Programmable Modification of DNA

Abstract

A self-reconfiguring genome uses a cassette having operons or DNA sequences that code for guide RNA, reverse transcriptase, donor RNA, and a CRISPR cleavage enzyme. A self-reconfiguring genome may be based on lambda recombineering of in situ generated oligonucleotides. A method for programmable self-modification of a cellular genome includes transcribing guide RNA from a self-reconfiguring cassette, associating the transcribed guideRNA with the CRISPR enzyme, intercalating a region of complimentary sequence within an integration site of the genome, cutting upstream of a PAM site within the integration site; transcribing the donorRNA, translating donorRNA to double-stranded DNA, and recombining the double-stranded DNA via homologous recombination at the cut site of the integration site. A set of cascadable and multiplexable genetic logic gates with a universal RNA input/output based on single-strand annealing or non-homologous end joining, comprises transcription promoters or terminators, homologous regions, DNA sequences, RNA, and enzymes from the CRISPR system.

Claims

1. A method for programmable modification of a cellular genome, the method comprising the steps of: programming a genetic cassette to effect a desired genomic modification, the cassette comprising operons or DNA sequences that code for a guide RNA, a reverse transcriptase, donor RNA, and a cleavage enzyme from a CRISPR system, the step of programming comprising selecting or designing the guide RNA and the donor RNA to have an ability to target promoters or ribosome binding sites that have been selected in accordance with the desired genomic modification; introducing the programmed cassette into a cell having a target cellular genome; and causing expression of the cassette by the cell in order to effect the desired genomic modification, wherein the expression of the cassette is controlled so that the cell is caused to self-modify the target cellular genome by performing the steps of: transcribing the guide RNA from the cassette; associating the transcribed guideRNA with the CRISPR enzyme; intercalating a region of complimentary sequence within an integration site of the cellular genome; cutting, using the CRISPR enzyme, upstream of a PAM site located within the integration site; transcribing the donor RNA from the cassette; reverse transcribing the donor RNA to double-stranded DNA using the reverse transcriptase; and recombining the double-stranded DNA via homologous recombination at the cut site of the integration site, thereby producing the desired genomic modification within the integration site of the target cellular genome.

2. The method of claim 1, further comprising the step of repeating the step of causing expression of the cassette a plurality of times in order to create serial insertions at the integration site, thereby producing further modification of the cellular genome.

3. The method of claim 1, wherein the modified genome is configured to comprise a counter.

4. The method of claim 1, wherein the modified genome is configured to comprise a data logger.

5. The method of claim 4, wherein the data logger is configured to log the presence at least one of: small molecule, peptide, protein, DNA, RNA, heat, or light.

6. The method of claim 1, wherein the modified genome is configured to reconfigure one or more of an organism's metabolic pathways.

7. A self-reconfiguring genome based on a self-reconfiguring cassette, the cassette comprising operons or DNA sequences that code for: a guide RNA; a reverse transcriptase; donor RNA; and a cleavage enzyme from the CRISPR system, wherein the guide RNA and the donor RNA are selected or designed to have an ability to target promoters or ribosome binding sites that have been selected in accordance with a desired genomic self-reconfiguration.

8. The genome of claim 7, configured to comprise a counter.

9. The genome of claim 7, configured to comprise a data logger.

10. The genome of claim 9, wherein the data logger is configured to log the presence at least one of: small molecule, peptide, protein, DNA, RNA, heat, or light.

11. The genome of claim 7, configured to reconfigure one or more of an organism's metabolic pathways.

12. The genome of claim 7, configured to reconfigure one or more of an organism's metabolic pathways.

13. A set of cascadable and multiplexable genetic logic gates with a universal RNA input/output based on single-strand annealing or non-homologous end joining, comprising transcription promoters or terminators, homologous regions, DNA sequences, RNA, and enzymes from the CRISPR system.

