub.10e is I, N, Q, or R; X.sub.11e is F, I, L, M, Q, or S; X.sub.14e is A, G, K, N, or S; X.sub.15e is K, M, Q, R, S, T, or V; X.sub.18e is A, E, G, K, M, N, S, T, or Y; and X.sub.5e, X.sub.12e, X.sub.13e, X.sub.16e, and X.sub.17e are each individually selected from any amino acid; 6) WX.sub.2fX.sub.3fX.sub.4fX.sub.5fX.sub.6fX.sub.7fX.sub.8fX.sub.9fX.sub.10fX.sub.11fX.sub.12fX.sub.13fX.sub.14fX.sub.15fX.sub.16fGX.sub.18fX.sub.19fX.sub.20fX.sub.21fX.sub.22fX.sub.23f, wherein: X.sub.2f is A, E, H, N, R, S, T, or V; X.sub.4f is A, G, N, S, or T; X.sub.5f is F, G, L, M, N, Q, S, T, or V; X.sub.6f is I, L, P, or V; X.sub.9f is I, L, T, or V; X.sub.14f is A, C, G, M, Q, R, S, or T; X.sub.16f is I, L, V, or Y; X.sub.18f is D, E, H, N, Q, or S; X.sub.20f is E, H, I, L, M, Q, R, or T; X.sub.21f is A, E, F, H, L, N, P, or Y; X.sub.22f is C, F, H, K, M, N, Q, R, T, or Y; X.sub.23f is D, E, F, I, K, L, N, Q, R, S, T, or V; and X.sub.3f, X.sub.7f, X.sub.8f X.sub.10f, X.sub.11f, X.sub.12f, X.sub.13f, X.sub.15f, and X.sub.19f are each individually selected from any amino acid; 7) X.sub.1gX.sub.2gX.sub.3gX.sub.4gX.sub.5gEX.sub.7gX.sub.8gX.sub.9gX.sub.10gX.sub.11gX.sub.12gRX.sub.14gX.sub.15gX.sub.16gX.sub.17gX.sub.18gX.sub.19gX.sub.20gX.sub.21g, wherein: X.sub.1g is A, G, I, N, S, T, or V; X.sub.3g is A, I, or S; X.sub.5g is F, I, L, M, or Y; X.sub.7g is I or R; X.sub.10g is D, I, L, or T; X.sub.12g is A, E, I, K, M, Q, or S; X.sub.14g is I, T, or V; X.sub.16g is A, D, G, R, S, or T; X.sub.18g is F, K, L, M, or Y; X.sub.19g is A, E, H, I, K, L, M, N, Q, R, V, W, or Y; X.sub.21g is A, I, K, L, M, or R; and X.sub.2g, X.sub.4g, X.sub.8g, X.sub.9g, X.sub.11g, X.sub.15g, X.sub.17g, and X.sub.20g are each individually selected from any amino acid; 8) X.sub.1hX.sub.2hX.sub.3hX.sub.4hX.sub.5hX.sub.6hX.sub.7hX.sub.8hX.sub.9hX.sub.10hX.sub.11h, wherein: X.sub.1h is F or Y; X.sub.2h is D, E, K, Q, or S; X.sub.3h is E, K, L, M, or Q; X.sub.4h is K, L, or R: X.sub.5h is K, L, or V; X.sub.7h is G or N; X.sub.8h is D, E, H, K, L, M, or R; X.sub.9h is S or T; X.sub.11h is F, H, I, Q, S, T, V, or W; and X.sub.6h and X.sub.10h are each individually selected from any amino acid; 9) X.sub.1iX.sub.2iX.sub.3iX.sub.4iX.sub.5iX.sub.6iX.sub.7iX.sub.8iX.sub.9iX.sub.10iX.sub.11iX.sub.13iX.sub.14iX.sub.15iX.sub.16iX.sub.17iX.sub.18iX.sub.19iX.sub.20iX.sub.21iX.sub.22iX.sub.23iX.sub.24iX.sub.25iX.sub.26iX.sub.27i, wherein: X.sub.1i is I, L, or V; X.sub.4i is A, D, F, H, I, L, M, N, Q, S, V, or Y; X.sub.8i is A, G, or S; X.sub.10i is D, E, I, K, N, Q, R, or S; X.sub.11i is E or Q; X.sub.15i is A or K; X.sub.16i is A, Q, R, or S; X.sub.18i is L, M, or R; X.sub.19i is I, L, Q, R, S, or V; X.sub.21i is A, D, E, G, H, I, Q, R, or S; X.sub.22i is A, K, N, Q, S, T, or V; X.sub.23i is A, H, K, R, W, or Y; X.sub.25i is A, G, H, I, K, Q, R, S, or T; X.sub.27i is C, H, I, K, L, R, or V; and X.sub.2i, X.sub.3i, X.sub.5i, X.sub.6i, X.sub.7i, X.sub.9i, X.sub.13i, X.sub.14i, X.sub.17i, X.sub.20i, X.sub.24i, and X.sub.26i are each individually selected from any amino acid; 10) RX.sub.2jX.sub.3jX.sub.4jW, wherein: X.sub.2j is L, M, Q, or R; X.sub.3j is A, N, or S; and X.sub.4j is N, P, S, or T; 11) X.sub.1kX.sub.2kX.sub.3kX.sub.4kX.sub.5kX.sub.6kX.sub.7kX.sub.8kF, wherein: X.sub.1k is I, L, or V; X.sub.2k is A or V; X.sub.4k is A, F, H, I, L, Q, W, or Y; X.sub.5k is I, M, or V; X.sub.7k is E, L, Q, or T: X.sub.8k is A, I, or V; and X.sub.3k and X.sub.6k are each individually selected from any amino acid; 12) RX.sub.2lX.sub.3lX.sub.4lX.sub.5lX.sub.6lX.sub.7lX.sub.8lX.sub.9lX.sub.10lX.sub.11lX.sub.12lX.sub.13l, wherein: X.sub.2l is D, K, N, R, S, or V; X.sub.3l is A, D, E, F, G, K, P, Q, or S; X.sub.4l is A, E, I, K, L, S, T, or V; X.sub.5l is any amino acid: X.sub.6l is F, G, I, L, N, or V; X.sub.7l is A, F, I, L, Q, R, V, or Y; X.sub.8l is D, E, I, L, M, N, Q, S, T, or V; X.sub.9l is D, E, F, I, L, M, Q, T, V, or Y; X.sub.10l is I, K, L, R, or V; X.sub.11l is D, E, K, N, Q, or R; X.sub.12l is D, E, F, K, L, N, Q, W, or Y; and X.sub.13l is F or L; and 13) X.sub.1mX.sub.2mX.sub.3mX.sub.4mX.sub.5mX.sub.6mX.sub.7mX.sub.8mX.sub.9mX.sub.10mX.sub.11mX.sub.12mX.sub.13mX.sub.14mX.sub.15mX.sub.16mX.sub.17mX.sub.18mX.sub.19mX.sub.20mX.sub.21mX.sub.22mX.sub.23mX.sub.24m, wherein: X.sub.1m is A, E, F, I, L, M, N, Q, S, T, V, or Y; X.sub.2m is A, F, G, I, L, M, R, S, T, or V; X.sub.6m is A, D, E, F, G, H, L, M, N, S, or T; X.sub.9m is D, M, N, or S; X.sub.10m is D, E, or Q; X.sub.12m is C, F, H, L, T, V, or Y; X.sub.14m is A, E, K, L, R, or Y; X.sub.17m is A, L, or S; X.sub.19m is D, E, K, N, Q, R, or S; X.sub.20m is G, I, M, Q, R, T, or V; X.sub.21m is D, H, K, N, Q, or R; X.sub.23m is A, G, I, L, N, S, T, or V; X.sub.24m is F, H, I, K, L, M, N, Q, V, W, or Y; and X.sub.3m, X.sub.4m, X.sub.5m, X.sub.7m, X.sub.8m, X.sub.11m, X.sub.13m, X.sub.15m, X.sub.16m, X.sub.18m, and X.sub.22m, are each individually selected from any amino acid, or active fragments thereof, or a nucleic acid encoding thereof; and a first polynucleotide comprising a donor recognition sequence for the recombinase.
4. A system for DNA modification comprising: a polypeptide comprising a recombinase having an amino acid sequence having at least 70% identity to any of SEQ ID NOs: 88-1183, or a nucleic acid encoding thereof; and a first polynucleotide comprising a donor recognition sequence for the recombinase.
5. The system of any of claims 1-4, wherein the donor recognition sequence comprises a donor attachment site configured to bind the recombinase.
6. The system of any of claims 1-5, wherein the first polynucleotide further comprises a cargo DNA sequence.
7. The system of claim 6, wherein the cargo DNA sequence is greater than 1 kilobase pair.
8. The system of claim 6 or claim 7, wherein the cargo DNA sequence is greater than 5 kilobase pairs.
9. The system of any of claims 1-8, wherein the first polynucleotide further comprises a recipient recognition sequence for the recombinase.
10. The system of any of claims 1-8, wherein the system further comprises a second polynucleotide comprising a recipient recognition sequence for the recombinase.
11. The system of claim 9 or claim 10, wherein the recipient recognition sequence comprises a recipient attachment sequence configured to bind to the recombinase.
12. The system of any of claims 1-11, wherein the donor recognition sequence, the recipient recognition sequence, or both are pseudo-recognition sequences.
13. The system of any of claims 1-12, wherein the system is a cell free system.
14. A composition comprising the system of any one of claims 1-12.
15. A cell comprising the system of any one of claims 1-12.
16. The cell of claim 15, wherein the cell is a eukaryotic cell.
17. A method of altering a target DNA comprising contacting the target DNA with a polypeptide comprising a recombinase having an amino acid sequence having at least 70% identity to any of SEQ ID NOs: 1-74, or a nucleic acid encoding thereof.
18. A method of altering a target DNA comprising contacting the target DNA with a polypeptide comprising a recombinase having an amino acid sequence with at least 70% identity to one or more of the following: 1) X.sub.1aX.sub.2aX.sub.3aX.sub.4aX.sub.5aX.sub.6aX.sub.7aX.sub.8aX.sub.9aX.sub.10aX.sub.11aX.sub.12aX.sub.13aX.sub.14aX.sub.15aX.sub.16aX.sub.17aX.sub.18aX.sub.19aX.sub.20aX.sub.21aX.sub.22aX.sub.23aX.sub.24aX.sub.25aX.sub.26aX.sub.27aX.sub.28aX.sub.29aX.sub.30aX.sub.31aX.sub.32aX.sub.33aX.sub.34a, wherein: X.sub.1a is A, E, I, L, S, T, V, or Y; X.sub.2a is A, D, E, G, K, Q, R, S, or T; X.sub.6a is E or G; X.sub.8a is A, C, F, L, M, or V; X.sub.10a is A, F, I, L, M, T, or V; X.sub.13a is F, H, I, L, M, N, or V; X.sub.14a is A, G, S, or V; X.sub.15a is A, D, I, L, S, T, or V; X.sub.17a is A, G, or S; X.sub.21a is K, R, S, or V; X.sub.22a is A, D, E, G, K, N, S, or T; X.sub.23a is A, E, I, K, M, N, Q, S, or T; X.sub.24a is F, I, L, M, S, or T; X.sub.26a is D, E, L, Q, S, or V; X.sub.27a is E, N, Q, or R; X.sub.32a is A, F, H, I, K, L, M, N, Q, R, S, or V X.sub.34a is A, E, G, H, K, L, M, N, Q, R, S, or V; and X.sub.3a, X.sub.4a, X.sub.5a, X.sub.7a, X.sub.9a, X.sub.11a, X.sub.12a, X.sub.16a, X.sub.18a, X.sub.19a, X.sub.20a, X.sub.25a, X.sub.28a, X.sub.29a, X.sub.30a, X.sub.31a, and X.sub.33a are each individually selected from any amino acid; 2) X.sub.1bX.sub.2bX.sub.3bX.sub.4bX.sub.5bX.sub.6bX.sub.7bX.sub.8bX.sub.9bX.sub.10bX.sub.11bX.sub.12bX.sub.13bX.sub.14bX.sub.15bX.sub.16bX.sub.17bX.sub.18b, wherein X.sub.1b is A, G, or I; X.sub.2b is D, E, G, N, P, S, T, or V; X.sub.3b is D, G, N, Q, or S; X.sub.4b is A, H, N, Q, R, T, V, or Y; X.sub.6b is A, D, E, H, I, L, P, Q, R, T, or Y; X.sub.7b is A, D, E, Q, or R; X.sub.8b is F, I, K, or L; X.sub.10b is D, E, F, G, N, Q, R, S, T, or V; X.sub.11b is A, I, L, S, T, or V; X.sub.12b is D, E, I, K, L, N, Q, R, S, T, or V; X.sub.13b is A, D, E, K, M, N, R, S, T, or V; X.sub.14b is A, G, Q, R, S, or T; X.sub.16b is A, D, E, K, L, Q, R, or T; and X.sub.18b is A, L, M, or V; and X.sub.5b, X.sub.9b, X.sub.15b, and X.sub.17b are each individually selected from any amino acid; 3) X.sub.1cX.sub.2cX.sub.3cX.sub.4cX.sub.5cX.sub.6cX.sub.7cX.sub.8cX.sub.9cX.sub.10cX.sub.11cX.sub.12cX.sub.13cESX.sub.16cX.sub.17cKX.sub.19cX.sub.20cX.sub.21cX.sub.22cX.sub.23cX.sub.24cX.sub.25cX.sub.26c, wherein X.sub.1c is A, D, F, I, L, M, N, S, or Y; X.sub.4c is A, I, K, M, S, or V; X.sub.6c is A, F, G, I, L, M, or V; X.sub.10c is Q, R, or T; X.sub.11c is A, G, or S; X.sub.13c is D, E, G, N, Q, or S; X.sub.17c is A, H, K, N, R, S, T, or V; X.sub.21c is L, M, R, or Y; X.sub.22c is A, I, N, Q, S, T, or V; X.sub.23c is A, E, F, I, K, L, N, R, T, or V; X.sub.25c is A, F, H, L, N, Q, S, T, or Y; X.sub.26c is A, I, L, M, N, R, S, T, V, or Y; and X.sub.2c, X.sub.3c, X.sub.5c, X.sub.7c, X.sub.8c, X.sub.9c, X.sub.12c, X.sub.16c, X.sub.19c, X.sub.20c, and X.sub.24c are each individually selected from any amino acid; 4) X.sub.1dX.sub.2dX.sub.3dX.sub.4dX.sub.5dX.sub.6dX.sub.7dX.sub.8dX.sub.9dX.sub.10dX.sub.11dX.sub.12dX.sub.13dX.sub.14dX.sub.15dX.sub.16dX.sub.17dX.sub.18dX.sub.19dX.sub.20dX.sub.21dX.sub.22dX.sub.23dX.sub.24dX.sub.25dX.sub.26dX.sub.27dX.sub.28d, wherein: X.sub.1d is E, K, N, T, G, S, L, D, V, A, R, or P; X.sub.2d is E, H, I, T, G, S, L, D, V, A, or P; X.sub.4d is M, I, T, S, L, V, A, R or P; X.sub.5d is E, K, N, I, T, G, S, D, Q, V, A, R, or P; X.sub.6d is E, G, S, D, A, R, or P; X.sub.7d is I, L, D, A, or R; X.sub.8d is M, H, K, T, L, V, Q, D, A, or R; X.sub.9d is E, K, I, T, G, S, L, D, Q, V, or A; X.sub.10d is E, K, H, D, Q, V, A, or R; X.sub.11d is M, H, I, S, L, V, Q, A, or R; X.sub.12d is Q, E, K, N, M, S, L, D, V, A, or R; X.sub.13d is E, K, H, G, S, L, D, Q, A, or R; X.sub.14d is E, Y, K, N, I, H, L, V, or A; X.sub.16d is E, K, I, T, G, S, L, D, Q, A, or R; X.sub.17d is E, K, H, T, G, D, Q, A, or R: X.sub.19d is Q, E, K, N, T, G, S, D, V, A, or R; X.sub.20d is Q, E, K, N, T, G, S, V, D, A, or R; X.sub.21d is I, S, W, L, V, F, A, or R; X.sub.22d is Q, E, M, T, G, S, L, V, D, or A; X.sub.23d is E, K, N, I, T, G, S, D, A, R, or P; X.sub.24d is E, M, I, L, D, Q, or A: X.sub.25d is E, Y, I, L, V, F, A, or R; X.sub.26d is E, M, T, G, S, L, D, V, A, or R; X.sub.27d is E, K, N, G, S, L, D, Q, A, or R; X.sub.28d is Q, E, G, V, D, A, R, or P; and X.sub.3d, X.sub.15d, and X.sub.18d are each individually selected from any amino acid; 5) X.sub.1eX.sub.2eX.sub.3eX.sub.4eX.sub.5eX.sub.6eX.sub.7eX.sub.8eX.sub.9eX.sub.10eX.sub.11eX.sub.12eX.sub.13eX.sub.14eX.sub.15eX.sub.16eX.sub.17eX.sub.18e, wherein: X.sub.1e is A, D, E, H, K, N, Q, R, or S; X.sub.2e is A, D, E, F, G, H, K, M, N, Q, R, S, W, or Y; X.sub.3e is E, F, or Y; X.sub.4e is F, H, L, W, or Y; X.sub.6e is A, D, E, F, I, K, L, M, N, Q, R, S, T, or Y; X.sub.7e is F, I, Q, S, T, or V; X.sub.8e is A, G, K, L, N, R, S, T, or V; X.sub.9e is A, D, E, H, K, N, Q, R, T, or Y; X.sub.10e is I, N, Q, or R; X.sub.11e is F, I, L, M, Q, or S; X.sub.14e is A, G, K, N, or S; X.sub.15e is K, M, Q, R, S, T, or V; X.sub.18e is A, E, G, K, M, N, S, T, or Y; and X.sub.5e, X.sub.12e, X.sub.13e, X.sub.16e, and X.sub.17e are each individually selected from any amino acid; 6) WX.sub.2fX.sub.3fX.sub.4fX.sub.5fX.sub.6fX.sub.7fX.sub.8fX.sub.9fX.sub.10fX.sub.11fX.sub.12fX.sub.13fX.sub.14fX.sub.15fX.sub.16fGX.sub.18fX.sub.19fX.sub.20fX.sub.21fX.sub.22fX.sub.23f, wherein: X.sub.2f is A, E, H, N, R, S, T, or V; X.sub.4f is A, G, N, S, or T; X.sub.5f is F, G, L, M, N, Q, S, T, or V; X.sub.6f is I, L, P, or V; X.sub.9f is I, L, T, or V; X.sub.14f is A, C, G, M, Q, R, S, or T; X.sub.16f is I, L, V, or Y; X.sub.18f is D, E, H, N, Q, or S; X.sub.20f is E, H, I, L, M, Q, R, or T; X.sub.21f is A, E, F, H, L, N, P, or Y; X.sub.22f is C, F, H, K, M, N, Q, R, T, or Y; X.sub.23f is D, E, F, I, K, L, N, Q, R, S, T, or V; and X.sub.3f, X.sub.7f, X.sub.8f X.sub.10f, X.sub.11f, X.sub.12f, X.sub.13f, X.sub.15f, and X.sub.19f are each individually selected from any amino acid; 7) X.sub.1gX.sub.2gX.sub.3gX.sub.4gX.sub.5gEX.sub.7gX.sub.8gX.sub.9gX.sub.10gX.sub.11gX.sub.12gRX.sub.14gX.sub.15gX.sub.16gX.sub.17gX.sub.18gX.sub.19gX.sub.20gX.sub.21g, wherein: X.sub.1g is A, G, I, N, S, T, or V; X.sub.3g is A, I, or S; X.sub.5g is F, I, L, M, or Y; X.sub.7g is I or R; X.sub.10g is D, I, L, or T; X.sub.12g is A, E, I, K, M, Q, or S; X.sub.14g is I, T, or V; X.sub.16g is A, D, G, R, S, or T; X.sub.18g is F, K, L, M, or Y; X.sub.19g is A, E, H, I, K, L, M, N, Q, R, V, W, or Y; X.sub.21g is A, I, K, L, M, or R; and X.sub.2g, X.sub.4g, X.sub.8g, X.sub.9g, X.sub.11g, X.sub.15g, X.sub.17g, and X.sub.20g are each individually selected from any amino acid; 8) X.sub.1hX.sub.2hX.sub.3hX.sub.4hX.sub.5hX.sub.6hX.sub.7hX.sub.8hX.sub.9hX.sub.10hX.sub.11h, wherein: X.sub.1h is F or Y; X.sub.2h is D, E, K, Q, or S; X.sub.3h is E, K, L, M, or Q; X.sub.4h is K, L, or R: X.sub.5h is K, L, or V; X.sub.7h is G or N; X.sub.8h is D, E, H, K, L, M, or R; X.sub.9h is S or T; X.sub.11h is F, H, I, Q, S, T, V, or W; and X.sub.6h and X.sub.10h are each individually selected from any amino acid; 9) X.sub.1iX.sub.2iX.sub.3iX.sub.4iX.sub.5iX.sub.6iX.sub.7iX.sub.8iX.sub.9iX.sub.10iX.sub.11iX.sub.13iX.sub.14iX.sub.15iX.sub.16iX.sub.17iX.sub.18iX.sub.19iX.sub.20iX.sub.21iX.sub.22iX.sub.23iX.sub.24iX.sub.25iX.sub.26iX.sub.27i, wherein: X.sub.1i is I, L, or V; X.sub.4i is A, D, F, H, I, L, M, N, Q, S, V, or Y; X.sub.8i is A, G, or S; X.sub.10i is D, E, I, K, N, Q, R, or S; X.sub.11i is E or Q; X.sub.15i is A or K; X.sub.16i is A, Q, R, or S; X.sub.18i is L, M, or R; X.sub.19i is I, L, Q, R, S, or V; X.sub.21i is A, D, E, G, H, I, Q, R, or S; X.sub.22i is A, K, N, Q, S, T, or V; X.sub.23i is A, H, K, R, W, or Y; X.sub.25i is A, G, H, I, K, Q, R, S, or T; X.sub.27i is C, H, I, K, L, R, or V; and X.sub.2i, X.sub.3i, X.sub.5i, X.sub.6i, X.sub.7i, X.sub.9i, X.sub.13i, X.sub.14i, X.sub.17i, X.sub.20i, X.sub.24i, and X.sub.26i are each individually selected from any amino acid; 10) RX.sub.2jX.sub.3jX.sub.4jW, wherein: X.sub.2j is L, M, Q, or R; X.sub.3j is A, N, or S; and X.sub.4j is N, P, S, or T; 11) X.sub.1kX.sub.2kX.sub.3kX.sub.4kX.sub.5kX.sub.6kX.sub.7kX.sub.8kF, wherein: X.sub.1k is I, L, or V; X.sub.2k is A or V; X.sub.4k is A, F, H, I, L, Q, W, or Y; X.sub.5k is I, M, or V; X.sub.7k is E, L, Q, or T: X.sub.8k is A, I, or V; and X.sub.3k and X.sub.6k are each individually selected from any amino acid; 12) RX.sub.2lX.sub.3lX.sub.4lX.sub.5lX.sub.6lX.sub.7lX.sub.8lX.sub.9lX.sub.10lX.sub.11lX.sub.12lX.sub.13l, wherein: X.sub.2l is D, K, N, R, S, or V; X.sub.3l is A, D, E, F, G, K, P, Q, or S; X.sub.4l is A, E, I, K, L, S, T, or V; X.sub.5l is any amino acid: X.sub.6l is F, G, I, L, N, or V; X.sub.7l is A, F, I, L, Q, R, V, or Y; X.sub.8l is D, E, I, L, M, N, Q, S, T, or V; X.sub.9l is D, E, F, I, L, M, Q, T, V, or Y; X.sub.10l is I, K, L, R, or V; X.sub.11l is D, E, K, N, Q, or R; X.sub.12l is D, E, F, K, L, N, Q, W, or Y; and X.sub.13l is F or L; and 13) X.sub.1mX.sub.2mX.sub.3mX.sub.4mX.sub.5mX.sub.6mX.sub.7mX.sub.8mX.sub.9mX.sub.10mX.sub.11mX.sub.12mX.sub.13mX.sub.14mX.sub.15mX.sub.16mX.sub.17mX.sub.18mX.sub.19mX.sub.20mX.sub.21mX.sub.22mX.sub.23mX.sub.24m, wherein: X.sub.1m is A, E, F, I, L, M, N, Q, S, T, V, or Y; X.sub.2m is A, F, G, I, L, M, R, S, T, or V; X.sub.6m is A, D, E, F, G, H, L, M, N, S, or T; X.sub.9m is D, M, N, or S; X.sub.10m is D, E, or Q; X.sub.12m is C, F, H, L, T, V, or Y; X.sub.14m is A, E, K, L, R, or Y; X.sub.17m is A, L, or S; X.sub.19m is D, E, K, N, Q, R, or S; X.sub.20m is G, I, M, Q, R, T, or V; X.sub.21m is D, H, K, N, Q, or R; X.sub.23m is A, G, I, L, N, S, T, or V; X.sub.24m is F, H, I, K, L, M, N, Q, V, W, or Y; and X.sub.3m, X.sub.4m, X.sub.5m, X.sub.7m, X.sub.8m, X.sub.11m, X.sub.13m, X.sub.15m, X.sub.16m, X.sub.18m, and X.sub.22m, are each individually selected from any amino acid, or a nucleic acid encoding thereof.
19. A method of altering a target DNA comprising contacting the target DNA with a polypeptide comprising a recombinase having an amino acid sequence having at least 70% identity to any of SEQ ID NOs: 88-1183, or a nucleic acid encoding thereof.
20. The method of any of claims 17-19, wherein the target DNA comprises a donor recognition sequence, a recipient recognition sequence, or both.
21. The method of any of claims 17-20, further comprising contacting the target DNA with a first polynucleotide comprising a donor recognition sequence for the recombinase.
22. The method of claim 21, wherein the first polynucleotide further comprises a cargo DNA sequence.
23. The method of claim 22, wherein the cargo DNA sequence is greater than 1 kilobase pair.
24. The method of claim 22 or claim 23, wherein the cargo DNA sequence is greater than 5 kilobase pairs.
25. The method of any of claims 21-24, wherein the target DNA comprises a recipient attachment sequence configured to bind to the recombinase.
26. The method of any of claims 20-25, wherein the donor recognition sequence, the recipient recognition sequence or both are pseudo-recognition sequences.
27. The method of any of claims 17-26, wherein the target DNA sequence encodes a gene product.
28. The method of any of claims 17-27, wherein the target DNA is in a cell.
29. The method of claim 28, wherein the cell is a eukaryotic cell.
30. The method of claim 29, wherein the eukaryotic cell is a human cell.
31. The method of claim 28, wherein the cell is a prokaryotic cell.
32. The method of any of claims, 28-31, wherein the target DNA sequence is a genomic DNA sequence.
33. The method of any of claims 28-31, wherein the contacting comprises introducing into the cell.
34. The method of claim 33, wherein introducing into the cell comprises administering to a subject.
35. The method of claim 34, wherein the subject is a human.
36. The method of claim 34 or 35, wherein the administering comprises in vivo administration.
37. The method of claim 34 or 35, wherein the administering comprises transplantation of ex vivo treated cells comprising the system.
38. The method of any of claims 33-37, wherein the recombinase, or the nucleic acid encoding thereof, is introduced into the cell before, concurrently with, or after the introduction of the donor polynucleotide.
39. Use of the system of any of claims 1-12 or a composition of claim 13 to alter a target nucleic acid sequence.
40. The use of claim 39, wherein the target DNA comprises a donor recognition sequence, a recipient recognition sequence, or both.
41. The use of claim 39 or 40, further comprising contacting the target DNA with a first polynucleotide comprising a donor recognition sequence for the recombinase.
42. The use of claim 41, wherein the first polynucleotide further comprises a cargo DNA sequence.
43. The use of claim 42, wherein the cargo DNA sequence is greater than 1 kilobase pair.
44. The use of claim 42 or claim 43, wherein the cargo DNA sequence is greater than 5 kilobase pairs.
45. The use of any of claims 41-44, wherein the target DNA comprises a recipient attachment sequence configured to bind to the recombinase.
46. The use of any of claims 40-45, wherein the donor recognition sequence, the recipient recognition sequence or both are pseudo-recognition sequences.
47. The use of any of claims 39-46, wherein the target DNA sequence encodes a gene product.
48. The use of any of claims 39-47, wherein the target DNA is in a cell.
49. The use of claim 48, wherein the cell is a eukaryotic cell.
50. The use of claim 49, wherein the eukaryotic cell is a human cell.
51. The use of claim 48, wherein the cell is a prokaryotic cell.
52. The use of any of claims, 48-51, wherein the target DNA sequence is a genomic DNA sequence.
53. The use of any of claims 48-51, wherein the contacting comprises introducing into the cell.
54. The use of claim 53, wherein introducing into the cell comprises administering to a subject.
55. The use of claim 54, wherein the subject is a human.
56. The use of claim 54 or 55, wherein the administering comprises in vivo administration.
57. The use of claim 54 or 55, wherein the administering comprises transplantation of ex vivo treated cells comprising the system.
58. The use of any of claims 53-57, wherein the recombinase, or the nucleic acid encoding thereof, is introduced into the cell before, concurrently with, or after the introduction of the donor polynucleotide.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
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DETAILED DESCRIPTION
[0413] Described herein are large serine recombinases (LSRs) identified along with their cognate DNA attachment sites using a computational workflow. The LSRs were characterized according to three separate technological applications: 1) landing-pad LSRs that can integrate efficiently at a pre-installed integration site, 2) multi-targeting LSRs that can integrate efficiently at many different loci in a target genome, and 3) genome-targeting LSRs that can integrate at one or several specific target sites in a given target genome. Several candidates in all three of these categories were validated in human cells. For landing-pad LSRs, many candidates were identified that recombined at orthogonal attachment sites at high efficiency when compared to Bxb1, the existing gold standard. For multi-targeting LSRs, which have not previously been developed as an integration tool in human cells, several were identified that can integrate at high efficiency in human cell lines relative to C31. For genome-targeting LSRs, several candidates that integrate DNA cargos into predicted human genome target sites without pre-installation of an attachment site were identified and validated.
[0414] Recombinases have vast applications as genome engineering tools. However, efficient genome integration of large donor sequences into the human genome is an outstanding problem in the field of human genome engineering. One major hurdle is the cargo size limit of adeno-associated virus (AAV) vector, the most successful vector available for human genome engineering, which is around 4.7 kilobase pairs (kb). CRISPR-Cas9 can be used to introduce double-stranded breaks at programmable locations, but when followed by homologous recombination to introduce new DNA, the efficiency of integration decreases exponentially as the size of the insertion increases, with reported maximum insertion sizes of 3-6 kb. By contrast, for recombinases, there is no obvious upper limit on the size of the donor DNA to be integrated, which is a major advantage of recombinases over other technologies.
[0415] Section headings as used in this section and the entire disclosure herein are merely for organizational purposes and are not intended to be limiting.
1. DEFINITIONS
[0416] The terms comprise(s), include(s), having, has, can, contain(s), and variants thereof, as used herein, are intended to be open-ended transitional phrases, terms, or words that do not preclude the possibility of additional acts or structures. The singular forms a, and and the include plural references unless the context clearly dictates otherwise. The present disclosure also contemplates other embodiments comprising, consisting of and consisting essentially of, the embodiments or elements presented herein, whether explicitly set forth or not. As used herein, comprising a certain sequence or a certain SEQ ID NO usually implies that at least one copy of said sequence is present in recited peptide or polynucleotide. However, two or more copies are also contemplated.
[0417] For the recitation of numeric ranges herein, each intervening number there between with the same degree of precision is explicitly contemplated. For example, for the range of 6-9, the numbers 7 and 8 are contemplated in addition to 6 and 9, and for the range 6.0-7.0, the number 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are explicitly contemplated.
[0418] Unless otherwise defined herein, scientific, and technical terms used in connection with the present disclosure shall have the meanings that are commonly understood by those of ordinary skill in the art. The meaning and scope of the terms should be clear; in the event, however of any latent ambiguity, definitions provided herein take precedent over any dictionary or extrinsic definition. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular.
[0419] As used herein, a nucleic acid or a nucleic acid sequence refers to a polymer or oligomer of pyrimidine and/or purine bases, preferably cytosine, thymine, and uracil, and adenine and guanine, respectively (See Albert L. Lehninger, Principles of Biochemistry, at 793-800 (Worth Pub. 1982)). The present technology contemplates any deoxyribonucleotide, ribonucleotide, or peptide nucleic acid component, and any chemical variants thereof, such as methylated, hydroxymethylated, or glycosylated forms of these bases, and the like. The polymers or oligomers may be heterogenous or homogenous in composition and may be isolated from naturally occurring sources or may be artificially or synthetically produced. In addition, the nucleic acids may be DNA or RNA, or a mixture thereof, and may exist permanently or transitionally in single-stranded or double-stranded form, including homoduplex, heteroduplex, and hybrid states. In some embodiments, a nucleic acid or nucleic acid sequence comprises other kinds of nucleic acid structures such as, for instance, a DNA/RNA helix, peptide nucleic acid (PNA), morpholino nucleic acid (see, e.g., Braasch and Corey, Biochemistry, 41(14): 4503-4510 (2002)) and U.S. Pat. No. 5,034,506), locked nucleic acid (LNA: see Wahlestedt et al., Proc. Natl. Acad. Sci. U.S.A., 97: 5633-5638 (2000)), cyclohexenyl nucleic acids (see Wang, J. Am. Chem. Soc., 122: 8595-8602 (2000)), and/or a ribozyme. Hence, the term nucleic acid or nucleic acid sequence may also encompass a chain comprising non-natural nucleotides, modified nucleotides, and/or non-nucleotide building blocks that can exhibit the same function as natural nucleotides (e.g., nucleotide analogs); further, the term nucleic acid sequence as used herein refers to an oligonucleotide, nucleotide or polynucleotide, and fragments or portions thereof, and to DNA or RNA of genomic or synthetic origin, which may be single or double-stranded, and represent the sense or antisense strand. The terms nucleic acid, polynucleotide, nucleotide sequence, and oligonucleotide are used interchangeably. They refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof.
[0420] A peptide or polypeptide is a linked sequence of two or more amino acids linked by peptide bonds. The peptide or polypeptide can be natural, synthetic, or a modification or combination of natural and synthetic. Polypeptides include proteins such as binding proteins, receptors, and antibodies. The proteins may be modified by the addition of sugars, lipids or other moieties not included in the amino acid chain. The terms polypeptide and protein, are used interchangeably herein.
[0421] As used herein, the term percent sequence identity refers to the percentage of nucleotides or nucleotide analogs in a nucleic acid sequence, or amino acids in an amino acid sequence, that is identical with the corresponding nucleotides or amino acids in a reference sequence after aligning the two sequences and introducing gaps, if necessary, to achieve the maximum percent identity. Hence, in case a nucleic acid according to the technology is longer than a reference sequence, additional nucleotides in the nucleic acid, that do not align with the reference sequence, are not taken into account for determining sequence identity. A number of mathematical algorithms for obtaining the optimal alignment and calculating identity between two or more sequences are known and incorporated into a number of available software programs. Examples of such programs include CLUSTAL-W, T-Coffee, and ALIGN (for alignment of nucleic acid and amino acid sequences), BLAST programs (e.g., BLAST 2.1, BL2SEQ, and later versions thereof) and FASTA programs (e.g., FASTA3, FAS, and SSEARCH) (for sequence alignment and sequence similarity searches). Sequence alignment algorithms also are disclosed in, for example, Altschul et al., J. Molecular Biol., 215(3): 403-410 (1990), Beigert et al., Proc. Natl. Acad. Sci. USA, 106(10): 3770-3775 (2009), Durbin et al., eds., Biological Sequence Analysis: Probabilistic Models of Proteins and Nucleic Acids, Cambridge University Press, Cambridge, UK (2009), Soding, Bioinformatics, 21(7): 951-960 (2005), Altschul et al., Nucleic Acids Res., 25(17): 3389-3402 (1997), and Gusfield, Algorithms on Strings, Trees and Sequences, Cambridge University Press, Cambridge UK (1997)).
[0422] The term amino acid or any amino acid as used here refers to any and all amino acids, including naturally occurring amino acids (e.g., a-amino acids), unnatural amino acids, modified amino acids, and non-natural amino acids. It includes both D- and L-amino acids. Natural amino acids include those found in nature, such as, e.g., the 23 amino acids that combine into peptide chains to form the building-blocks of a vast array of proteins. These are primarily L stereoisomers, although a few D-amino acids occur in bacterial envelopes and some antibiotics. The non-standard, natural amino acids include, for example, pyrolysine (found in methanogenic organisms and other eukaryotes), selenocysteine (present in many non-eukaryotes as well as most eukaryotes), and N-formylmethionine (encoded by the start codon AUG in bacteria, mitochondria, and chloroplasts). Unnatural or non-natural amino acids are non-proteinogenic amino acids (e.g., those not naturally encoded or found in the genetic code) that either occur naturally or are chemically synthesized. Over 140 unnatural amino acids are known and thousands of more combinations are possible. Examples of unnatural amino acids include -amino acids (.sup.3 and .sup.2), homo-amino acids, proline and pyruvic acid derivatives, 3-substituted alanine derivatives, glycine derivatives, ring-substituted phenylalanine and tyrosine derivatives, linear core amino acids, diamino acids, D-amino acids, alpha-methyl amino acids and N-methyl amino acids. Unnatural or non-natural amino acids also include modified amino acids. Modified amino acids include amino acids (e.g., natural amino acids) that have been chemically modified to include a group, groups, or chemical moiety not naturally present on the amino acid.
[0423] For the most part, the names of naturally occurring and non-naturally occurring aminoacyl residues used herein follow the naming conventions suggested by the IUPAC Commission on the Nomenclature of Organic Chemistry and the IUPAC-IUB Commission on Biochemical Nomenclature as set out in Nomenclature of -Amino Acids (Recommendations, 1974) Biochemistry, 14(2), (1975). To the extent that the names and abbreviations of amino acids and aminoacyl residues employed in this specification and appended claims differ from those suggestions, they will be made clear.
[0424] Throughout the present specification, unless naturally occurring amino acids are referred to by their full name (e.g., alanine, arginine, etc.), they are designated by their conventional three-letter or single-letter abbreviations (e.g., Ala or A for alanine, Arg or R for arginine, etc.). The term L-amino acid, as used herein, refers to the L isomeric form of a peptide, and conversely the term D-amino acid refers to the D isomeric form of a peptide (e.g., Dphe, (D)Phe, D-Phe, or .sup.DF for the D isomeric form of Phenylalanine). Amino acid residues in the D isomeric form can be substituted for any L-amino acid residue, as long as the desired function is retained by the peptide.