14. A genetic logic device comprising a plurality of genetic logic gates from the set of claim 13.

15. The logic device of claim 14, wherein the genetic logic gates are cascaded.

16. The logic device of claim 14, wherein the genetic logic gates are multiplexed.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Other aspects, advantages and novel features of the invention will become more apparent from the following detailed description of the invention when considered in conjunction with the accompanying drawings, wherein:

[0019] FIG. 1 illustrates the prior art CRISPR—Cas9 system (SEQ ID No: 1, SEQ ID No: 2, SEQ ID No: 3) for programmable double stranded cutting of an integration site;

[0020] FIGS. 2A-C depict prior art examples of transcription factor based logic;

[0021] FIG. 3 depicts prior art examples of recombinase based logic;

[0022] FIG. 4 illustrates the prior art process of directed nuclease assisted homologous recombination;

[0023] FIG. 5 illustrates the prior art process of deletion by single-strand annealing (SSA) homologous recombination;

[0024] FIGS. 6A-C together provide a schematic drawing of an exemplary embodiment of a self-reconfiguring genetic cassette (SEQ ID Nos: 4-11) according to one aspect of the invention;

[0025] FIGS. 7A-H together provide a schematic drawing of an exemplary embodiment of the generation of double stranded DNA donors from mRNA (SEQ ID Nos. 12-15) according to one aspect of the invention;

[0026] FIGS. 8 (SEQ ID Nos: 16-18) and 9 (SEQ ID Nos: 19-22) are schematic drawings of parts of an exemplary embodiment of a counter or data logger that adds segments of DNA to the genome as a function of time or stimulus, according to one aspect of the invention;

[0027] FIG. 10 illustrates an exemplary embodiment of a self-reconfiguring system based on lambda recombination, according to one aspect of the invention;

[0028] FIG. 11 is illustrates an alternate embodiment of a self-reconfiguring system based on lambda recombination, according to one aspect of the invention;

[0029] FIG. 12 is a schematic drawing of an exemplary embodiment of genetic logic gates that cascade, according to one aspect of the invention;

[0030] FIG. 13 is a schematic drawing of an exemplary embodiment of genetic logic gates that multiplex, according to one aspect of the invention;

[0031] FIG. 14 is a schematic drawing of an exemplary embodiment of alternative genetic logic gates that cascade, according to one aspect of the invention;

[0032] FIG. 15 depicts the sequence (SEQ ID No: 23) resulting from experimentally cloning a reporter with the T7 promoter followed by the first 171 bases of GFP, a protospacer and protospacer adjacent sequence, transcription terminator, and the entire GFP gene into BL21 E. coli; and

[0033] FIG. 16 depicts an experimentally produced sequence (SEQ ID No: 24) consistent with SSA repair, resulting from introducing the corresponding guide RNA and Cas9 to the sequence (SEQ ID No.: 23) of FIG. 15.

DETAILED DESCRIPTION

[0034] In some embodiments, means based on Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRs) allow the cell to self-reconfigure its own genome. A self-reconfiguring cassette according to one aspect of the invention comprises operons or DNA sequences which code for i) a guide RNA to recognize and cleave at an integration site, ii) the CRISPR protein Cas9, iii) reverse transcriptase, and iv) Donor RNA, which is reverse transcribed into double stranded donor DNA.

[0035] In some embodiments, the cassette operates in the following manner. Guide RNA (guideRNA) is transcribed from the cassette, associates with the protein CAS9 and intercalates a region of complimentary sequence within the Integration site. Once intercalated, the Cas9 cuts upstream of a PAM site also located within the Integration site. In parallel, donor RNA, whose termini are homologous to the integration site cut site, is transcribed from the cassette by RNA polymerase and then translated to double stranded DNA by means of reverse transcriptase. The double stranded DNA is recombined via homologous recombination at the integration site cut site to produce a genomic modification within the integration site. This serves as a general means for the cell to modify its own genome.