[0425] In the case of less common or non-naturally occurring amino acids, unless they are referred to by their full name (e.g. sarcosine, ornithine, etc.), frequently employed three- or four-character codes are employed for residues thereof, including, Sar or Sarc (sarcosine, i.e. N-methylglycine), Aib (-aminoisobutyric acid), Dab (2,4-diaminobutanoic acid). Dapa (2,3-diaminopropanoic acid), -Glu (-glutamic acid), Gaba (-aminobutanoic acid), -Pro (pyrrolidine-3-carboxylic acid), and 8Ado (8-amino-3,6-dioxaoctanoic acid), Abu (2-amino butyric acid), hPro (-homoproline), hPhe (-homophenylalanine) and Bip (,diphenylalanine), and Ida (Iminodiacetic acid).
[0426] The term pharmaceutically acceptable salt in the context of the present invention
[0427] The terms non-naturally occurring, engineered, and synthetic are used interchangeably and indicate the involvement of the hand of man. The terms, when referring to nucleic acid molecules or polypeptides mean that the nucleic acid molecule or the polypeptide is at least substantially free from at least one other component with which they are naturally associated in nature and as found in nature.
[0428] A vector or expression vector is a replicon, such as plasmid, phage, virus, or cosmid, to which another DNA segment, e.g., an insert, may be attached or incorporated so as to bring about the replication of the attached segment in a cell.
[0429] A cell has been genetically modified, transformed, or transfected by exogenous DNA, e.g., a recombinant expression vector, when such DNA has been introduced inside the cell. The presence of the exogenous DNA results in permanent or transient genetic change. The transforming DNA may or may not be integrated (covalently linked) into the genome of the cell. In prokaryotes, yeast, and mammalian cells for example, the transforming DNA may be maintained on an episomal element such as a plasmid. With respect to eukaryotic cells, a stably transformed cell is one in which the transforming DNA has become integrated into a chromosome so that it is inherited by daughter cells through chromosome replication. This stability is demonstrated by the ability of the eukaryotic cell to establish cell lines or clones that comprise a population of daughter cells containing the transforming DNA. A clone is a population of cells derived from a single cell or common ancestor by mitosis. A cell line is a clone of a primary cell that is capable of stable growth in vitro for many generations.
[0430] The term contacting as used herein refers to bring or put in contact, to be in or come into contact. The term contact as used herein refers to a state or condition of touching or of immediate or local proximity. Contacting a system to a target destination, such as, but not limited to, an organ, tissue, cell, or tumor, may occur by any means of administration known to the skilled artisan.
[0431] As used herein, the terms providing. administering. introducing, are used interchangeably herein and refer to the placement of the systems, recombinases, or nucleic acids of the disclosure into a cell, organism, or subject by a method or route which results in at least partial localization of the system to a desired site. The systems, recombinases, or nucleic acids can be administered by any appropriate route which results in delivery to a desired location in the cell, organism, or subject.
[0432] A subject or patient may be human or non-human and may include, for example, animal strains or species used as model systems for research purposes, such a mouse model as described herein. Likewise, patient may include either adults or juveniles (e.g., children). Moreover, patient may mean any living organism, preferably a mammal (e.g., human or non-human) that may benefit from the administration of compositions contemplated herein. Examples of mammals include, but are not limited to, any member of the Mammalian class: humans, non-human primates such as chimpanzees, and other apes and monkey species: farm animals such as cattle, horses, sheep, goats, swine; domestic animals such as rabbits, dogs, and cats; laboratory animals including rodents, such as rats, mice and guinea pigs, and the like. Examples of non-mammals include, but are not limited to, birds, fish, and the like. In one embodiment of the methods and compositions provided herein, the mammal is a human.
[0433] Preferred methods and materials are described below, although methods and materials similar or equivalent to those described herein can be used in practice or testing of the present disclosure. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety. The materials, methods, and examples disclosed herein are illustrative only and not intended to be limiting.
2. RECOMBINASE SYSTEMS
[0434] The present disclosure provides systems for DNA modification comprising: a polypeptide comprising a recombinase (e.g., a large serine recombinase) having an amino acid sequence having at least 70% identity (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100%) to any of SEQ ID NOs: 1-74, or a nucleic acid encoding thereof; and a first polynucleotide comprising a donor recognition sequence for the recombinase. Also provided herein are enzymatically active fragments thereof (e.g., C- or N-terminal truncations or containing internal deletions, but retaining the desired enzymatic activity). The active fragment may contain at least 20 amino acids, at least 30 amino acids, at least 40 amino acids, at least 50 amino acids, at least 100 amino acids, or more of SEQ ID NOs: 1-74 or sequences at least 70% identity to at least 20 amino acids, at least 30 amino acids, at least 40 amino acids, at least 50 amino acids, at least 100 amino acids, or more of SEQ ID NOs: 1-74. In some embodiments, the recombinase has an amino acid sequence having at least 70% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100%) identity to any of SEQ ID NOs: 2, 6, 10, 12, 18, 19, 26, 29, 61, 65, or 66, or an active fragment thereof. In select embodiments, the recombinase has an amino acid sequence of SEQ ID NOs: 2, 6, 10, 12, 18, 19, 26, 29, 61, 65, or 66, or an active fragment thereof.
[0435] The present disclosure also provides systems for DNA modification comprising: a polypeptide comprising a recombinase (e.g., a large serine recombinase), or a nucleic acid encoding thereof; and a first polynucleotide comprising a donor recognition sequence for the recombinase, wherein the recombinase (e.g., a large serine recombinase) comprises one or more of the following amino acid motifs, written in the common Prosite format, where the potential amino acids at any one position are in square brackets, x is any amino acid and x(n) represents n number of any amino acid (e.g., x(3) is xxx or 3 consecutive amino acids):
TABLE-US-00001 Motif1: [AEILSTVY]-[ADEGKQRST]-x(3)-[EG]-x-[ACFLMV]-x-[AFILMTV]-x(2)-[FHILMNV]- [AGSV]-[ADILSTV]-x-[AGS]-x(3)-[KRSV]-[ADEGKNST]-[AEIKMNQST]-[FILMST]-x- [DELQSV]-[ENQR]-x(4)-[AFHIKLMNQRSV]-x-[AEGHKLMNQRSV] Motif2: [AGI]-[DEGNPSTV]-[DGNQS]-[AHNQRTVY]-x-[ADEHILPQRTY]-[ADEQR]-[FIKL]-x- [DEFGNQRSTV]-[AILSTV]-[DEIKLNQRSTV]-[ADEKMNRSTV]-[AGQRST]-x- [ADEKLQRT]-x-[ALMV] Motif3: [ADFILMNSY]-x(2)-[AIKMSV]-x-[AFGILMV]-x(3)-[QRT]-[AGS]-x-[DEGNQS]-E-S-x- [AHKNRSTV]-K-x(2)-[LMRY]-[AINQSTV]-[AEFIKLNRTV]-x-[AFHLNQSTY]- [AILMNRSTVY] Motif4: [EKNTGSLDVARP]-[EHITGSLDVAP]-x-[MITSLVARP]-[EKNITGSDQVARP]- [EGSDARP]-[ILDAR]-[MHKTLVQDAR]-[EKITGSLDQVA]-[EKHDQVAR]- [MHISLVQAR]-[QEKNMSLDVAR]-[EKHGSLDQAR]-[EYKNIHLVA]-x- [EKITGSLDQAR]-[EKHTGDQAR]-x-[QEKNTGSDVAR]-[QEKNTGSVDAR]- [SWLVFAR]-[QEMTGSLVDA]-[EKNITGSDARP]-[EMILDQA]-[EYILVFAR]- [EMTGSLDVAR]-[EKNGSLDQAR]-[QEGVDARP] Motif5: [ADEHKNQRS]-[ADEFGHKMNQRSWY]-[EFY]-[FHLWY]-x-[ADEFIKLMNQRSTY]- [FIQSTV]-[AGKLNRSTV]-[ADEHKNQRTY]-[INQR]-[FILMQS]-x(2)-[AGKNS]- [KMQRSTV]-x(2)-[AEGKMNSTY] Motif6: W-[AEHNRSTV]-x-[AGNST]-[FGLMNQSTV]-[ILPV]-x(2)-[ILTV]-x(4)-[ACGMQRST]-x- [ILVY]-G-[DEHNQS]-x-[EHILMQRT]-[AEFHLNPY]-[CFHKMNQRTY]-[DEFIKLNQRSTV] Motif7: [AGINSTV]-x-[AIS]-x-[FILMY]-E-[IR]-x(2)-[DILT]-x-[AEIKMQS]-R-[ITV]-x- [ADGRST]-x-[FKLMY]-[AEHIKLMNQRVWY]-x-[AIKLMR] Motif8: [FY]-[DEKQS]-[EKLMQ]-[KLR]-[KLV]-x-[GN]-[DEHKLMR]-[ST]-x-[FHIQSTVW] Motif9: [ILV]-x(2)-[ADFHILMNQSVY]-x(3)-[AGS]-x-[DEIKNQRS]-[EQ]-S-x(2)-[AK]-[AQRS]- x-[LMR]-[ILQRSV]-x-[ADEGHIQRS]-[AKNQSTV]-[AHKRWY]-x-[AGHIKQRST]-x- [CHIKLRV] Motif10: R-[LMQR]-[ANS]-[NPST]-W Motif11: [LV]-[AV]-x-[AFHILQWY]-[IMV]-x-[ELQT]-[AIV]-F Motif12: R-[DKNRSV]-[ADEFGKPQS]-[AEIKLSTV]-x-[FGILNV]-[AFILQRVY]-[DEILMNQSTV]- [DEFILMQTVY]-[IKLRV]-[DEKNQR]-[DEFKLNQWY]-[FL] Motif13: [AEFILMNQSTVY]-[AFGILMRSTV]-x(3)-[ADEFGHLMNST]-x(2)-[DMNS]-[DEQ]-x- [CFHLTVY]-x-[AEKLRY]-x(2)-[ALS]-x-[DEKNQRS]-[GIMQRTV]-[DHKNQR]-x- [AGILNSTV]-[FHIKLMNQVWY]
[0436] Alternatively, the motifs can be written as the following, where each position is defined by a designated amino acid or X, wherein Xis the amino acid options in brackets, or any amino acid, as indicated.
Motif 1:
X.sub.1aX.sub.2aX.sub.3aX.sub.4aX.sub.5aX.sub.6aX.sub.7aX.sub.8aX.sub.9aX.sub.10aX.sub.11aX.sub.12aX.sub.13aX.sub.14aX.sub.15aX.sub.16aX.sub.17aX.sub.18aX.sub.19aX.sub.20aX.sub.21aX.sub.22aX.sub.23aX.sub.24aX.sub.25aX.sub.26aX.sub.27aX.sub.28aX.sub.29aX.sub.30aX.sub.31aX.sub.32aX.sub.33aX.sub.34a, wherein:
[0437] X.sub.3a, X.sub.4a, X.sub.5a, X.sub.7a, X.sub.9a, X.sub.11a, X.sub.12a, X.sub.16a, X.sub.18a, X.sub.19a, X.sub.20a, X.sub.25a, X.sub.28a, X.sub.29a, X.sub.30a, X.sub.31a, and X.sub.33a are each individually selected from any amino acid;
[0438] X.sub.1a is A, E, I, L, S, T, V, or Y;
[0439] X.sub.2a is A, D, E, G, K, Q, R, S, or T;
[0440] X.sub.6a is E or G;
[0441] X.sub.8a is A, C, F, L, M, or V;
[0442] X.sub.10a is A, F, I, L, M, T, or V;
[0443] X.sub.13a is F, H, I, L, M, N, or V;
[0444] X.sub.14a is A, G, S, or V;
[0445] X.sub.15a is A, D, I, L, S, T, or V;
[0446] X.sub.17a is A, G, or S;
[0447] X.sub.21a is K, R, S, or V;
[0448] X.sub.22a is A, D, E, G, K, N, S, or T;
[0449] X.sub.23a is A, E, I, K, M, N, Q, S, or T;
[0450] X.sub.24a is F, I, L, M, S, or T;
[0451] X.sub.26a is D, E, L, Q, S, or V;
[0452] X.sub.27a is E, N, Q, or R;
[0453] X.sub.32a is A, F, H, I, K, L, M, N, Q, R, S, or V
[0454] X.sub.34a is A, E, G, H, K, L, M, N, Q, R, S, or V
Motif 2:
X.sub.1bX.sub.2bX.sub.3bX.sub.4bX.sub.5bX.sub.6bX.sub.7bX.sub.8bX.sub.9bX.sub.10bX.sub.11bX.sub.12bX.sub.13bX.sub.14bX.sub.15bX.sub.16bX.sub.17bX.sub.18b, wherein
[0455] X.sub.5b, X.sub.9b, X.sub.15b, and X.sub.17b are each individually selected from any amino acid;
[0456] X.sub.1b is A, G, or I;
[0457] X.sub.2b is D, E, G, N, P, S, T, or V;
[0458] X.sub.3b is D, G, N, Q, or S;
[0459] X.sub.4b is A, H, N, Q, R, T, V, or Y;
[0460] X.sub.6b is A, D, E, H, I, L, P, Q, R, T, or Y;
[0461] X.sub.7b is A, D, E, Q, or R;
[0462] X.sub.8b is F, I, K, or L;
[0463] X.sub.10b is D, E, F, G, N, Q, R, S, T, or V;
[0464] X.sub.11b is A, I, L, S, T, or V;
[0465] X.sub.12b is D, E, I, K, L, N, Q, R, S, T, or V;
[0466] X.sub.13b is A, D, E, K, M, N, R, S, T, or V;
[0467] X.sub.14b is A, G, Q, R, S, or T;
[0468] X.sub.16b is A, D, E, K, L, Q, R, or T; and
[0469] X.sub.18b is A, L, M, or V
Motif 3:
X.sub.1cX.sub.2cX.sub.3cX.sub.4cX.sub.5cX.sub.6cX.sub.7cX.sub.8cX.sub.9cX.sub.10cX.sub.11cX.sub.12cX.sub.13cESX.sub.16cX.sub.17cKX.sub.19cX.sub.20cX.sub.21cX.sub.22cX.sub.23cX.sub.24cX.sub.25cX.sub.26c, wherein
[0470] X.sub.2c, X.sub.3c, X.sub.5c, X.sub.7c, X.sub.8c, X.sub.9c, X.sub.12c, X.sub.16c, X.sub.19c, X.sub.20c, and X.sub.24c are each individually selected from any amino acid;
[0471] X.sub.1c is A, D, F, I, L, M, N, S, or Y;
[0472] X.sub.4c is A, I, K, M, S, or V;
[0473] X.sub.6c is A, F, G, I, L, M, or V;
[0474] X.sub.10c is Q, R, or T;
[0475] X.sub.11c is A, G, or S;
[0476] X.sub.13c is D, E, G, N, Q, or S;
[0477] X.sub.17c is A, H, K, N, R, S, T, or V;
[0478] X.sub.21c is L, M, R, or Y;
[0479] X.sub.22c is A, I, N, Q, S, T, or V;
[0480] X.sub.23c is A, E, F, I, K, L, N, R, T, or V;
[0481] X.sub.25c is A, F, H, L, N, Q, S, T, or Y;
[0482] X.sub.26c is A, I, L, M, N, R, S, T, V, or Y
Motif 4:
X.sub.1dX.sub.2dX.sub.3dX.sub.4dX.sub.5dX.sub.6dX.sub.7dX.sub.8dX.sub.9dX.sub.10dX.sub.11dX.sub.12dX.sub.13dX.sub.14dX.sub.15dX.sub.16dX.sub.17dX.sub.18dX.sub.19dX.sub.20dX.sub.21dX.sub.22dX.sub.23dX.sub.24dX.sub.25dX.sub.26dX.sub.27dX.sub.28d, wherein:
[0483] X.sub.3d, X.sub.15d, and X.sub.18d are each individually selected from any amino acid;
[0484] X.sub.1d is E, K, N, T, G, S, L, D, V, A, R, or P;
[0485] X.sub.2d is E, H, I, T, G, S, L, D, V, A, or P;
[0486] X.sub.4d is M, I, T, S, L, V, A, R or P;
[0487] X.sub.5d is E, K, N, I, T, G, S, D, Q, V, A, R, or P;
[0488] X.sub.6d is E, G, S, D, A, R, or P;
[0489] X.sub.7d is I, L, D, A, or R;
[0490] X.sub.8d is M, H, K, T, L, V, Q, D, A, or R;
[0491] X.sub.9d is E, K, I, T, G, S, L, D, Q, V, or A;
[0492] X.sub.10d is E, K, H, D, Q, V, A, or R;
[0493] X.sub.11d is M, H, I, S, L, V, Q, A, or R;
[0494] X.sub.12d is Q, E, K, N, M, S, L, D, V, A, or R;
[0495] X.sub.13d is E, K, H, G, S, L, D, Q, A, or R;
[0496] X.sub.14d is E, Y, K, N, I, H, L, V, or A;
[0497] X.sub.16d is E, K, I, T, G, S, L, D, Q, A, or R;
[0498] X.sub.17d is E, K, H, T, G, D, Q, A, or R:
[0499] X.sub.19d is Q, E, K, N, T, G, S, D, V, A, or R;
[0500] X.sub.20d is Q, E, K, N, T, G, S, V, D, A, or R;
[0501] X.sub.21d is I, S, W, L, V, F, A, or R;
[0502] X.sub.22d is Q, E, M, T, G, S, L, V, D, or A;
[0503] X.sub.23d is E, K, N, I, T, G, S, D, A, R, or P;
[0504] X.sub.24d is E, M, I, L, D, Q, or A:
[0505] X.sub.25d is E, Y, I, L, V, F, A, or R;
[0506] X.sub.26d is E, M, T, G, S, L, D, V, A, or R;
[0507] X.sub.27d is E, K, N, G, S, L, D, Q, A, or R;
[0508] X.sub.28d is Q, E, G, V, D, A, R, or P; and
Motif 5:
X.sub.1eX.sub.2eX.sub.3eX.sub.4eX.sub.5eX.sub.6eX.sub.7eX.sub.8eX.sub.9eX.sub.10eX.sub.11eX.sub.12eX.sub.13eX.sub.14eX.sub.15eX.sub.16eX.sub.17eX.sub.18e, wherein:
[0509] X.sub.5e, X.sub.12e, X.sub.13e, X.sub.16e, and X.sub.17e are each individually selected from any amino acid;
[0510] X.sub.1e is A, D, E, H, K, N, Q, R, or S;
[0511] X.sub.2e is A, D, E, F, G, H, K, M, N, Q, R, S, W, or Y;
[0512] X.sub.3e is E, F, or Y;
[0513] X.sub.4e is F, H, L, W, or Y;
[0514] X.sub.6e is A, D, E, F, I, K, L, M, N, Q, R, S, T, or Y;
[0515] X.sub.7e is F, I, Q, S, T, or V;
[0516] X.sub.8e is A, G, K, L, N, R, S, T, or V;
[0517] X.sub.9e is A, D, E, H, K, N, Q, R, T, or Y;
[0518] X.sub.10e is I, N, Q, or R;
[0519] X.sub.11e is F, I, L, M, Q, or S;
[0520] X.sub.14e is A, G, K, N, or S;
[0521] X.sub.15e is K, M, Q, R, S, T, or V;
[0522] X.sub.18e is A, E, G, K, M, N, S, T, or Y;
Motif 6:
WX.sub.2fX.sub.3fX.sub.4fX.sub.5fX.sub.6fX.sub.7fX.sub.8fX.sub.9fX.sub.10fX.sub.11fX.sub.12fX.sub.13fX.sub.14fX.sub.15fX.sub.16fGX.sub.18fX.sub.19fX.sub.20fX.sub.21fX.sub.22fX.sub.23f, wherein:
[0523] X.sub.3f, X.sub.7f, X.sub.8f X.sub.10f, X.sub.11f, X.sub.12f, X.sub.13f, X.sub.15f, and X.sub.19f are each individually selected from any amino acid;
[0524] X.sub.2f is A, E, H, N, R, S, T, or V;
[0525] X.sub.4f is A, G, N, S, or T;
[0526] X.sub.5f is F, G, L, M, N, Q, S, T, or V;
[0527] X.sub.6f is I, L, P, or V;
[0528] X.sub.9f is I, L, T, or V;
[0529] X.sub.14f is A, C, G, M, Q, R, S, or T;
[0530] X.sub.16f is I, L, V, or Y;
[0531] X.sub.18f is D, E, H, N, Q, or S;
[0532] X.sub.20f is E, H, I, L, M, Q, R. or T;
[0533] X.sub.21f is A, E, F, H, L, N, P, or Y;
[0534] X.sub.22f is C, F, H, K, M, N, Q, R, T, or Y;
[0535] X.sub.23f is D, E, F, I, K, L, N, Q, R, S, T, or V;
Motif 7:
X.sub.1gX.sub.2gX.sub.3gX.sub.4gX.sub.5gEX.sub.7gX.sub.8gX.sub.9gX.sub.10gX.sub.11gX.sub.12gRX.sub.14gX.sub.15gX.sub.16gX.sub.17gX.sub.18gX.sub.19gX.sub.20gX.sub.21g, wherein:
[0536] X.sub.2g, X.sub.4g, X.sub.8g, X.sub.9g, X.sub.11g, X.sub.15g, X.sub.17g, and X.sub.20g are each individually selected from any amino acid;
[0537] X.sub.1g is A, G, I, N, S, T, or V;
[0538] X.sub.3g is A, I, or S;
[0539] X.sub.5g is F, I, L, M, or Y;
[0540] X.sub.7g is I or R;
[0541] X.sub.10g is D, I, L, or T;
[0542] X.sub.12g is A, E, I, K, M, Q, or S;
[0543] X.sub.14g is I, T, or V;
[0544] X.sub.16g is A, D, G, R, S, or T;
[0545] X.sub.18g is F, K, L, M, or Y;
[0546] X.sub.19g is A, E, H, I, K, L, M, N, Q, R, V, W, or Y;
[0547] X.sub.21g is A, I, K, L, M, or R
Motif 8:
X.sub.1hX.sub.2hX.sub.3hX.sub.4hX.sub.5hX.sub.6hX.sub.7hX.sub.8hX.sub.9hX.sub.10hX.sub.11h, wherein:
[0548] X.sub.6h and X.sub.10h are each individually selected from any amino acid;
[0549] X.sub.1h is F or Y;
[0550] X.sub.2h is D, E, K, Q, or S;
[0551] X.sub.3h is E, K, L, M, or Q;
[0552] X.sub.4h is K, L, or R:
[0553] X.sub.5h is K, L, or V;
[0554] X.sub.7h is G or N;
[0555] X.sub.8h is D, E, H, K, L, M, or R;
[0556] X.sub.9h is S or T;
[0557] X.sub.11h is F, H, I, Q, S, T, V, or W
Motif 9:
X.sub.1iX.sub.2iX.sub.3iX.sub.4iX.sub.5iX.sub.6iX.sub.7iX.sub.8iX.sub.9iX.sub.10iX.sub.11iX.sub.13iX.sub.14iX.sub.15iX.sub.16iX.sub.17iX.sub.18iX.sub.19iX.sub.20iX.sub.21iX.sub.22iX.sub.23iX.sub.24iX.sub.25iX.sub.26iX.sub.27i, wherein:
[0558] X.sub.2i, X.sub.3i, X.sub.5i, X.sub.6i, X.sub.7i, X.sub.9i, X.sub.13i, X.sub.14i, X.sub.17i, X.sub.20i, X.sub.24i, and X.sub.26i are each individually selected from any amino acid;
[0559] X.sub.1i is I, L, or V;
[0560] X.sub.4i is A, D, F, H, I, L, M, N, Q, S, V, or Y;
[0561] X.sub.8i is A, G, or S;
[0562] X.sub.10i is D, E, I, K, N, Q, R, or S;
[0563] X.sub.11i is E or Q;
[0564] X.sub.15i is A or K;
[0565] X.sub.16i is A, Q, R, or S;
[0566] X.sub.18i is L, M, or R;
[0567] X.sub.19i is I, L, Q, R, S, or V;
[0568] X.sub.21i is A, D, E, G, H, I, Q. R, or S;
[0569] X.sub.22i is A, K, N, Q, S, T, or V;
[0570] X.sub.23i is A, H, K, R, W, or Y;
[0571] X.sub.25i is A, G, H, I, K, Q, R, S, or T;
[0572] X.sub.27i is C, H, I, K, L, R, or V
Motif 10:
RX.sub.2jX.sub.3jX.sub.4jW, wherein:
[0573] X.sub.2j is L, M, Q, or R;
[0574] X.sub.3j is A, N, or S; and
[0575] X.sub.4j is N, P, S, or T
Motif 11:
X.sub.1kX.sub.2kX.sub.3kX.sub.4kX.sub.5kX.sub.6kX.sub.7kX.sub.8kF, wherein:
[0576] X.sub.3k and X.sub.6k are each individually selected from any amino acid;
[0577] X.sub.1k is I, L, or V;
[0578] X.sub.2k is A or V;
[0579] X.sub.4k is A, F, H, I, L, Q, W, or Y;
[0580] X.sub.5k is I, M, or V;
[0581] X.sub.7k is E, L, Q, or T:
[0582] X.sub.8k is A, I, or V
Motif 12:
RX.sub.2lX.sub.3lX.sub.4lX.sub.5lX.sub.6lX.sub.7lX.sub.8lX.sub.9lX.sub.10lX.sub.11lX.sub.12lX.sub.13l, wherein:
[0583] X.sub.2l is D, K, N, R, S, or V;
[0584] X.sub.3l is A, D, E, F, G, K, P, Q, or S;
[0585] X.sub.4l is A, E, I, K, L, S, T, or V;
[0586] X.sub.5l is any amino acid:
[0587] X.sub.6l is F, G, I, L, N, or V;
[0588] X.sub.7l is A, F, I, L, Q, R, V, or Y;
[0589] X.sub.8l is D, E, I, L, M, N, Q, S, T, or V;
[0590] X.sub.9l is D, E, F, I, L, M, Q, T, V, or Y;
[0591] X.sub.10l is I, K, L, R, or V;
[0592] X.sub.11l is D, E, K, N, Q, or R;
[0593] X.sub.12l is D, E, F, K, L, N, Q, W, or Y; and
[0594] X.sub.13l is F or L
Motif 13:
X.sub.1mX.sub.2mX.sub.3mX.sub.4mX.sub.5mX.sub.6mX.sub.7mX.sub.8mX.sub.9mX.sub.10mX.sub.11mX.sub.12mX.sub.13mX.sub.14mX.sub.15mX.sub.16mX.sub.17mX.sub.18mX.sub.19mX.sub.20mX.sub.21mX.sub.22mX.sub.23mX.sub.24m, wherein:
[0595] X.sub.3m, X.sub.4m, X.sub.5m, X.sub.7m, X.sub.8m, X.sub.11m, X.sub.13m, X.sub.15m, X.sub.16m, X.sub.18m, and X.sub.22m, are each individually selected from any amino acid,
[0596] X.sub.1m is A, E, F, I, L, M, N, Q, S, T, V, or Y;
[0597] X.sub.2m is A, F, G, I, L, M, R, S, T, or V;
[0598] X.sub.6m is A, D, E, F, G, H, L, M, N, S, or T;
[0599] X.sub.9m is D, M, N, or S;
[0600] X.sub.10m is D, E, or Q;
[0601] X.sub.12m is C, F, H, L, T, V, or Y;
[0602] X.sub.14m is A, E, K, L, R, or Y;
[0603] X.sub.17m is A, L, or S;
[0604] X.sub.19m is D, E, K, N, Q, R, or S;
[0605] X.sub.20m is G, I, M, Q, R, T, or V;
[0606] X.sub.21m is D, H, K, N, Q, or R;
[0607] X.sub.23m is A, G, I, L, N, S, T, or V;
[0608] X.sub.24m is F, H, I, K, L, M, N, Q, V, W, or Y
[0609] In some embodiments, the recombinase may comprise an amino acid sequence having at least 70% identity (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100%) to any of amino acid motifs 1-13. The recombinase may also comprise enzymatically active fragments of the recited amino acid motifs (e.g., C- or N-terminal truncations or containing internal deletions, but retaining the desired enzymatic activity).
[0610] In some embodiments, the systems comprise a polypeptide comprising a recombinase having an amino acid sequence having at least 70% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100%) identity to any of SEQ ID NOs: 88-1183 (those listed in Tables 4 and 5). Also provided herein are enzymatically active fragments of SEQ ID NOs: 88-1183, from those sequences listed in Tables 4 and 5 (e.g., C- or N-terminal truncations or containing internal deletions, but retaining the desired enzymatic activity). The active fragment may contain at least 20 amino acids, at least 30 amino acids, at least 40 amino acids, at least 50 amino acids, at least 100 amino acids, or more of SEQ ID NOs: 88-1183 (Tables 4 and 5) or sequences at least 70% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100%) identity to at least 20 amino acids, at least 30 amino acids, at least 40 amino acids, at least 50 amino acids, at least 100 amino acids, or more of SEQ ID NOs: 88-1183 (Tables 4 and 5).
[0611] The term recombinase, as used herein, refers to a site-specific enzyme that mediates the recombination of DNA between recombinase recognition sequences, which results in the excision, integration, inversion, or exchange (e.g., translocation) of DNA fragments between the recombinase recognition sequences. In some embodiments, the recombinase is a large serine recombinase.
[0612] Large serine recombinases (LSRs) are site-specific recombinases that are commonly found on microbial mobile genetic elements and within phage genomes, allowing an invading phage to insert into the host genome and thus enter into their prophage state. The typical LSR is composed of distinct domains: an N-terminal resolvase domain that contains the active site; a recombinase domain that determines the DNA binding specificity of the enzyme; and a zinc beta ribbon domain and a coiled-coil motif implicated in additional binding specificity and irreversibility of forward integration reaction without excision cofactors. Based on detailed studies of the C31 LSR, the following mechanism has been proposed: two LSR monomers bind to the donor attachment site and two bind to the acceptor attachment sitethe four monomers come together to form a tetramer (
[0613] The first polynucleotide may be a part of a bacterial plasmid, bacteriophage, plant virus, retrovirus. DNA virus, autonomously replicating extra chromosomal DNA element, linear plasmid, mitochondrial or other organellar DNA, chromosomal DNA, and the like. In some embodiments, the first polynucleotide comprises a human nucleic acid sequence. In some embodiments, the first polynucleotide is an exogenous or synthetic polynucleotide (e.g., a vector or engineered plasmid).
[0614] The first polynucleotide may comprise a donor recognition site for the recombinase. Recognition sites are specific polynucleotide sequences that are recognized by the recombinase enzymes described herein. The terms attB and attP, which refer to attachment (or recombination) sites originally from a bacterial target and a phage donor, respectively, are used herein although recombination sites for particular enzymes may have different names (e.g., attD and attA). The recombination sites typically include left and right arms separated by a core or spacer region.
[0615] In some embodiments, the first polynucleotide further comprises a cargo nucleic acid. The cargo nucleic acid may encode a gene product including but not limited to RNAs (e.g., non-coding RNA, such as tRNA, rRNA, micro RNA (miRNA), and small interfering RNA (siRNA), and coding RNA, such as messenger RNA (mRNA)) or proteins or polypeptides. The cargo nucleic acid may encode a transcription or translational control element (e.g., promoter elements, response elements (e.g., activator/repressor sequences)). In some embodiments, the cargo nucleic acid encodes a therapeutic protein. In some embodiments, the cargo nucleic acid encodes a therapeutic RNA.
[0616] The donor DNA, and by extension the cargo nucleic acid, may of any suitable length to facilitate recombination and delivery of the full cargo nucleic acid, including, for example, about 50-100 bp (base pairs), about 100-1000 bp, at least or about 10 bp, at least or about 20 bp, at least or about 25 bp, at least or about 30 bp, at least or about 35 bp, at least or about 40 bp, at least or about 45 bp, at least or about 50 bp, at least or about 55 bp, at least or about 60 bp, at least or about 65 bp, at least or about 70 bp, at least or about 75 bp, at least or about 80 bp, at least or about 85 bp, at least or about 90 bp, at least or about 95 bp, at least or about 100 bp, at least or about 200 bp, at least or about 300 bp, at least or about 400 bp, at least or about 500 bp, at least or about 600 bp, at least or about 700 bp, at least or about 800 bp, at least or about 900 bp, at least or about 1 kb (kilobase pair), at least or about 2 kb, at least or about 3 kb, at least or about 4 kb, at least or about 5 kb, at least or about 6 kb, at least or about 7 kb, at least or about 8 kb, at least or about 9 kb, at least or about 10 kb, or less than 10 kb, in length or greater. The donor DNA, and the cargo nucleic acid, may be at least or about 10 kb, at least or about 50 kb, at least or about 100 kb, between 20 kb and 60 kb, between 20 kb and 100 kb.
[0617] In essence, by contacting a set of corresponding recombination recognition sites with a corresponding recombinase, the recombinase mediates recombination between the sites. In some embodiments, the first polynucleotide further comprises a recipient recognition sequence for the recombinase.
[0618] In some embodiments, the system further comprises a second polynucleotide comprising a recipient recognition sequence for the recombinase. The second polynucleotide may be a part of a bacterial plasmid, bacteriophage, plant virus, retrovirus, DNA virus, autonomously replicating extra chromosomal DNA element, linear plasmid, mitochondrial or other organellar DNA, chromosomal DNA, and the like. In some embodiments, the second polynucleotide comprises a human nucleic acid sequence.
[0619] The type of recognition site will vary depending on the recombinase. In some embodiments, the recombinase is a landing-pad LSRs that can integrate efficiently at a pre-installed recognition site. Examples of landing-pad LSRs are shown in Table 1 along with their corresponding recombination attachment sites. In some embodiments, the recombinase is a multi-targeting LSRs that can integrate efficiently at many different loci in a target genome. Examples of a multi-targeting LSRs are shown in Table 3 along with their corresponding recombination attachment sites. In some embodiments, the recombinase is genome-targeting LSRs that can integrate at one or several target sites in a given target (e.g., target genome). Examples of genome-targeting LSRs are shown in Table 2 along with their corresponding recombination attachment sites. Attachment sites can be determined by mapping the edges of mobile genetic elements, as described herein.
[0620] In some embodiments, the donor recognition sequence, the recipient recognition sequence, or both are pseudo-recognition sequences or pseudosites. Pseudo-recognition sequences or pseudosites refer to a recognition sequences which is not necessarily that which is the native recognition sequence for a given recombinase but rather is sufficient to promote recombination. The pseudo-recognition sequence differs in one or more nucleotides from the corresponding native recombinase recognition sequence (e.g., due to insertions, deletions, or substitutions). In some embodiments, the pseudo-recognition sequence may be less than 50% identical to the native sequence. Pseudo-recognition sequences may also be those sequences present as an endogenous sequence in a genome that differs from the sequence of a genome where the wild-type recognition sequence for the recombinase resides. Identification of pseudo-recognition sequences can be accomplished, for example, by using sequence alignment and analysis, where the query sequence is the recognition sequence of interest, as described herein.
[0621] Depending upon the relative locations of the recombination attachment sites, any one of a number of events can occur as a result of the recombination. For example, if the recombination attachment sites are present on different nucleic acid molecules, the recombination can result in integration of one nucleic acid molecule into a second molecule.
[0622] The recombination attachment sites can also be present on the same nucleic acid molecule. In such cases, the resulting product typically depends upon the relative orientation of the attachment sites. For example, recombination between sites that are in the parallel or direct orientation will generally result in excision of any DNA that lies between the recombination attachment sites. In contrast, recombination between attachment sites that are in the reverse orientation can result in inversion of the intervening DNA.
[0623] The present disclosure also provides nucleic acids encoding the recombinases disclosed herein. The present disclosure further provides nucleic acids encoding the first polynucleotide and the second polynucleotide. The recombinase and the first polynucleotide may be encoded by the same or different nucleic acids (e.g., vectors). In some embodiments, a nucleic acid sequence encoding a recombinase is transiently or stable integrated into a cell, tissue, or organism so that the cell, tissue, or organism expresses the heterologous recombinase.
[0624] Nucleic acids of the present disclosure can comprise any of a number of promoters known to the art, wherein the promoter is constitutive, regulatable or inducible, cell type specific, tissue-specific, or species specific. In addition to the sequence sufficient to direct transcription, a promoter sequence of the invention can also include sequences of other regulatory elements that are involved in modulating transcription (e.g., enhancers, Kozak sequences and introns). Many promoter/regulatory sequences useful for driving constitutive expression of a gene are available in the art and include, but are not limited to, for example, CMV (cytomegalovirus promoter), EF1a (human elongation factor 1 alpha promoter), SV40 (simian vacuolating virus 40 promoter), PGK (mammalian phosphoglycerate kinase promoter), Ubc (human ubiquitin C promoter), human beta-actin promoter, rodent beta-actin promoter, CBh (chicken beta-actin promoter), CAG (hybrid promoter contains CMV enhancer, chicken beta actin promoter, and rabbit beta-globin splice acceptor), TRE (Tetracycline response element promoter), HI (human polymerase III RNA promoter), U6 (human U6 small nuclear promoter), and the like. Additional promoters that can be used for expression of the components of the present system, include, without limitation, cytomegalovirus (CMV) intermediate early promoter, a viral LTR such as the Rous sarcoma virus LTR, HIV-LTR, HTLV-1 LTR, Maloney murine leukemia virus (MMLV) LTR, myeoloproliferative sarcoma virus (MPSV) LTR, spleen focus-forming virus (SFFV) LTR, the simian virus 40 (SV40) early promoter, herpes simplex tk virus promoter, elongation factor 1-alpha (EF1-) promoter with or without the EF1- intron. Additional promoters include any constitutively active promoter. Alternatively, any regulatable promoter may be used, such that its expression can be modulated within a cell.
[0625] Moreover, inducible expression can be accomplished by placing the nucleic acid encoding such a molecule under the control of an inducible promoter/regulatory sequence. Promoters that are well known in the art can be induced in response to inducing agents such as metals, glucocorticoids, tetracycline, hormones, and the like, are also contemplated for use with the invention. Thus, it will be appreciated that the present disclosure includes the use of any promoter/regulatory sequence known in the art that is capable of driving expression of the desired protein operably linked thereto.
[0626] The present disclosure also provides for vectors containing the nucleic acids or system and cells containing the nucleic acids or vectors, thereof. Thus, the disclosure further provides for cells comprising the serine recombinases or systems, as disclosed herein.
[0627] The vectors may be used to propagate the nucleic acid in an appropriate cell and/or to allow expression from the nucleic acid (e.g., an expression vector). The person of ordinary skill in the art would be aware of the various vectors available for propagation and expression of a nucleic acid sequence.
[0628] To construct cells that express the present system described herein, expression vectors for stable or transient expression of the present system may be constructed via conventional methods and introduced into cells. For example, nucleic acids may be cloned into a suitable expression vector, such as a plasmid or a viral vector in operable linkage to a suitable promoter. The selection of expression vectors/plasmids/viral vectors should be suitable for integration and replication in eukaryotic cells.