[0036] Serial insertions at the integration site can act as a counter. Serial insertions triggered by a stimuli, such as, but not limited to, light small molecular protein, or RNA/DNA, comprise a data logger. Structuring guide RNA sequences and donor DNAs to target promoters or ribosome binding sites within metabolic pathways may comprise a system for carrying out synthetic evolution, diversity or library generation and genomic engineering.

[0037] In some other embodiments, means based on CRISPRs allow the cell to carry out cascadable and multiplexable digital logic. In such embodiments, input RNA combines with the Cas9 protein to cut a protospacer sequence, complementary to a spacer sequence in the RNA, followed by a PAM sequence in DNA of the genetic logic gate. This DNA break results in deletion of a transcription promoter or terminator by means of single-strand annealing (SSA) homologous recombination or non-homologous end joining (NHEJ). Output RNA either self-cleaves or is cleaved by Csy4 at CRISPR repeat sequences to improve its affinity for Cas9, thus serving as input for the next layer of gates. The sequence space of such RNA prevents interaction between gates.

[0038] FIGS. 6A-C together provide a schematic drawing of an exemplary embodiment of a self-reconfiguring genetic cassette according to one aspect of the invention. Referring to FIG. 6A, a self-reconfiguring DNA cassette 605 based on Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRs) comprises operons or DNA sequences which code for i) a guide RNA 610 (SEQ ID No: 4, SEQ ID No: 5) to recognize and cleave at an integration site 615 (SEQ ID No: 6, SEQ ID No: 7), ii) the CRISPR protein Cas9 620, iii) reverse transcriptase 625, and iv) Donor RNA 630 which is reverse transcribed into double stranded donor DNA. Guide RNA 610 (guideRNA) is transcribed from cassette 605, associates with the protein Cas9 620 and intercalates a region of complimentary sequence within Integration site 615.

[0039] Referring to FIG. 6B, once intercalated, the Cas9 620 cuts upstream of a Proto-spacer Adjacent Motif (PAM) site 640 also located within integration site 615. In parallel, donor RNA 630 whose termini are homologous to the integration site cut site, is transcribed from the cassette by RNA polymerase and then translated to double stranded donor DNA 650 (SEQ ID No: 8, SEQ ID No: 9) by means of reverse transcriptase 620. This reverse transcription may take place by the normal mechanism of reverse transcription employed by retroviruses, which leaves over-flanking heterologous (non-homologous) sequence or by a novel approach, depicted in FIGS. 7A-H, which can generate double stranded donor DNA without heterologous flanking sequence.

[0040] Referring to FIG. 6C, double stranded donor DNA 650 is recombined via the cell's homologous recombination system at integration site cut site 640 to produce a DNA sequence modification (SEQ ID No: 10, SEQ ID No: 11) within integration site 615. Such homologous recombination efficiency in bacteria is greatly enhanced by engineering the λ prophage Red recombination system [Zhang, Yongwei, Uwe Werling, and Winfried Edelmann, “SLiCE: a novel bacterial cell extract-based DNA cloning method”, Nucleic Acids Research 40.8, pp. e55-e55 (2012)]. In the strain termed PPY, such homologous recombination can take place at high efficiency, either without heterologous flanking sequence or with short (<˜45 bp) heterologous flanking sequence, although the efficiency is greater without appreciable heterologous flanking sequence.