[0629] In certain embodiments, vectors of the present disclosure can drive the expression of one or more sequences in mammalian cells using a mammalian expression vector. Examples of mammalian expression vectors include pCDM8 (Seed, Nature (1987) 329:840, incorporated herein by reference) and pMT2PC (Kaufman, et al., EMBO J. (1987) 6:187, incorporated herein by reference). When used in mammalian cells, the expression vector's control functions are typically provided by one or more regulatory elements. For example, commonly used promoters are derived from polyoma, adenovirus 2, cytomegalovirus, simian virus 40, and others disclosed herein and known in the art. For other suitable expression systems for both prokaryotic and eukaryotic cells see, e.g., Chapters 16 and 17 of Sambrook, et al., MOLECULAR CLONING: A LABORATORY MANUAL. 2nd eds., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989, incorporated herein by reference.
[0630] The vectors of the present disclosure may direct the expression of the nucleic acid in a particular cell type (e.g., tissue-specific regulatory elements are used to express the nucleic acid). Such regulatory elements include promoters that may be tissue specific or cell specific. The term tissue specific as it applies to a promoter refers to a promoter that is capable of directing selective expression of a nucleotide sequence of interest to a specific type of tissue (e.g., seeds) in the relative absence of expression of the same nucleotide sequence of interest in a different type of tissue. The term cell type specific as applied to a promoter refers to a promoter that is capable of directing selective expression of a nucleotide sequence of interest in a specific type of cell in the relative absence of expression of the same nucleotide sequence of interest in a different type of cell within the same tissue. The term cell type specific when applied to a promoter also means a promoter capable of promoting selective expression of a nucleotide sequence of interest in a region within a single tissue. Cell type specificity of a promoter may be assessed using methods well known in the art, e.g., immunohistochemical staining.
[0631] Additionally, the vector may contain, for example, some or all of the following: a selectable marker gene for selection of stable or transient transfectants in host cells; transcription termination and RNA processing signals; 5- and 3-untranslated regions; internal ribosome binding sites (IRESes), versatile multiple cloning sites; and reporter gene for assessing expression of the chimeric receptor. Suitable vectors and methods for producing vectors containing transgenes are well known and available in the art. Selectable markers include chloramphenicol resistance, tetracycline resistance, spectinomycin resistance, neomycin, streptomycin resistance, erythromycin resistance, rifampicin resistance, bleomycin resistance, thermally adapted kanamycin resistance, gentamycin resistance, hygromycin resistance, trimethoprim resistance, dihydrofolate reductase (DHFR), GPT; the URA3, HIS4, LEU2, and TRP1 genes of S. cerevisiae.
[0632] Conventional viral and non-viral based gene transfer methods can be used to introduce the nucleic acids into cells, tissues, or a subject. Such methods can be used to administer the nucleic acids to cells in culture, or in a host organism. Non-viral vector delivery systems include DNA plasmids, cosmids, RNA (e.g., a transcript of a vector described herein), a nucleic acid, and a nucleic acid complexed with a delivery vehicle.
[0633] The nucleic acids may be delivered by any suitable means. In certain embodiments, the nucleic acids or proteins thereof are delivered in vivo. In other embodiments, the nucleic acids or proteins thereof are delivered to isolated/cultured cells in vitro or ex vivo to provide modified cells useful for in vivo delivery to patients afflicted with a disease or condition.
[0634] Vectors according to the present disclosure can be transformed, transfected, or otherwise introduced into a wide variety of host cells. Transfection refers to the taking up of a vector by a cell whether or not any coding sequences are in fact expressed. Numerous methods of transfection are known to the ordinarily skilled artisan, for example, lipofectamine, calcium phosphate co-precipitation, electroporation, DEAE-dextran treatment, microinjection, viral infection, and other methods known in the art. Transduction refers to entry of a virus into the cell and expression (e.g., transcription and/or translation) of sequences delivered by the viral vector genome. In the case of a recombinant vector, transduction generally refers to entry of the recombinant viral vector into the cell and expression of a nucleic acid of interest delivered by the vector genome.
[0635] Methods of delivering vectors to cells are well known in the art and may include DNA or RNA electroporation, transfection reagents such as liposomes or nanoparticles to delivery DNA or RNA; delivery of DNA, RNA, or protein by mechanical deformation (see, e.g., Sharei et al. Proc. Natl. Acad. Sci. USA (2013) 110(6): 2082-2087, incorporated herein by reference. Nucleic acids can be delivered as part of a larger construct, such as a plasmid or viral vector, or directly, e.g., by electroporation, lipid vesicles, viral transporters, microinjection, and biolistics (high-speed particle bombardment).
[0636] Additionally, delivery vehicles such as nanoparticle- and lipid-based delivery systems can be used. Further examples of delivery vehicles include lentiviral vectors, ribonucleoprotein (RNP) complexes, lipid-based delivery system, gene gun, hydrodynamic, electroporation or nucleofection microinjection, and biolistics. Various gene delivery methods are discussed in detail by Nayerossadat et al. (Adv Biomed Res. 2012: 1: 27) and Ibraheem et al. (Int J Pharm. 2014 Jan. 1; 459(1-2):70-83), incorporated herein by reference.
[0637] As such, the disclosure provides an isolated cell comprising the vector(s) or nucleic acid(s) disclosed herein. Preferred cells are those that can be easily and reliably grown, have reasonably fast growth rates, have well characterized expression systems, and can be transformed or transfected easily and efficiently. Examples of suitable prokaryotic cells include, but are not limited to, cells from the genera Bacillus (such as Bacillus subtilis and Bacillus brevis), Escherichia (such as E. coli), Pseudomonas, Streptomyces, Salmonella, and Envinia. Suitable eukaryotic cells are known in the art and include, for example, yeast cells, insect cells, and mammalian cells. Examples of suitable yeast cells include those from the genera Kluyveromyces, Pichia, Rhino-sporidium, Saccharomyces, and Schizosaccharomyces. Exemplary insect cells include Sf-9 and HIS (Invitrogen, Carlsbad, Calif.) and are described in, for example, Kitts et al., Biotechniques, 14: 810-817 (1993); Lucklow, Curr. Opin. Biotechnol., 4: 564-572 (1993); and Lucklow et al., J. Virol., 67: 4566-4579 (1993), incorporated herein by reference. Desirably, the cell is a mammalian cell, and in some embodiments, the cell is a human cell. A number of suitable mammalian and human host cells are known in the art, and many are available from the American Type Culture Collection (ATCC, Manassas, Va.). Examples of suitable mammalian cells include, but are not limited to, Chinese hamster ovary cells (CHO) (ATCC No. CCL61), CHO DHFR-cells (Urlaub et al., Proc. Natl. Acad. Sci. USA, 97: 4216-4220 (1980)), human embryonic kidney (HEK) 293 or 293T cells (ATCC No. CRL1573), and 3T3 cells (ATCC No. CCL92). Other suitable mammalian cell lines are the monkey COS-1 (ATCC No. CRL1650) and COS-7 cell lines (ATCC No. CRL1651), as well as the CV-1 cell line (ATCC No. CCL70). Further exemplary mammalian host cells include primate, rodent, and human cell lines, including transformed cell lines. Normal diploid cells, cell strains derived from in vitro culture of primary tissue, as well as primary explants, are also suitable. Other suitable mammalian cell lines include, but are not limited to, mouse neuroblastoma N2A cells, HeLa, HEK, A549, HepG2, mouse L-929 cells, and BHK or HaK hamster cell lines.
[0638] Methods for selecting suitable mammalian cells and methods for transformation, culture, amplification, screening, and purification of cells are known in the art.
[0639] The present invention is also directed to compositions comprising a recombinase, a system, a nucleic acid, a vector, or a cell, as described herein.
[0640] Further disclosed herein are methods for identifying recombinases for use in the systems and methods disclosed herein. In some embodiments, the methods comprise: acquiring bacterial genome sequences; identifying putative recombinase genes in the bacterial genome sequences based on predicted recombinase domain; comparing genomes encoding the putative recombinase genes with those without the putative recombinase genes; mapping boundaries of a mobile genetic element comprising the putative recombinase genes: determine recombinase recognition sequences and/or attachment sites. In some embodiments, the predicted recombinase domain is a Pfam domain. In some embodiments, the method further comprises isolating mobile genetic elements from the bacterial genome sequences prior to identifying the putative recombinase genes. Mapping boundaries of a mobile genetic element may comprise determining 3 and 5 flanking sequences of the mobile genetic element termini and, if present, the duplication sites created upon insertion of the mobile genetic element.
3. METHODS OF ALTERING DNA
[0641] Applications of genetic engineering through alteration of DNA has yielded impactful results including CAR-T cell therapies, genetically modified crops, and cells producing diverse compounds and medicines. In many of these applications, genomic integration is highly preferred over plasmid-based methods for maintaining heterologous genes in engineered cells, due to improved stability in the genome, better control of copy numbers, and regulatory concerns regarding biocontainment of recombinant DNA. However, generation of modified cells with kilobases of changes across the genome remains practically challenging, often requiring inefficient, multi-step processes that are time and resource intensive. The systems and methods described herein allow integration of a large (e.g., kilobase or larger) exogenous donor polynucleotide into a DNA sequence. The methods may be used in vitro, ex vivo, or in vivo and allow alteration of a target DNA strand in solution, in a cell, in a tissue, or in a subject.
[0642] The disclosure provides a method of altering a target nucleic acid sequence. The phrases altering a DNA sequence or altering a target DNA, as used herein, refer to modifying at least one physical feature of a DNA sequence of interest. DNA alterations include, for example, single or double strand DNA breaks, deletion, or insertion of one or more nucleotides, and other modifications that affect the structural integrity or nucleotide sequence of the DNA sequence.
[0643] In some embodiments, the methods comprise contacting a target nucleic acid sequence with a system disclosed herein or with a polypeptide comprising a recombinase having an amino acid sequence having at least 70% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100%) identity to any of SEQ ID NOs: 1-74, an enzymatically active fragment thereof, or a nucleic acid encoding thereof.
[0644] In some embodiments, the recombinase has an amino acid sequence having at least 70% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100%) identity to any of SEQ ID NOs: 2, 6, 10, 12, 18, 19, 26, 29, 61, 65, or 66. In select embodiments, the recombinase has an amino acid sequence of SEQ ID NOs: 2, 6, 10, 12, 18, 19, 26, 29, 61, 65, or 66.
[0645] In some embodiments, the methods comprise contacting a target nucleic acid sequence with a system disclosed herein or with a polypeptide comprising a recombinase having an amino acid sequence having at least 70% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100%) identity to any of motifs 1-13 as disclosed above, an enzymatically active fragment thereof, or a nucleic acid encoding thereof.
[0646] In some embodiments, the systems comprise a polypeptide comprising a recombinase having an amino acid sequence having at least 70% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100%) identity to any of SEQ ID NOs: 88-1183, those listed in Tables 4 and 5. Also provided herein are enzymatically active fragments of SEQ ID NOs: 88-1183, those sequences listed in Tables 4 and 5 (e.g., C- or N-terminal truncations or containing internal deletions, but retaining the desired enzymatic activity). The active fragment may contain at least 20 amino acids, at least 30 amino acids, at least 40 amino acids, at least 50 amino acids, at least 100 amino acids, or more of SEQ ID NOs: 88-1183 (Tables 4 and 5) or sequences at least 70% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100%) identity to at least 20 amino acids, at least 30 amino acids, at least 40 amino acids, at least 50 amino acids, at least 100 amino acids, or more of SEQ ID NOs: 88-1183 (Tables 4 and 5).
[0647] In some embodiments, the target DNA comprises a donor recognition sequence, a recipient recognition sequence, or both.
[0648] In some embodiments, the methods further comprise contacting the target DNA with a first polynucleotide comprising a donor recognition sequence for the recombinase. In some embodiments, the first polynucleotide further comprises a cargo DNA sequence. In some embodiments, the donor recognition sequence, the recipient recognition sequence, or both are pseudo-recognition sequences.
[0649] The descriptions and embodiments provided above for the disclosed system, recombinase, first and second polynucleotide, donor and recipient recognition sequences, and cargo DNA sequence are applicable to the methods described herein.
[0650] In some embodiments, the methods may comprise introducing the disclosed systems or recombinase, or a nucleic acid encoding thereof, and a donor polynucleotide into a cell. In some embodiments, the recombinase, or the nucleic acid encoding thereof, is introduced into the cell before the introduction of the donor polynucleotide. In some embodiments, the recombinase, or the nucleic acid encoding thereof, is introduced into the cell after the introduction of the donor polynucleotide. In some embodiments, the recombinase, or the nucleic acid encoding thereof, and the donor polynucleotide may be introduced, in any order, with a time period separating each introduction.
[0651] In some embodiments, the recombinase is part of a system comprising a Cas protein, a reverse transcriptase, or active fragments or combinations thereof. In some embodiments, the recombinase is in a fusion protein with a Cas protein (e.g., Cas 9) and a reverse transcriptase, or active fragments thereof. For example, a Programmable Addition via Site-specific Targeting Elements (PASTE) system which integrates large cargos in a single delivery. See, Eleonora I. Ioannidi, et al., bioRxiv 2021.11.01.466786, incorporated herein by reference in its entirety.
[0652] In some embodiments, the recombinase, or the nucleic acid encoding thereof, is introduced into the cell concurrently with the introduction of the donor polynucleotide. For example, the recombinase, or the nucleic acid encoding thereof, and the donor polynucleotide are introduced simultaneously or nearly simultaneously.
[0653] The cell can be a mitotic and/or post-mitotic cell from any eukaryotic cell or organism (e.g. a cell of a single-cell eukaryotic organism, a plant cell, an algal cell, a fungal cell (e.g., a yeast cell), an animal cell, a cell from an invertebrate animal (e.g. fruit fly, cnidarian, echinoderm, nematode, an insect, an arachnid, etc.), a cell from a vertebrate animal (e.g., fish, amphibian, reptile, bird, mammal), a cell from a mammal, a cell from a rodent, a cell from a human, etc.), or a protozoan cell. Any type of cell may be of interest (e.g. a stem cell, e.g. an embryonic stem (ES) cell, an induced pluripotent stem (iPS) cell, a germ cell; a somatic cell, e.g. a fibroblast, a hematopoietic cell, a neuron, a muscle cell, a bone cell, a hepatocyte, a pancreatic cell, a liver cell, a lung cell, a skin cell; an in vitro or in vivo embryonic cell of an embryo at any stage, e.g., a 1-cell, 2-cell, 4-cell, 8-cell, etc. stage zebrafish embryo; etc.). Cells may be from established cell lines or they may be primary cells, where primary cells, primary cell lines, and primary cultures are used interchangeably herein to refer to cells and cells cultures that have been derived from a subject and allowed to grow in vitro for a limited number of passages.
[0654] In some embodiments, the one or more cells are animal cells. The present disclosure provides for a modified animal cell produced by the present system and method, an animal comprising the animal cell, a population of cells comprising the cell, tissues, and at least one organ of the animal. The present disclosure further encompasses the progeny, clones, cell lines or cells of the genetically modified animal. The present cells may be used for transplantation (e.g., hematopoietic stem cells or bone marrow).
[0655] Non-limiting examples of animal cells that may be genetically modified using the systems and methods include, but are not limited to, cells from: mammals such as primates (e.g., ape, chimpanzee, macaque), rodents (e.g., mouse, rabbit, rat), canine or dog, livestock (cow/bovine, donkey, sheep/ovine, goat or pig), fowl or poultry (e.g., chicken), and fish (e.g., zebra fish). The present methods and systems may be used for cells from other eukaryotic model organisms, e.g., Drosophila, C. elegans, etc. In certain embodiments, the mammal is a human, a non-human primate (e.g., marmoset, rhesus monkey, chimpanzee), a rodent (e.g., mouse, rat, gerbil, Guinea pig, hamster, cotton rat, naked mole rat), a rabbit, a livestock animal (e.g., goat, sheep, pig, cow, cattle, buffalo, horse, camelid), a pet mammal (e.g., dog, cat), a zoo mammal, a marsupial, an endangered mammal, and an outbred or a random bred population thereof.
[0656] In some embodiments, the one or more cells comprise plant cells. Suitable plant cells may be from a number of different plants including, but are not limited to, monocotyledonous and dicotyledonous plants, such as crops including grain crops (e.g., wheat, maize, rice, millet, barley), fruit crops (e.g., tomato, apple, pear, strawberry, orange), forage crops (e.g., alfalfa), root vegetable crops (e.g., carrot, potato, sugar beets, yam), leafy vegetable crops (e.g., lettuce, spinach); flowering plants (e.g., petunia, rose, chrysanthemum), conifers and pine trees (e.g., pine fir, spruce); plants used in phytoremediation (e.g., heavy metal accumulating plants); oil crops (e.g., sunflower, rapeseed) and plants used for experimental purposes (e.g., Arabidopsis). Thus, the disclosed methods and compositions have use over a broad range of plants, including, but not limited to, species from the genera Asparagus, Avena, Brassica, Citrus, Citrullus, Capsicum, Cucurbita, Daucus, Glycine, Hordeum, Lactuca, Lycopersicon, Malus, Manihot, Nicotiana, Oryza, Persea, Pisum, Pyrus, Prunus, Raphanus, Secale, Solanum, Sorghum, Triticum, Vitis, Vigna, and Zea.
[0657] In some embodiments, the one or more cells comprise microbial cells. In some embodiments, the microbial cells are Gram-negative bacterial cells, Gram-positive bacterial cells, or a combination thereof. In some embodiments, the microbial cells are pathogenic bacterial cells. In some embodiments, the microbial cells are non-pathogenic bacterial cells (e.g., probiotic and/or commensal bacterial cells). In some embodiments, the microbial cells form microbial flora (e.g., natural human microbial flora). In some embodiments, the microbial cells are used in industrial or environmental bioprocesses (e.g., bioremediation).
[0658] The cell can be a cancer cell. An appropriate cancer cell can be derived from a breast cancer, lung cancer, colon cancer, pancreatic cancer, renal cancer, stomach cancer, liver cancer, bone cancer, hematological cancer (e.g., leukemia or lymphoma), neural tissue cancer, melanoma, ovarian cancer, testicular cancer, prostate cancer, cervical cancer, vaginal cancer, or bladder cancer.
[0659] The systems and methods may be used to modify a stem cell. The term stem cell is used herein to refer to a cell that has the ability both to self-renew and to generate a differentiated cell type (see Morrison et al. (1997) Cell 88:287-298, incorporated herein by reference). Stem cells may be characterized by both the presence of specific markers (e.g., proteins, RNAs, etc.) and the absence of specific markers. Stem cells may also be identified by functional assays both in vitro and in vivo, particularly assays relating to the ability of stem cells to give rise to multiple differentiated progeny. Examples of stem cells include pluripotent, multipotent and unipotent stem cells. Examples of pluripotent stem cells include embryonic stem cells, embryonic germ cells, embryonic carcinoma cells and induced pluripotent stem cells (iPSCs). The cell may be an induced pluripotent stem cell (iPSC), e.g., derived from a fibroblast of a subject. In another embodiment, the cell can be a fibroblast. In some embodiments, the cell may be a cancer stem cell.
[0660] The present disclosure further provides progeny of a genetically modified cell, where the progeny can comprise the same genetic modification as the genetically modified cell from which it was derived. The present disclosure further provides a composition comprising a genetically modified cell. In some embodiments, a genetically modified host cell can generate a genetically modified organism. For example, the genetically modified host cell is a pluripotent stem cell, it can generate a genetically modified organism. Methods of producing genetically modified organisms are known in the art.
[0661] In some embodiments, the cell is in an organism or host, such that introducing the disclosed recombinases, systems, compositions, nucleic acids, or vectors into the cell comprises administration to a subject. The method may comprise providing or administering to the subject, in vivo, or by transplantation of ex vivo treated cells, a recombinase, nucleic acid, vector, composition, or system as described herein.
[0662] Cell replacement therapy can be used to prevent, correct, or treat a disease or condition, where the methods of the present disclosure are applied to isolated subject's cells (ex vivo), which is then followed by the administration of the genetically modified cells into the patient.
[0663] The cell may be autologous or allogeneic to the subject who is administered the cell. As described herein, the genetically modified cells may be autologous to the subject, e.g., the cells are obtained from the subject in need of the treatment, genetically engineered, and then administered to the same subject. Alternatively, the host cells are allogeneic cells, e.g., the cells are obtained from a first subject, genetically engineered, and administered to a second subject that is different from the first subject but of the same species. In some embodiments, the genetically modified cells are allogeneic cells and have been further genetically engineered to reduced graft-versus-host disease.
[0664] A subject may be human or non-human and may include, for example, animal strains or species used as model systems for research purposes, such a mouse model as described herein. Likewise, subject may include either adults or juveniles (e.g., children). Moreover, subject may mean any living organism, preferably a mammal (e.g., human or non-human) that may benefit from the administration of compositions contemplated herein. Examples of mammals include, but are not limited to, any member of the Mammalian class: humans, non-human primates such as chimpanzees, and other apes and monkey species; farm animals such as cattle, horses, sheep, goats, swine; domestic animals such as rabbits, dogs, and cats; laboratory animals including rodents, such as rats, mice and guinea pigs, and the like. Examples of non-mammals include, but are not limited to, birds, fish, and the like. In one embodiment of the methods and compositions provided herein, the mammal is a human.
[0665] The methods find use in inactivating a gene of interest or deleting a nucleic acid sequence. In some embodiments, the disclosed methods alter a target genomic DNA sequence in a host cell, tissue, or subject so as to modulate expression of the target DNA sequence, e.g., expression of the target DNA sequence is increased, decreased, or completely eliminated (e.g., via deletion of a gene or insertion or inversion of a promoter element). In some embodiments, the systems and methods described herein may be used to introduce an exogenous donor polynucleotide into a target DNA sequence.
[0666] In some embodiments, the target DNA encodes a gene product. The term gene product, as used herein, refers to any biochemical product resulting from expression of a gene. Gene products may be RNA or protein. RNA gene products include non-coding RNA, such as tRNA, rRNA, micro RNA (miRNA), and small interfering RNA (siRNA), and coding RNA, such as messenger RNA (mRNA). In some embodiments, the target genomic DNA sequence encodes a protein or polypeptide. However, the invention is not limited to editing of gene products. Any target DNA sequence may be edited, as desired. For example, in some embodiments, target DNA comprises non-coding DNA or comprises regions which are responsible for producing RNA. In some embodiments, the gene of interest is located chromosomally. In some embodiments, the gene of interest is located episomally, e.g., in bacterial cells.
[0667] Methods for inactivating a gene of interest comprise introducing into one or more cells the recombinases, systems, nucleic acids, or vectors described herein, wherein the target nucleic acid sequence comprises at least a portion of the gene of interest. The gene of interest may comprise any gene of interest to inactivate. In some embodiments, the gene of interest comprises an antibiotic resistance gene, a virulence gene, a metabolic gene, a toxin gene, a remodeling gene, a gene or gene variant responsible for a disease, or a mutant gene.
[0668] In select embodiments, the systems and methods described herein may be used to correct one or more defects or mutations in a gene (referred to as gene correction). In such cases, the cell or target sequence encodes a defective version of a gene, and the disclosed system further comprises a cargo nucleic acid molecule which encodes a wild-type or corrected version of the gene. Thus, in other words, the cell expresses a disease-associated gene. The term disease-associated gene, refers to any gene or polynucleotide whose gene products are expressed at an abnormal level or in an abnormal form in cells obtained from a disease-affected individual as compared with tissues or cells obtained from an individual not affected by the disease. A disease-associated gene may be expressed at an abnormally high level or at an abnormally low level, where the altered expression correlates with the occurrence and/or progression of the disease. A disease-associated gene also refers to a gene, the mutation or genetic variation of which is directly responsible or is in linkage disequilibrium with a gene(s) that is responsible for the etiology of a disease. Examples of genes responsible for such single gene or monogenic diseases include, but are not limited to, adenosine deaminase, -1 antitrypsin, cystic fibrosis transmembrane conductance regulator (CFTR), -hemoglobin (HBB), oculocutaneous albinism II (OCA2), Huntingtin (HTT), dystrophia myotonica-protein kinase (DMPK), low-density lipoprotein receptor (LDLR), apolipoprotein B (APOB), neurofibromin 1 (NF1), polycystic kidney disease 1 (PKD1), polycystic kidney disease 2 (PKD2), coagulation factor VIII (F8), dystrophin (DMD), phosphate-regulating endopeptidase homologue, X-linked (PHEX), methyl-CpG-binding protein 2 (MECP2), and ubiquitin-specific peptidase 9Y, Y-linked (USP9Y). Other single gene or monogenic diseases are known in the art and described in, e.g., Chial, H. Rare Genetic Disorders: Learning About Genetic Disease Through Gene Mapping, SNPs, and Microarray Data, Nature Education 1(1):192 (2008); Online Mendelian Inheritance in Man (OMIM); and the Human Gene Mutation Database (HGMD). In another embodiment, the target genomic DNA sequence can comprise a gene, the mutation of which contributes to a particular disease in combination with mutations in other genes. Diseases caused by the contribution of multiple genes which lack simple (i.e., Mendelian) inheritance patterns are referred to in the art as a multifactorial or polygenic disease. Examples of multifactorial or polygenic diseases include, but are not limited to, asthma, diabetes, epilepsy, hypertension, bipolar disorder, and schizophrenia. Certain developmental abnormalities also can be inherited in a multifactorial or polygenic pattern and include, for example, cleft lip/palate, congenital heart defects, and neural tube defects.
4. KITS
[0669] Also within the scope of the present disclosure are kits including a recombinase, or nucleic acid encoding thereof, a donor or first polynucleotide, a composition, or system as described herein, or a cell comprising a system as described herein or a recombinase as described herein.
[0670] The kits can also comprise instructions for using the components of the kit. The instructions are relevant materials or methodologies pertaining to the kit. The materials may include any combination of the following: background information, list of components, brief or detailed protocols for using the compositions, trouble-shooting, references, technical support, and any other related documents. Instructions can be supplied with the kit or as a separate member component, either as a paper form or an electronic form which may be supplied on computer readable memory device or downloaded from an internet website, or as recorded presentation.
[0671] It is understood that the disclosed kits can be employed in connection with the disclosed methods. The kit may include instructions for use in any of the methods described herein. The instructions can comprise a description of use of the components for the methods of identifying recombinases or methods of altering DNA.
[0672] The kits provided herein are in suitable packaging. Suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packaging, and the like.
[0673] Kits optionally may provide additional components such as buffers and interpretive information. Normally, the kit comprises a container and a label or package insert(s) on or associated with the container. In some embodiment, the disclosure provides articles of manufacture comprising contents of the kits described above.
[0674] The kit may further comprise a device for holding or administering the present recombinase, nucleic acids, system, or composition. The device may include an infusion device, an intravenous solution bag, a hypodermic needle, a vial, and/or a syringe.
[0675] The present disclosure also provides for kits for performing the methods or producing the components in vitro. The kit may include the components of the present system. Optional components of the kit include one or more of the following: (1) buffer constituents. (2) control plasmid, (3) transfection or transduction reagents.
5. EXAMPLES
[0676] Cell lines and cell culture. K562 (ATCC CCL-243) cells were cultured in a controlled humidified incubator at 37 C. and 5% CO.sub.2, in RPMI 1640 (Gibco) media supplemented with 10% FBS (Hyclone), penicillin (10,000 I.U./mL), streptomycin (10,000 g/mL), and L-glutamine (2 mM). HEK-293T cells, as well as HEK-293FT and HEK-293T-LentiX cells used to produce lentivirus, as described below, were grown in DMEM (Gibco) media supplemented with 10% FBS (Hyclone), penicillin (10,000 I.U./mL), and streptomycin (10,000 g/mL).
[0677] Selecting large serine recombinases (LSRs) for initial pilot experiments. LSRs for the pilot experiments were identified by searching for the Recombinase Pfam domain among the mobile genetic elements (MGEs) previously identified (See Durrant et al. (2020) Cell Host & Microbe 28(5): 767 and El-Gebali et al., Nucleic Acids Res. 47, D427-D432 (2019), incorporated herein by reference in their entirety). The identity of the attachment site was inferred from the boundaries of the MGE that contained each LSR. For example, if a sequence had the following structure:
B.sub.1-D-P.sub.1-E-P.sub.2-D-B.sub.2
where B.sub.1 indicates the sequence flanking the MGE insertion on the 5 end, D indicates the target site duplication created upon insertion (if it exists), P.sub.1 indicates the sequence flanking the 5 integration boundary that is included in the MGE, E is the intervening MGE, P.sub.2 indicates the sequence flanking the 3 integration boundary that is included in the MGE, and B2 indicates the sequence flanking the MGE insertion on the 3 end, then the attB and attP sequences can be reconstructed as:
attB=B.sub.1+D+B.sub.2
attP=P.sub.2+D+P.sub.1
where the + operator in this case indicates nucleotide sequence concatenation.
[0678] Candidates were then annotated to determine features such as: 1) whether or not the element was predicted to be a phage element, 2) how many isolates contain the integrated MGE, and 3) how often MGEs containing distinct LSRs will integrate at the same location in the genome. Candidates were then given higher priority if they were contained within predicted phage elements, if they appeared in multiple isolates, and if the attachment sites were targeted by multiple distinct LSRs.
[0679] Computational workflow to identify thousands of LSRs and cognate attachment sites. The LSR-identification workflow was implemented as described schematically in
[0680] After this initial round of LSR mining was complete, a modified approach was taken to further expand the database and avoid redundant searches. First, bacterial species with a high number of isolate genomes available in the first round of LSR mining were analyzed to determine if further mining of these genomes would be necessary. Rarefaction curves representing the number of new LSR families identified with each additional genome analyzed were estimated for these common species, and species that appeared saturated (e.g., less than 1 new cluster per 1000 genomes analyzed) were considered complete, meaning no further genomes belonging to this species would be analyzed. Next, 48,557 genomes that met these filtering criteria were downloaded from the GenBank database and prepared for further analysis. The analysis was very similar to round 1, but with some notable differences. First, a database of over 496,133 isolate genomes from the RefSeq and GenBank genomes was constructed. PhyloPhlAn marker genes were then extracted from all of these genomes. Next, for each genome that was found to contain a given LSR, closely related isolates found in the database were selected according to marker gene homology were then selected for the comparative genomics analysis and further LSR discovery. This marker gene search approach was made available in a public github repository (github(dot)com(backslash)bhattlab(backslash)GenomeSearch). This second round of LSR and attachment site mining increased the total number of candidates by approximately 32%.
[0681] Predicting LSR target site specificity. LSR protein sequences were clustered at 90% and 50% identity using MMseqs2. Protein sequences that overlapped with predicted attachment sites were extracted from their genome of origin and clustered with all other target proteins at 50% identity using MMseqs2. LSR-attachment site combinations that were found to meet intermediate quality control filters were considered. To identify site-specific LSRs, only LSRs clustered at 50% identity and target proteins clustered at 50% identity were considered. Next, LSR-target pairs were filtered to only include target protein clusters that were targeted by 3 or more LSR clusters. Next, only LSR clusters that targeted a single target protein cluster were considered. The remaining sets of LSR clusters were considered to be single-targeting, meaning that they likely site-specifically targeted only one protein cluster. Multi-targeting, or transposable LSRs with minimal site-specificity, were identified. Only LSRs clustered at 90% identity and target proteins clustered at 50% identity were considered. Next, the total number of target protein clusters that were targeted by each LSR cluster were counted, and LSR clusters that targeted only one protein cluster were removed from consideration. Next, the remaining LSRs were binned according to the number of protein clusters that they targeted, where 2 indicates two target proteins, 3 indicates three target proteins, and >3 indicates more than three target proteins. As referred to herein, 2 and 3 are considered moderately multi-targeting, while >3 are considered fully multi-targeting. Each 50% identity cluster was then assigned to a multi-targeting bin according to the highest bin attained by any one 90% cluster found within the 50% identity cluster.
[0682] Phylogenetic analysis of site-specific integrases targeting a conserved attachment site. One example of several site-specific integrases targeting a conserved attachment site is shown in
[0683] Identifying target site motifs from attachment sites in the LSR database. Multi-targeting LSRs in the database were analyzed at the level of individual proteins, at the level of 90% amino acid identity clusters, and at the level of 50% amino acid identity clusters. For each of these levels, only candidates that were found to target more than 10 unique attB sequences or 10 target genes clustered at 50% amino acid identity were kept. Then all of the corresponding attB sequences were extracted, with only one attachment site per target gene cluster being extracted to avoid redundancy. These attB sequences were then initially aligned using MAFFT-LINSI. Next, possible core dinucleotides were identified in each alignment by extracting all dinucleotides in the alignment, and ranking them by the conservation of their most frequent nucleotides and their proximity to the center of the attB sequences, using a custom score that equally weighted high nucleotide conservation and normalized distance to the attB center. Candidates were then re-aligned only with respect to these predicted dinucleotide cores, rather than using an alignment algorithm such as MAFFT. These alignments were then visualized in using ggseqlogo to identify conserved target site motifs.
[0684] Quality controls and selection criteria for LSRs. LSRs with large attachment site cores, above 20 base pairs in length, were removed. The attachment site core is the portion of the attB and the attP that are predicted to be perfectly homologous. LSRs with attachment sites with more than 5% of their nucleotides being ambiguous in the original genome assemblies were removed. Only LSRs between 400 amino acids and 650 amino acids were kept. Next, only predicted LSRs that contained at least one of the three main LSR Pfam domains were retained (Resolvase. Recombinase, and Zn_ribbon_recom). Next, LSRs were removed from consideration if their sequences contained more than 5% ambiguous amino acids. Only LSRs that were found on integrative mobile genetic elements that were less than 200 kilobases in length were retained. And finally, only LSRs that were within 500 nucleotides of their predicted attachment sites were retained. Candidates that met all of these filters were considered to meet quality-control thresholds.
[0685] Plasmid recombination assay to validate LSR-attD-attA predictions. Three plasmids were designed for each LSR candidate. The effector plasmid contained the EF1a promoter, followed by the recombinase coding sequence (codon optimized for human cells), a 2A self-cleaving peptide, and an eGFP coding sequence. The attA plasmid contained an EF1a promoter, followed by the attA sequence, followed by mTagBFP2 coding sequence, which should constitutively express the mTagBFP2 protein in human cells. The attD plasmid included only the attD sequence followed by the mCherry coding sequence, which should produce no fluorescent mCherry prior to integration. HEK-293T cells were plated into 96 well plates and transfected one day later with 200 ng of effector plasmid, 70 ng of attA plasmid, and 50 ng of attD plasmid using Lipofectamine 2000 (Invitrogen). 2-3 days after transfection of cells with all three plasmids, cells were then measured using flow cytometry on an Attune NxT Flow Cytometer (ThermoFisher). HEK-293T cells were lifted from the plate using TrypLE (Gibco), and resuspended in Stain Buffer (BD). These experiments were conducted in triplicate transfections. Cells were gated for single cells using forward and side scatter, and then on cells expressing fluorescent eGFP. Next, mTagBFP2 fluorescence was measured to indicate the amount of un-recombined attD plasmids, and mCherry fluorescence was measured to indicate the amount of recombinant plasmid.
[0686] An experiment testing recombinases with matched and unmatched attD plasmids was performed similarly, following the above protocol for K562 cells. 3 days after transfection, cells were measured by flow cytometry on a BD Accuri C6 cytometer.
[0687] Landing pad cell line production. Landing pad LSR candidates were cloned into lentiviral plasmids under the expression of the strong pEF1a promoter, with their attB site in between the promoter and start codon, and with a 2A-EGFP fluorescent marker downstream the LSR coding sequence. Lentivirus production and spinfection of K562 cells were performed as follows: HEK-293T cells were plated on 6-well tissue culture plates. On each plate, 510.sup.5 HEK-293T cells were plated in 2 mL of DMEM, grown overnight, and then transfected with 0.75 g of an equimolar mixture of the three third-generation packaging plasmids (pMD2.G, psPAX2, pMDLg/pRRE) and 0.75 g of LSR vectors using 10 l of polyethyleneimine (PEI, Polysciences #23966) and 200 l of cold serum free DMEM. pMD2.G (Addgene plasmid #12259: RRID:Addgene_12259), psPAX2 (Addgene plasmid #12260; RRID:Addgene_12260), and pMDLg/pRRE (Addgene plasmid #12251; RRID:Addgene_12251). After 24 hours, 3 mL of DMEM was added to the cells, and after 72 hours of incubation, lentivirus was harvested. The pooled lentivirus was filtered through a 0.45-m PVDF filter (Millipore) to remove any cellular debris. 110.sup.5 K562 cells were infected with the lentiviruses by spinfection for 2 hours at 1000g at 33 C. Lentivirus doses of 50, 100, and 200 l were used for each vector, in order to find a condition with low multiplicity of infection wherein each transduced cell would be likely to contain only a single integrated copy of the landing pad. Infected cells grew for 3 days and then infection efficiency was measured using flow cytometry to measure EGFP (BD Accuri C6); the dose that gave rise to 5-15% EGFP+cells was selected for each LSR for further experiments. Ten days later, these EGFP+cells were sorted into a 96-well plate with a single cell in each well, in order to derive clonal lines with a single landing pad location. Two weeks later, 4 clones for each LSR with a unimodal high EGFP expression level were selected for expansion and subsequent experiments.
[0688] Landing pad integration efficiency assay. Clonal landing pad lines were electroporated with the promoterless mCherry donor containing the matching attP at a dose of either 1000 or 2000 ng donor plasmid. At timepoints from 3-11 days post-electroporation, the cells were subjected to flow cytometry to measure mCherry (BD Accuri C6).
[0689] Pseudosite integration efficiency assay to measure integration percent into the WT genome. To determine the percentage of integration of attD donors into pseudosites in the human genome, attD sequences were cloned into a plasmid containing an Ef1a promoter followed by mCherry, and p2a self-cleaving peptide, and a puromycin resistance marker. 1.010.sup.6 K562 cells were electroporated in Amaxa solution (Lonza Nucleofector SF, program FF-120), with 3000 ng LSR plasmid and 2000 ng pseudosite attD plasmid. As a non-matching LSR control, 3000 ng of Bxb1 was substituted for the correct LSR plasmid. The cells were cultured between 210.sup.5 cells/mL and 110.sup.6 cells/mL for 2-3 weeks. 100 uL of each sample was run on the Attune NxT Flow Cytometer every 3-4 days to measure the mCherry signal. After 2-3 weeks, transiently transfected plasmid was nearly fully diluted out in the non-matching LSR control, and the efficiency of the LSR was determined by the difference in mCherry percentage between the non-matching LSR control and the experimental condition.