[0041] FIGS. 7A-H together outline the steps for an exemplary embodiment of the generation of double stranded DNA donors from mRNA transcripts according to one aspect of the invention. In FIGS. 7A-H, darker lines 710 represents DNA and lighter lines 720 represent RNA. FIG. 7A depicts an mRNA transcript 730 (SEQ ID No: 12) designed to be self-priming by including hairpin sequences at both the 3′ and 5′ ends. FIG. 7B depicts the mRNA 730 having formed hairpins 740 at both the 3′ end and 5′ end. FIG. 7C (SEQ ID No: 13) depicts Reverse Transcriptase transcribing the mRNA 730 into DNA in the 3′ to 5′ direction. FIG. 7D (SEQ ID No: 14) depicts Reverse Transcriptase displacement of the 5′ end mRNA hairpin and continuation of the DNA transcript in the 3′ direction. FIG. 7E depicts digestion of the mRNA by an RNAse which may be the native RNAse activity of reverse transcriptase. FIG. 7F depicts hairpinning and self-priming of the DNA transcript. FIG. 7G depicts extension of the DNA transcript by DNA polymerase or the DNA polymerase activity of Reverse Transcriptase. FIG. 7G (SEQ ID No: 15) depicts optional restriction enzyme cleavage of the hairpin region of the DNA transcript producing a clean double stranded donor DNA 750.

[0042] FIGS. 8 and 9 are schematic drawings of parts of an exemplary embodiment of a counter or data logger that adds segments of DNA to the genome as a function of time or stimulus. These added segments may be read out by sequencing of the resultant modified genome. Referring to FIG. 8, a guide RNA 810 (SEQ ID No: 16) which targets integration site 820 (SEQ ID No: 17, SEQ ID No: 18) is expressed either as a function of time or as a function of an input stimulus (e.g., a small molecule such a tetracycline) that activates the promoter for the guide RNA 810. As described previously with respect to FIGS. 1 and 6A-C, the guide RNA 810 complexes with Cas 9 and induces a double stranded break 830 near the PAM sequence of the integration site 820.

[0043] Referring to FIG. 9, as discussed with respect to FIGS. 6A-C, double stranded (ds) donor DNA 910 (SEQ ID No: 19, SEQ ID No: 20) can now template the repair of the ds break 830 and add additional DNA sequence 920 to cleaved integration site 820, thus producing modified integration site 930 (SEQ ID No: 21, SEQ ID No: 22) and recording a stimulus event or the passage of time. This process may be continued by having a second guide RNA that now targets and cleaves the newly modified integration site near its PAM site and a second ds donor DNA which templates the repair of that new break and adds additional genetic sequence. If it is arranged that the second ds donor DNA has the same sequence as the original integration site, then this process will circle back on itself with the first guide RNA now targeting the integration site again and so on.

[0044] Designing guide RNA sequences and donor DNAs to target promoters or ribosome binding sites within metabolic pathways comprises a system for carrying out self-evolution, diversity or library generation, and self-genomic engineering analogous to the evolution, library generation, and genomic engineering carried out in the process known as MAGE, using exogenously introduced oligonucleotides [Wang, Harris H., et al., “Programming cells by multiplex genome engineering and accelerated evolution”, Nature 460.7257, pp. 894-898 (2009)].

[0045] Lambda phage protein (red locus) mediated recombineering can be used to incorporate exogenous oligonucleotides into a chromosome, a form of in vivo site-directed mutagenesis [D. Court et. al., “Genetic Engineering Using Homologous Recombination”, Annual Review of Genetics, Vol. 36, p. 361 (2002)]. The efficiency of this process can be high enough that antibiotic selection is unnecessary, as one can simply screen for recombinants. However, when multiple exogenous oligos are introduced into the cell simultaneously, such as by electroporation or chemical competency, the efficiency of incorporation of each oligo decreases substantially. One limiting factor can be the supply of available β protein. Another can be the amount of each oligo available in the cell. To remedy the second concern, the production of oligos intracellularly, from a plasmid template, is employed. The large plasmid (or BAC) is produced in vivo using gene synthesis techniques, and then transformed into the host. The plasmid is then induced to manufacture large numbers of each desired oligo, which in turn self-reconfigures the genome of the cell.