[0690] Integration site mapping assay to determine human genome integration specificity. Utilizing the same protocol as above, K562s were electroporated with LSR and pseudosite attD plasmids. After 5 days in culture, puromycin was added to the media at 1 ug/mL. The cells were cultured for 1.5 more weeks, and then the gDNA was harvested using the Quick-DNA Miniprep Kit (Zymo) and quantified by Qubit HS dsDNA Assay (Thermo). A modified version of the UDiTaS sequencing assay was used as described in Giannoukos et al. BMC Genomics 19, 212 (2018), and Danner, 2020 Protocols.io.(doi(dot)org(backslash)10.17504(backslash)protocols.io.7k2hkye). Tn5 was purified and stored at 7.5 mg/mL. Adaptors were assembled by combining 50 uL of 100 uM top and bottom strand, heating to 95 C. for 2 minutes, and slowly ramping down to 25 C. over 12 hours. Next, the transposome was assembled by combining 85.7 uL of Tn5 transposase with 14.3 uL pre-annealed oligos, and incubated for 60 minutes at room temperature. Tagmentation was performed by adding 150 ng gDNA, 4 L of 5TAPS-DMF (50 mM TAPS NaOH, 25 mM MgCl2, 50% v/v DMF (pH 8.5) at 25 C.), 3 uL assembled transposome, and water for a 20 uL final reaction volume. The reaction was incubated at 55 C. for 10-15 minutes and then purified with Zymo DNA Clean and Concentrator-5. The tagmented products were run on Agilent Bioanalyzer HS DNA kit to confirm average fragment size of 2 kb. Next, PCR was performed with the outer primers for 12 cycles using 12.5 uL Platinum Superfi PCR Master Mix (Thermo), 1.5 uL of 0.5M TMAC, 0.5 uL of 10 uM outer nest GSP primer, 0.25 uL of 10 uM outer i5 primer, 9 ul of tagmented DNA, and 1.25 uL of DMSO. After Ampure XP 0.9 bead clean-up, a second PCR with the inner next primers, was performed for 18 cycles. The PCR contained 25 uL Platinum Superfi Master Mix (Thermo), 3 uL 0.5M TMAC, 2.5 uL DMSO, 2.5 uL of 10 uM i5 primer, 5 L of 10 uM i7 GSP primer, 10 uL of the purified 1 st round PCR product, and 2 uL water for a final reaction volume of 50 L. The final library was size selected on a 2% agarose gel for fragments between 300-800 bases, gel extracted with the Monarch DNA Gel Extraction Kit (NEB), quantified with Qubit HS dsDNA Assay (Thermo) and KAPA Library Quantification Kit, fragment analyzed with Agilent Bioanalyzer HS DNA kit, and sequenced on a MiSeq (Illumina).
[0691] Computational analysis of integration site mapping sequence assay. Snakemake workflows were constructed and used to analyze NGS data for the UDiTaS pseudosite sequencing assay. First, stagger sequences (filler sequences added for better discrimination of samples during sequencing) were added to primers were removed using custom python scripts. Next, fastp was used to trim nextera adaptors from reads and to remove reads with low PHRED scores. Next, reads were aligned to both the human genome (GRCh38) and a donor plasmid sequence containing the LSR-specific attD sequence in single-end mode using BWA. Reads were analyzed individually using custom python scripts to identify 1) if they aligned to the donor plasmid, human genome, or both, 2) whether or not the reads began at the predicted primer, and 3) whether or not the pre-integration attachment site was intact. Reads were then filtered to only include those reads that mapped to both the donor plasmid and the human genome, those that began at the primer site, and those that did not have an intact attD sequence (if this could be determined from the length of a particular read). This filtered read set was then aligned in paired-end mode to the human genome using default settings in BWA MEM. Alignments with a mapping quality score less than 30 were removed, along with supplementary alignments and paired read alignments with an insert size longer than 1500 bp. The samtools markdup tool was used to remove potential PCR duplicates and identify unique reads for downstream analysis. Next, MGEfinder was used to extract clipped end sequences from reads aligned to the human genome and generate a consensus sequence of the clipped ends, which represent the crossover from the human genome into the integrated attD sequence. Using custom python scripts, k-mers of length 9 base pairs were extracted from these consensus sequences and compared with a subsequence of the attD plasmid extending from the original primer to 25 bp after the end of the attD attachment site. If there were no shared 9-mers, the candidate was discarded. Otherwise, consensus sequences were clipped to begin at the primer site, and these consensus sequences were then aligned back to the original attD subsequence using the biopython local alignment tool. Two aligned portions were extractedthe full local alignment of the consensus sequence to the attD (called the full local alignment), and the longest subset of the alignment that included no ambiguous bases and no gaps (called the contiguous alignment). To filter a final set of true insertion sites, only sites with at least 80% nucleotide identity shared between the consensus sequence and the attD subsequence in either the full local alignment or the contiguous alignment were kept. Finally, only sites with a crossover point within 15 base pairs of the predicted dinucleotide core were kept.
[0692] This approach could precisely predict integration sites, but errors in sequencing reads led to some variability in this prediction. To account for this, integration sites were combined into integration loci by merging all sites that were within 500 base pairs of each other, using bedtools. This approach would merge integration events that occurred at the same site but in opposite orientations, for example. When pooling reads across biological or technical replicates, these loci were also merged if they overlapped. When measuring the relative frequency of insertion across different loci, all uniquely aligned reads (deduplicated using samtools markdup) found within each locus were counted. These were then converted into percentages for each locus by dividing by the total number of unique reads aligned to all integration loci.
[0693] Target site motifs for different LSRs could be determined from precise predictions of dinucleotide cores for all integration sites. For each integration locus, only one integration site was chosen if there were multiple, and integration sites with more reads supporting them were prioritized. Up to 30 base pairs of human genome sequence around the predicted dinucleotide core were extracted using bedtools, choosing the forward or reverse strand depending on the orientation of the integration. All such target sites, or a subset of these target sites if desired, were then analyzed for conservation at each nucleotide position using the ggseqlogo package in R.
[0694] Phylogenetic tree construction. Representative amino acid sequences of each quality-controlled 50% identity LSR cluster were used to construct the phylogenetic tree. LSRs were aligned using MAFFT in G-INS-i mode, and IQ-TREE was then used to generate a consensus tree using 1000 bootstrap replicates and automatic model selection.
Example 1
Systematic Identification of Recombinases and Predicted Attachment Sites Revealed Site-Specific and Multi-Targeting/Transposable Clades
[0695] LSRs such as Bxb1 and PhiC31 catalyze an integration reaction that recombines two DNA sequences at specific attachment sites, referred to as attP (the DNA sequence found in the phage) and attB (the DNA sequence found in the bacteria). Using a comparative genomics approach built to identify precise boundaries of integrative elements (
[0696] To predict the site-specificity of candidate LSRs using only the constructed database, the network of LSRs and associated attachment sites were inspected, and LSRs from a diverse set of 20 host phyla were recovered (
[0697] For each LSR cluster, the number of associated target gene clusters were estimated and visualized on the phylogenetic tree of representatives of each LSR cluster at the amino acid level. LSRs were binned into two groups: Site-specific integrases or Multi-targeting integrases (
[0698] Many examples of distantly related LSRs targeted the same gene clusters (
[0699]
Example 2
Characterization of Landing Pad LSRs
[0700] One valuable application for LSRs in biotechnology is specific delivery of genetic cargo to an introduced site or so-called landing pad that is not present elsewhere in the target genome. An ideal landing pad LSR is highly specific for an attB that does not exist in a target genome, but can efficiently integrate once the attB is installed.
[0701] Using previously identified MGEs for LSRs (Durrant et al. (2020) Cell Host & Microbe 28(5): 767, incorporated herein by reference in its entirety), a set of 17 LSR candidates with evidence for site-specificity was curated as an initial proof of concept. To validate that these recombinases were active in mammalian cells, an inter-plasmid recombination assay was developed in HEK293FT cell by synthesizing three plasmids: one for expression of the human codon-optimized LSRs, and separate plasmids containing their putative attP and attB sequences (
[0702] Integration into attB-containing landing pads that were pre-installed in the human genome were also tested (
[0703] Landing pad integration may be most useful when the landing pad is known to be at a single genomic site in all cells. To develop single position landing pad lines, landing pad LSR-GFP construct was integrated via low MOI lentivirus, resulting in a single copy of the landing pad per cell. Clonal cell lines which should contain a single landing pad site were then sorted, expanded, and electroporated with the attP-mCherry donor plasmid. Using this landing pad assay, four integrase candidates (Ec03, Ec04, Kp03, and Pa01) were tested and Pa01 performed better than Bxb1 in terms of the percentage of cells that were stably fluorescent after 11 days (
[0704] Previous characterization of the Bxb1 attB identified a sequence as short as 38 bp as being necessary for integration, but the computational pipeline conservatively predicted 100 bp attB sequences initially. A minimum 33 base pair attB for efficient Pa01 recombination was determined, but efficient recombination for Kp03 was seen down to a 25 base pair attB (
[0705] Efficient landing pads could be especially useful for multiplex gene integration, which could be achieved by using several of LSRs in parallel, given that they do not operate on each other's attachment sites (
[0706] The specificity of these LSRs was tested by transfecting attP-pEF1a-mCherry donors with or without co-transfected LSR into wildtype K562 cells and measuring mCherry expression 18 days later, by which point episomal donor plasmid is no longer detectable. Pa01 showed no evidence of mCherry integration above background, while Kp03 did have elevated mCherry+fluorescence, suggesting it has off-target pseudosites (
[0707] This assay detected off-target integration for all LSRs, including Bxb1 (3.48%+/2.98%, 9 unique reads across 9 integration loci) and Pa01 (0.47%+/0.46%, 13 unique reads across 10 loci), but Kp03 had significantly more than the others at 15.5%+/2.43%, with 312 unique reads detected across 83 different loci, confirming a relatively high percentage of off-target integrations. Wild-type cells that were transfected with Kp03 and Pa01 were sequenced using the integration site mapping assay at high coverage, and 79 off-target genome integration loci were detected for Pa01, and 2,415 off-target integration loci were detected for Kp03. From these integration sites, the target site motifs targeted by these LSRs were identified, and the motifs showed conservation at the dinucleotide core and flanking sequence, indicating that these are bona fide integrations rather than random plasmid integrations (
[0708] A second batch of 21 LSRs were selected from the database, prioritizing those with low BLAST similarity between their attB/P sites and the human genome, and applying stringent quality thresholds. 17 out of 21 (81%) of them were functional in the plasmid recombination assay, providing validation of the computational pipeline for identifying functional candidates. Promisingly, 16 candidates had higher mCherry+MFI values than PhiC31, and 11 candidates had higher MFI values than Bxb1 (
Example 3
Genome-Targeting LSRs Integrate into Human Genome at Predicted Target Sites
[0709] A particularly useful LSR would be one that integrates directly into only one, or very few, pseudosites in safe locations in the human genome and does so with appreciable efficiency. Historically, LSRs with pseudosites such as that for PhiC31 had to be experimentally discovered by transfecting the LSR into human cells and searching for the integration sites. While effective in demonstrating proof of concept, this approach has not yielded highly efficient and specific human genome-targeting LSRs. BLAST was used to search all attB/P sequences against the GRCh38 human genome assembly (
[0710] All 103 candidates were tested in the plasmid recombination assay, and 27 candidates recombined at predicted attachment sites (one-tailed t-test, P<0.05;
[0711] To determine if these LSRs could target the chromosomes directly in human K562 cells, another plasmid recombination assay was performed, replacing the native attA with the human pseudosite (or attH) instead of the native attachment site and found 4 of the candidates recombined with their predicted attH: Sp56, Pf80, Ps45, and Enc3 (
[0712] Of these four candidates, Pf80 had the highest predicted specificity, with 34.3% of unique reads mapping to the predicted target site, an exon of the gene FKBP2 at position 64,243,293 on chromosome 11 (
Example 4
Multi-Targeting LSRs Directly Integrate DNA into the Human Genome
[0713] An LSR is considered to be a good multi-targeting candidate if it has relaxed specificity requirements, if it appears in the multi-targeting clade (
[0714] One such multi-targeting LSR found in Clostridium perfringens, named Cp36, was characterized. This LSR is 544 amino acids in length, and it contains a predicted DUF4368 domain at its C-terminus. This LSR can integrate an mCherry donor cargo into the genome of K562 cells at up to 40% efficiency without pre-installation of a landing pad or antibiotic selection (
[0715] Using these precise prediction of human integration sites, a sequence motif targeted by Cp36 was reconstructed (
[0716] To compare the efficiency of Cp36 to the PiggyBac (PB) transposase, a commonly used tool for delivering DNA cargos at random into TTAA tetranucleotides found in a target genome, a plasmid construct was designed that included a Cp36 attD (donor attachment site), PB ITR sequences, and an mCherry reporter (
[0717] To test if Cp36 could be re-used to integrate a second gene, a pure population of mCherry+cells was generated via Cp36-mediated integration and puromycin selection, and re-electroporated with Cp36 and a donor containing BFP. After 13 days, 9% of the cells were double positive (mCherry+ and BFP+) (
[0718] Additionally, two other orthologs (Pc01 and Enc9) were found in the database that also functioned as multi-targeters in human cells with efficiencies of 13% and 35% (
Example 5
Biological Role of LSR Target Genes
[0719] Genes that were targeted and disrupted upon LSR integration could indicate an evolved strategy for LSR-carrying MGEs. Pfam domains that were enriched among target genes were identified (
Example 6
Post Hoc Identification of Human Genome Integration
[0720] A post hoc analysis of the genome-targeting and multi-targeting candidates in this study was performed to determine how feasible a motif-based search would be. Starting with each experimentally characterized candidate, sequence motifs were built by iteratively adding natural attB sequences of the next most closely related LSR ortholog, only adding additional attB sequences if they were 95% identical or less to already selected attB sequences. Motifs of 20, 50 and 100 such attB sequences were built. Then these motifs were searched against the experimentally observed human integration sites, and approximately 30.000 randomly selected human genome sequences. Next, these sequences were iterated across motif score cutoffs and the true positive rate and the false positive rate were calculated at each cutoff, generating a ROC curve (
[0721] Sequence motifs belonging to the multi-targeting candidates performed quite well, with AUC values ranging from 0.94 for the Cp36 motif to 0.68 for the Bt24 motif. For the genome-targeting candidates the performance of the sequence motifs varied, ranging in AUC values from 0.65 for Dn29 to 0.44 for Enc3. All of these motifs assigned significantly higher scores to observed integration sites than randomly selected controls, except for Sp56 and Enc3, which did not differ significantly (Wilcoxon rank-sum test; P<0.0001 for Cp36, Enc9, Pc01, Bt24, and Dn29, P<0.01 for Pf80, P>0.05 for Sp56 and Enc3). Despite the relatively poor performance of the Pf80 motif and the Sp56 motif, they did assign the highest motif scores to the most frequently targeted human genome integration sites, suggesting that there is predictive value to their database-derived sequence motifs (
[0722] These results suggest that there is value in taking a motif-based sequence search when prioritizing multi-targeting and genome-targeting candidates. The potential targeting profile of multi-targeters could be better understood prior to experimental validation, as with Cp36 and Enc9, and genome-targeting candidates could be selected based on those that have high, outlier motif matches that could indicate higher specificity, such as for Pf80. The difference in performance between motifs may be explained by the different selection pressures placed on multi-targeting and single-targeting LSRs, where multi-targeting LSRs are more likely to maintain their relaxed sequence specificity across larger evolutionary distances due to a greater abundance of possible target sites, leading to more accurate sequence motifs. These results could also have been influenced by the efficiency of the LSR in human cells or epigenetic modifications such as those that influence chromatin accessibility (
[0723] All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein.
[0724] Preferred embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those preferred embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.
TABLE-US-00002 Table1: LandingPadIntegrases attP attB Protein sequence sequence sequence SEQID SEQID SEQID LSR NOs: NOs: NOs: Sh25 1184 1216 1 Si74 1185 1217 2 Bm99 1186 1218 3 Me99 1187 1219 4 Ma37 1188 1220 5 Nm60 1189 1221 6 Cc91 1190 1222 7 Vh19 1191 1223 8 Cs56 1192 1224 9 Bt24 1193 1225 10 No67 1194 1226 11 Fm04 1195 1227 12 Bu30 1196 1228 13 Ma05 1197 1229 14 Rh64 1198 1230 15 Cb16 1199 1231 16 uCb4 1200 1232 17 Ec03 1201 1233 18 Ec04 1202 1234 19 Ec05 1203 1235 20 Ec06 1204 1236 21 Ec07 1205 1237 22 Ef01 1206 1238 23 Ef02 1207 1239 24 Kp01 1208 1240 25 Kp03 1209 1241 26 Kp04 1210 1242 27 Kp05 1211 1243 28 Pa01 1212 1244 29 Pa03 1213 1245 30 Sa01 1214 1246 31 Sa02 1215 1247 32 Table2: Genome-TargetingIntegrases attD attA Protein sequence sequence sequence SEQID SEQID SEQID LSR NOs: NOs: NOs: Pf13 1248 1282 33 Td08 1249 1283 34 Se37 1250 1284 35 Ct03 1251 1285 36 Ps40 1252 1286 38 Sa10 1253 1287 39 Td01 1254 1288 40 Enc3 1255 1289 41 Fp10 1256 1290 42 Ph43 1257 1291 43 Sm18 1258 1292 44 Pf80 1259 1293 46 Bs46 1260 1294 47 Pf48 1261 1295 48 Rb27 1262 1296 49 Sa51 1263 1297 50 Bc30 1264 1298 51 Cd04 1265 1299 52 Sa34 1266 1300 54 Pp20 1267 1301 55 Efs2 1268 1302 57 Pf15 1269 1303 58 Ps45 1270 1304 59 Sp56 1271 1305 60 Dn29 1272 1306 61 Vh73 1273 1307 62 Em12 1274 1308 63 Pc64 1275 1309 64 Vp82 1276 1310 65 CMp1 1277 1311 69 Pa19 1278 1312 70 Pg17 1279 1313 71 Sall 1280 1314 72 El01 1281 1315 73 Table3: Multi-TargetingIntegrases attD attA Protein sequence sequence sequence SEQID SEQID SEQID LSR NOs: NOs: NOs: Cp36 1316 1324 66 Pc01 1317 1325 67 Enc9 1318 1326 68 Cd16 1319 1327 45 Cd15 1320 1328 53 Cd31 1321 1329 37 Rl09 1322 1330 56 Cd08 1323 1331 74 Table4: LSRProtein sequence attBsequence attPsequence SEQIDNOs: SEQIDNOs: SEQIDNOs: 88 1332 2413 89 1333 2414 90 1334 2415 91 1335 2416 92 1336 2417 93 1337 2418 94 1338 2419 95 1339 2420 96 1340 2421 97 1341 2422 98 1342 2423 99 1343 2424 100 1344 2425 101 1345 2426 102 1346 2427 103 1347 2428 104 1348 2429 105 1349 2430 106 1350 2431 107 1351 2432 108 1352 2433 109 1353 2434 110 1354 2435 111 1355 2436 112 1356 2437 113 1357 2438 114 1358 2439 115 1359 2440 116 1360 2441 117 1361 2442 118 1362 2443 119 1363 2444 120 1364 2445 121 1365 2446 122 1366 2447 123 1367 2448 124 1368 2449 125 1369 2450 126 1370 2451 127 1371 2452 128 1372 2453 129 1373 2454 130 1374 2455 131 1375 2456 132 1376 2457 133 1377 2458 134 1378 2459 135 1379 2460 136 1380 2461 137 1381 2462 138 1382 2463 139 1383 2464 140 1384 2465 141 1385 2466 142 1386 2467 143 1387 2468 144 1388 2469 145 1389 2470 146 1390 2471 147 1391 2472 148 1392 2473 149 1393 2474 150 139 2475 151 1395 2476 152 1396 2477 153 1397 2478 154 1398 2479 155 1399 2480 156 1400 2481 157 1401 2482 158 1402 2483 159 1403 2484 160 1404 2485 161 1405 2486 162 1406 2487 163 1407 2488 164 1408 2489 165 1409 2490 166 1410 2491 167 1411 2492 168 1412 2493 169 1413 2494 170 1414 2495 171 1415 2496 172 1416 2497 173 1417 2498 174 1418 2499 175 1419 2500 176 1420 2501 177 1421 2502 178 1422 2503 179 1423 2504 180 1424 2505 181 1425 2506 182 1426 2507 183 1427 2508 184 1428 2509 185 1429 2510 186 1430 2511 187 1431 2512 188 1432 2513 189 1433 2514 190 1434 2515 191 1435 2516 192 1436 2517 193 1437 2518 194 1438 2519 195 1439 2520 196 1440 2521 197 1441 2522 198 1442 2523 199 1443 2524 200 1444 2525 201 1445 2526 202 1446 2527 203 1447 2528 204 1448 2529 205 1449 2530 206 1450 2531 207 1451 2532 208 1452 2533 209 1453 2534 210 1454 2535 211 1455 2536 212 1456 2537 213 1457 2538 214 1458 2539 215 1459 2540 216 1460 2541 217 1461 2542 218 1462 2543 219 1463 2544 220 1464 2545 221 1465 2546 222 1466 2547 223 1467 2548 224 1468 2549 225 1469 2550 226 1470 2551 227 1471 2552 228 1472 2553 229 1473 2554 230 1474 2555 231 1475 2556 232 1476 2557 233 1477 2558 234 1478 2559 235 1479 2560 236 1480 2561 237 1481 2562 238 1482 2563 239 1483 2564 240 1484 2565 241 1485 2566 242 1486 2567 243 1487 2568 244 1488 2569 245 1489 2570 246 1490 2571 247 1491 2572 248 1492 2573 249 1493 2574 250 1494 2575 251 1495 2576 252 1496 2577 253 1497 2578 254 1498 2579 255 1499 2580 256 1500 2581 257 1501 2582 258 1502 2583 259 1503 2584 260 1504 2585 261 1505 2586 262 1506 2587 263 1507 2588 264 1508 2589 265 1509 2590 266 1510 2591 267 1511 2592 268 1512 2593 269 1513 2594 270 1514 2595 271 1515 2596 272 1516 2597 273 1517 2598 274 1518 2599 275 1519 2600 276 1520 2601 277 1521 2602 278 1522 2603 279 1523 2604 280 1524 2605 281 1525 2606 282 1526 2607 283 1527 2608 284 1528 2609 285 1529 2610 286 1530 2611 287 1531 2612 288 1532 2613 289 1533 2614 290 1534 2615 291 1535 2616 292 1536 2617 293 1537 2618 294 1538 2619 295 1539 2620 296 1540 2621 297 1541 2622 298 1542 2623 299 1543 2624 300 1544 2625 301 1545 2626 302 1546 2627 303 1547 2628 304 1548 2629 305 1549 2630 306 1550 2631 307 1551 2632 308 1552 2633 309 1553 2634 310 1554 2635 311 1555 2636 312 1556 2637 313 1557 2638 314 1558 2639 315 1559 2640 316 1560 2641 317 1561 2642 318 1562 2643 319 1563 2644 320 1564 2645 321 1565 2646 322 1566 2647 323 1567 2648 324 1568 2649 325 1569 2650 326 1570 2651 327 1571 2652 328 1572 2653 329 1573 2654 330 1574 2655 331 1575 2656 332 1576 2657 333 1577 2658 334 1578 2659 335 1579 2660 336 1580 2661 337 1581 2662 338 1582 2663 339 1583 2664 340 1584 2665 341 1585 2666 342 1586 2667 343 1587 2668 344 1588 2669 345 1589 2670 346 1590 2671 347 1591 2672 348 1592 2673 349 1593 2674 350 1594 2675 351 1595 2676 352 1596 2677 353 1597 2678 354 1598 2679 355 1599 2680 356 1600 2681 357 1601 2682 358 1602 2683 359 1603 2684 360 1604 2685 361 1605 2686 362 1606 2687 363 1607 2688 364 1608 2689 365 1609 2690 366 1610 2691 367 1611 2692 368 1612 2693 369 1613 2694 370 1614 2695 371 1615 2696 372 1616 2697 373 1617 2698 374 1618 2699 375 1619 2700 376 1620 2701 377 1621 2702 378 1622 2703 379 1623 2704 380 1624 2705 381 1625 2706 382 1626 2707 383 1627 2708 384 1628 2709 385 1629 2710 386 1630 2711 387 163 2712 388 1632 2713 389 1633 2714 390 1634 2715 391 1635 2716 392 1636 2717 393 1637 2718 394 1638 2719 395 1639 2720 396 1640 2721 397 1641 2722 398 1642 2723 399 1643 2724 400 1644 2725 401 1645 2726 402 1646 2727 403 1647 2728 404 1648 2729 405 1649 2730 406 1650 2731 407 1651 2732 408 1652 2733 409 1653 2734 410 1654 2735 411 1655 2736 412 1656 2737 413 1657 2738 414 1658 2739 415 1659 2740 416 1660 2741 417 1661 2742 418 1662 2743 419 1663 2744 420 1664 2745 421 1665 2746 422 1666 2747 423 1667 2748 424 1668 2749 425 1669 2750 426 1670 2751 427 1671 2752 428 1672 2753 429 1673 2754 430 1674 2755 431 1675 2756 432 1676 2757 433 1677 2758 434 1678 2759 435 1679 2760 436 1680 2761 437 1681 2762 438 1682 2763 439 1683 2764 440 1684 2765 441 1685 2766 442 1686 2767 443 1687 2768 444 1688 2769 445 1689 2770 446 1690 2771 447 1691 2772 448 1692 2773 449 1693 2774 450 1694 2775 451 1695 2776 452 1696 2777 453 1697 2778 454 1698 2779 455 1699 2780 456 1700 2781 457 1701 2782 458 1702 2783 459 1703 2784 460 1704 2785 461 1705 2786 462 1706 2787 463 1707 2788 464 1708 2789 465 1709 2790 466 1710 2791 467 1711 2792 468 1712 2793 469 1713 2794 470 1714 2795 471 1715 2796 472 1716 2797 473 1717 2798 474 1718 2799 475 1719 2800 476 1720 2801 477 1721 2802 478 1722 2803 479 1723 2804 480 1724 2805 481 1725 2806 482 1726 2807 483 1727 2808 484 1728 2809 485 1729 2810 486 1730 2811 487 1731 2812 488 1732 2813 489 1733 2814 490 1734 2815 491 1735 2816 492 1736 2817 493 1737 2818 494 1738 2819 495 1739 2820 496 1740 2821 497 1741 2822 498 1742 2823 499 1743 2824 500 1744 2825 501 1745 2826 502 1746 2827 503 1747 2828 504 1748 2829 505 1749 2830 506 1750 2831 507 1751 2832 508 1752 2833 509 1753 2834 510 1754 2835 511 1755 2836 512 1756 2837 513 1757 2838 514 1758 2839 515 1759 2840 516 1760 2841 517 1761 2842 518 1762 2843 519 1763 2844 520 1764 2845 521 1765 2846 522 1766 2847 523 1767 2848 524 1768 2849 525 1769 2850 526 1770 2851 527 1771 2852 528 1772 2853 529 1773 2854 530 1774 2855 531 1775 2856 532 1776 2857 533 1777 2858 534 1778 2859 535 1779 2860 536 1780 2861 537 1781 2862 538 1782 2863 539 1783 2864 540 1784 2865 541 1785 2866 542 1786 2867 543 1787 2868 544 1788 2869 545 1789 2870 546 1790 2871 547 1791 2872 548 1792 2873 549 1793 2874 550 1794 2875 551 1795 2876 552 1796 2877 553 1797 2878 554 1798 2879 555 1799 2880 556 1800 2881 557 1801 2882 558 1802 2883 559 1803 2884 560 1804 2885 561 1805 2886 562 1806 2887 563 1807 2888 564 1808 2889 565 1809 2890 566 1810 2891 567 1811 2892 568 1812 2893 569 1813 2894 570 1814 2895 571 1815 2896 572 1816 2897 573 1817 2898 574 1818 2899 575 1819 2900 576 1820 2901 577 1821 2902 578 1822 2903 579 1823 2904 580 1824 2905 581 1825 2906 582 1826 2907 583 1827 2908 584 1828 2909 585 1829 2910 586 1830 2911 587 1831 2912 588 1832 2913 589 1833 2914 590 1834 2915 591 1835 2916 592 1836 2917 593 1837 2918 594 1838 2919 595 1839 2920 596 1840 2921 597 1841 2922 598 1842 2923 599 1843 2924 600 1844 2925 601 1845 2926 602 1846 2927 603 1847 2928 604 1848 2929 605 1849 2930 606 1850 2931 607 1851 2932 608 1852 2933 609 1853 2934 610 1854 2935 611 1855 2936 612 1856 2937 613 1857 2938 614 1858 2939 615 1859 2940 616 1860 2941 617 1861 2942 618 1862 2943 619 1863 2944 620 1864 2945 621 1865 2946 622 1866 2947 623 1867 2948 624 1868 2949 625 1869 2950 626 1870 2951 627 1871 2952 628 1872 2953 629 1873 2954 630 1874 2955 631 1875 2956 632 1876 2957 633 1877 2958 634 1878 2959 635 1879 2960 636 1880 2961 637 1881 2962 638 1882 2963 639 1883 2964 640 1884 2965 641 1885 2966 642 1886 2967 643 1887 2968 644 1888 2969 645 1889 2970 646 1890 2971 647 1891 2972 648 1892 2973 649 1893 2974 650 1894 2975 651 1895 2976 652 1896 2977 653 1897 2978 654 1898 2979 655 1899 2980 656 1900 2981 657 1901 2982 658 1902 2983 659 1903 2984 660 1904 2985 661 1905 2986 662 1906 2987 663 1907 2988 664 1908 2989 665 1909 2990 666 1910 2991 667 1911 2992 668 1912 2993 669 1913 2994 670 1914 2995 671 1915 2996 672 1916 2997 673 1917 2998 674 1918 2999 675 1919 3000 676 1920 3001 677 1921 3002 678 1922 3003 679 1923 3004 680 1924 3005 681 1925 3006 682 1926 3007 683 1927 3008 684 1928 3009 685 1929 3010 686 1930 3011 687 1931 3012 688 1932 3013 689 1933 3014 690 1934 3015 691 1935 3016 692 1936 3017 693 1937 3018 694 1938 3019 695 1939 3020 696 1940 3021 697 1941 3022 698 1942 3023 699 1943 3024 700 1944 3025 701 1945 3026 702 1946 3027 703 1947 3028 704 1948 3029 705 1949 3030 706 1950 3031 707 1951 3032 708 1952 3033 709 1953 3034 710 1954 3035 711 1955 3036 712 1956 3037 713 1957 3038 714 1958 3039 715 1959 3040 716 1960 3041 717 1961 3042 718 1962 3043 719 1963 3044 720 1964 3045 721 1965 3046 722 1966 3047 723 1967 3048 724 1968 3049 725 1969 3050 726 1970 3051 727 1971 3052 728 1972 3053 729 1973 3054 730 1974 3055 731 1975 3056 732 1976 3057 733 1977 3058 734 1978 3059 735 1979 3060 736 1980 3061 737 1981 3062 738 1982 3063 739 1983 3064 740 1984 3065 741 1985 3066 742 1986 3067 743 1987 3068 744 1988 3069 745 1989 3070 746 1990 3071 747 1991 3072 748 1992 3073 749 1993 3074 750 1994 3075 751 1995 3076 752 1996 3077 753 1997 3078 754 1998 3079 755 1999 3080 756 2000 3081 757 2001 3082 758 2002 3083 759 2003 3084 760 2004 3085 761 2005 3086 762 2006 3087 763 2007 3088 764 2008 3089 765 2009 3090 766 2010 3091 767 2011 3092 768 2012 3093 769 2013 3094 770 2014 3095 771 2015 3096 772 2016 3097 773 2017 3098 774 2018 3099 775 2019 3100 776 2020 3101 777 2021 3102 778 2022 3103 779 2023 3104 780 2024 3105 781 2025 3106 782 2026 3107 783 2027 3108 784 2028 3109 785 2029 3110 786 2030 3111 787 2031 3112 788 2032 3113 789 2033 3114 790 2034 3115 791 2035 3116 792 2036 3117 793 2037 3118 794 2038 3119 795 2039 3120 796 2040 3121 797 2041 3122 798 2042 3123 799 2043 3124 800 2044 3125 801 2045 3126 802 2046 3127 803 2047 3128 804 2048 3129 805 2049 3130 806 2050 3131 807 2051 3132 808 2052 3133 809 2053 3134 810 2054 3135 811 2055 3136 812 2056 3137 813 2057 3138 814 2058 3139 815 2059 3140 816 2060 3141 817 2061 3142 818 2062 3143 819 2063 3144 820 2064 3145 821 2065 3146 822 2066 3147 823 2067 3148 824 2068 3149 825 2069 3150 826 2070 3151 827 2071 3152 828 2072 3153 829 2073 3154 830 2074 3155 831 2075 3156 832 2076 3157 833 2077 3158 834 2078 3159 835 2079 3160 836 2080 3161 837 2081 3162 838 2082 3163 839 2083 3164 840 2084 3165 841 2085 3166 842 2086 3167 843 2087 3168 844 2088 3169 845 2089 3170 846 2090 3171 847 2091 3172 848 2092 3173 849 2093 3174 850 2094 3175 851 2095 3176 852 2096 3177 853 2097 3178 854 2098 3179 855 2099 3180 856 21.00 3181 857 2101 3182 858 2102 3183 859 2103 3184 860 2104 3185 861 2105 3186 862 2106 3187 863 2107 3188 864 2108 3189 865 2109 3190 866 2110 3191 867 2111 3192 868 2112 3193 869 2113 3194 870 2114 3195 871 2115 3196 872 2116 3197 873 2117 3198 874 2118 3199 875 2119 3200 876 2120 3201 877 2121 3202 878 2122 3203 879 2123 3204 880 2124 3205 881 2125 3206 882 2126 3207 883 2127 3208 884 2128 3209 885 2129 3210 886 2130 3211 887 2131 3212 888 2132 3213 889 2133 3214 890 2134 3215 891 2135 3216 892 2136 3217 893 2137 3218 894 2138 3219 895 2139 3220 896 2140 3221 897 2141 3222 898 2142 3223 899 2143 3224 900 2144 3225 901 2145 3226 902 2146 3227 903 2147 3228 904 2148 3229 905 2149 3230 906 2150 3231 907 2151 3232 908 2152 3233 909 2153 3234 910 2154 3235 911 2155 3236 912 2156 3237 913 2157 3238 914 2158 3239 915 2159 3240 916 2160 3241 917 2161 3242 918 2162 3243 919 2163 3244 920 2164 3245 921 2165 3246 922 2166 3247 923 2167 3248 924 2168 3249 925 2169 3250 926 2170 3251 927 2171 3252 928 2172 3253 929 2173 3254 930 2174 3255 931 2175 3256 932 2176 3257 933 2177 3258 934 2178 3259 935 2179 3260 936 2180 3261 937 2181 3262 938 2182 3263 939 2183 3264 940 2184 3265 941 2185 3266 942 2186 3267 943 2187 3268 944 2188 3269 945 2189 3270 946 2190 3271 947 2191 3272 948 2192 3273 949 2193 3274 950 2194 3275 951 2195 3276 952 2196 3277 953 2197 3278 954 2198 3279 955 2199 3280 956 2200 3281 957 2201 3282 958 2202 3283 959 2203 3284 960 2204 3285 961 2205 3286 962 2206 3287 963 2207 3288 964 2208 3289 965 2209 3290 966 2210 3291 967 2211 3292 968 2212 3293 969 2213 3294 970 2214 3295 971 2215 3296 972 2216 3297 973 2217 3298 974 2218 3299 975 2219 3300 976 2220 3301 977 2221 3302 978 2222 3303 979 2223 3304 980 2224 3305 981 2225 3306 982 2226 3307 983 2227 3308 984 2228 3309 985 2229 3310 986 2230 3311 987 2231 3312 988 2232 3313 989 2233 3314 990 2234 3315 991 2235 3316 992 2236 3317 993 2237 3318 994 2238 3319 995 2239 3320 996 2240 3321 997 2241 3322 998 2242 3323 999 2243 3324 1000 2244 3325 1001 2245 3326 1002 2246 3327 1003 2247 3328 1004 2248 3329 1005 2249 3330 1006 2250 3331 1007 2251 3332 1008 2252 3333 1009 2253 3334 1010 2254 3335 1011 2255 3336 1012 2256 3337 1013 2257 3338 1014 2258 3339 1015 2259 3340 1016 2260 3341 1017 2261 3342 1018 2262 3343 1019 2263 3344 1020 2264 3345 1021 2265 3346 1022 2266 3347 1023 2267 3348 1024 2268 3349 1025 2269 3350 1026 2270 3351 1027 2271 3352 1028 2272 3353 1029 2273 3354 1030 2274 3355 1031 2275 3356 1032 2276 3357 1033 2277 3358 1034 2278 3359 1035 2279 3360 1036 2280 3361 1037 2281 3362 1038 2282 3363 1039 2283 3364 1040 2284 3365 1041 2285 3366 1042 2286 3367 1043 2287 3368 1044 2288 3369 1045 2289 3370 1046 2290 3371 1047 2291 3372 1048 2292 3373 1049 2293 3374 1050 2294 3375 1051 2295 3376 1052 2296 3377 1053 2297 3378 1054 2298 3379 1055 2299 3380 1056 2300 3381 1057 2301 3382 1058 2302 3383 1059 2303 3384 1060 2304 3385 1061 2305 3386 1062 2306 3387 1063 2307 3388 1064 2308 3389 1065 2309 3390 1066 2310 3391 1067 2311 3392 1068 2312 3393 1069 2313 3394 1070 2314 3395 1071 2315 3396 1072 2316 3397 1073 2317 3398 1074 2318 3399 1075 2319 3400 1076 2320 3401 1077 2321 3402 1078 2322 3403 1079 2323 3404 1080 2324 3405 1081 2325 3406 1082 2326 3407 1083 2327 3408 1084 2328 3409 1085 2329 3410 1086 2330 3411 1087 2331 3412 1088 2332 3413 1089 2333 3414 1090 2334 3415 1091 2335 3416 1092 2336 3417 1093 2337 3418 1094 2338 3419 1095 2339 3420 1096 2340 3421 1097 2341 3422 1098 2342 3423 1099 2343 3424 1100 2344 3425 1101 2345 3426 1102 2346 3427 1103 2347 3428 1104 2348 3429 1105 2349 3430 1106 2350 3431 1107 2351 3432 1108 2352 3433 1109 2353 3434 1110 2354 3435 1111 2355 3436 1112 2356 3437 1113 2357 3438 1114 2358 3439 1115 2359 3440 1116 2360 3441 1117 2361 3442 1118 2362 3443 1119 2363 3444 1120 2364 3445 1121 2365 3446 1122 2366 3447 1123 2367 3448 1124 2368 3449 1125 2369 3450 1126 2370 3451 1127 2371 3452 1128 2372 3453 1129 2373 3454 1130 2374 3455 1131 2375 3456 1132 2376 3457 1133 2377 3458 1134 2378 3459 1135 2379 3460 1136 2380 3461 1137 2381 3462 1138 2382 3463 1139 2383 3464 1140 2384 3465 1141 2385 3466 1142 2386 3467 1143 2387 3468 1144 2388 3469 1145 2389 3470 1146 2390 3471 1147 2391 3472 1148 2392 3473 1149 2393 3474 1150 2394 3475 1151 2395 3476 1152 2396 3477 1153 2397 3478 1154 2398 3479 1155 2399 3480 1156 2400 3481 1157 2401 3482 1158 2402 3483 1159 2403 3484 1160 2404 3485 1161 2405 3486 1162 2406 3487 1163 2407 3488 1164 2408 3489 1165 2409 3490 1166 2410 3491 1167 2411 3492 1168 2412 3493 Table5: LSRProtein sequence SEQID NOs: 1169 1170 1171 1172 1173 1174 1175 1176 1177 1178 1179 1180 1181 1182 1183