[0046] FIGS. 10 and 11 illustrate exemplary embodiments of a self-reconfiguring system based on lamda recombination, according to one aspect of the invention. Referring to FIG. 10, a DNA cassette 1010 is incorporated into the cell. DNA cassette 1010 comprises an RNA polymerase promoter 1020, a first oligonucleotide sequence 1030, a terminator/reverse primer 1040, and then a second oligonucleotide sequence. Additional oligonucleotide sequences may be incorporated, each separated by a terminator/reverse primer, such as shown in cassette 1110 in FIG. 11, so that the oligonucleotide sequence-terminator/reverse primer element is used repeatedly, there being one per oligo being produced. The oligonucleotides are designed to form a hairpin. The oligonucleotides are transcribed 1050 into RNA by RNA polymerase. Additionally, the cassette codes for reverse transcriptase, which makes 1060 a complimentary DNA strand primed by the RNA hairpin 1065 or by tRNA. Finally, RNAseH activity digests 1070 the RNA strand, yielding single stranded DNA oligonucleotides which are further incorporated into the host genome via lambda mediated recombineering [D. Court et. al., “Genetic Engineering Using Homologous Recombination”, Annual Review of Genetics, Vol. 36, p. 361 (2002)]. If the RNA polymerase promoter is activated by a small molecule, light, protein or other stimulus, then this system comprises a data logger in which the new lambda mediated recombineering modification of the genome records the presence of the stimulus.

[0047] Referring to FIG. 11, a DNA cassette 1110 is incorporated into the cell. DNA cassette 1110 comprises a rolling circle amplification (RCA) initiation site 1120, a first oligo sequence 1130, a universal separator 1140, and a second oligo sequence 1150. Additional oligonucleotide sequences may be incorporated, each separated by a universal separator 1140. Inside the cell, polymerase transcribes 1150 single stranded copies 1165 of the template, producing ssDNA 1165 by rolling circle (strand displacing) amplification. The universal separators 1140 are designed to form 1170 double stranded hairpins 1175, which in turn are cleaved by a hairpin nuclease, Y flap nuclease, or an exonuclease designed to cut the separator sequence, thus releasing 1180 single stranded DNA oligos 1185 that are further incorporated into the host genome via lambda mediated recombineering

[0048] FIG. 12 is a schematic drawing of an exemplary embodiment of genetic logic gates that can be cascaded, according to one aspect of the invention. FIG. 12 depicts all of the non-trivial gates (OR, NOR, XOR, XNOR, AND, NAND, X.fwdarw.Y, and X˜.fwdarw.Y) for a complete set of two-input-one-output logic based on Cas9-gRNA cleavage and SSA homologous recombination. In FIG. 12, “.fwdarw.” represents a promoter, “T” is a terminator, “R” is a CRISPR repeat for Csy4 cleavage or ribozyme RiboJ self-cleavage, “A”, “B”, and “C” are homologs for SSA, “X” and “Y” are protospacer and PAM cut sites, and “gRNA.sub.Z” represents output RNA. In the system of FIG. 12, gRNA serves as a universal input and output.

[0049] FIG. 13 is a schematic drawing of an exemplary embodiment of three-input-two-output genetic logic gates that multiplex, including OR, NOR, XOR, XNOR, AND, and NAND gates. In FIG. 13, “.fwdarw.” represents a promoter, “T” is a terminator, “R” is a CRISPR repeat for Csy4 cleavage or ribozyme RiboJ self-cleavage, “A”, “A′”, “A″”, and “A′″” are homologs for SSA, “X”, “X′”, and “X″” are protospacer and PAM cut sites, and “gRNA.sub.Y” and “gRNA.sub.Y,” represent output RNA.

[0050] FIG. 14 is a schematic drawing of an exemplary embodiment of alternative genetic logic gates that cascade. FIG. 14 depicts almost all of the non-trivial gates for a complete set of two-input-one-output logic based on Cas9-gRNA cleavage and non-homologous end joining (NHEJ), including OR, NOR, AND, NAND, X.fwdarw.Y, and X˜.fwdarw.Y gates. In FIG. 14, “ ” represents a promoter, “T” is a terminator, “R” is a CRISPR repeat for Csy4 cleavage or ribozyme RiboJ self-cleavage, “X” and “Y” are protospacer and PAM cut sites, and “gRNAZ” represents output RNA. In the system of FIG. 14, gRNA serves as a universal input and output.

[0051] Logic, universal input/output, and programmable gain are necessary properties for demonstrating computation by single-strand annealing (SSA) homologous recombination repair of CRISPR-induced cleavage. The elements for implementation of this logic have been described above. The parts that make up these elements are well defined: promoter, guide RNA, terminator, RNA processing, and homologous arm sequences.

[0052] To verify the ideal homologous arm length for instigating SSA, a reporter with the T7 promoter followed by the first 171 bases of GFP 1510 (highlighted), a protospacer and protospacer adjacent sequence 1520 (bold), transcription terminator 1530 (italicized), and the entire GFP gene 1540 were cloned into BL21 E. coli. The resulting construct 1550 (SEQ ID No. 23) is shown in FIG. 15.

[0053] Upon introducing the corresponding guide RNA and Cas9, all colonies were found to have sequence 1610 (SEQ ID No. 24) shown in FIG. 16, which is consistent with SSA repair. As hoped, no GFP expression was observed until guide RNA and Cas9 were introduced. To demonstrate universality of input/output and second-layer output, guide RNA will instead follow sequence 1510. In this experiment, second-layer guide RNA targets a sequence on the plasmid to enable quick readout by Surveyor. Gain can then be programmed by adding an array of redundant output guide RNA for increased gain or by adding mismatches to a guide RNA sequence for decreased gain.

[0054] Exemplary Implementations: This invention may be implemented in many ways. The items in the list of exemplary implementations that follows are not intended as patent claims. Instead, they are non-limiting examples of ways that this invention may be implemented or embodied. Following are some non-limiting examples of how this invention may be implemented:

[0055] Implementation 1. A self-reconfiguring genome based on a self-reconfiguring cassette comprising a guide RNA, a reverse transcriptase, a donor RNA, and a cleavage enzyme from the CRISPR system.

[0056] Implementation 2. The system of Implementation 1, configured to comprise a counter.

[0057] Implementation 3. The system of Implementation 1, configured to comprise a data logger.

[0058] Implementation 4. The system of Implementation 3, configured to comprise a data logger to log the presence of one or more of: small molecule, peptide, protein, DNA, RNA, heat, or light.

[0059] Implementation 5. The system of Implementation 1, configured to reconfigure one or more of an organism's metabolic pathways.

[0060] Implementation 6. A self-reconfiguring genome based on lambda recombineering of in-situ generated oligonucleotides.

[0061] Implementation 7. The system of Implementation 6, configured to reconfigure one or more of an organism's metabolic pathways.

[0062] Implementation 8. The system of Implementation 6, configured to comprise a data logger to log the presence of one or more of: small molecule, peptide, protein, DNA, RNA, heat, or light.

[0063] Implementation 9. The system of Implementation 6, in which the in situ generated oligonucleotides are generated by means of in situ reverse transcription of RNA.

[0064] Implementation 10. Cascadable and multiplexable genetic logic gates with a universal RNA input/output based on single-strand annealing or non-homologous end joining comprising transcription promoters or terminators, homologous regions, as well as DNA sequences, RNA, and enzymes from the CRISPR system.

[0065] Implementation 11. The system of Implementation 10, configured to cascade genetic logic gates.

[0066] Implementation 12. The system of Implementation 10, configured to multiplex genetic logic gates.

[0067] While preferred embodiments of the invention are disclosed herein, many other implementations will occur to one of ordinary skill in the art and are all within the scope of the invention. Each of the various embodiments described above may be combined with other described embodiments in order to provide multiple features. Furthermore, while the foregoing describes a number of separate embodiments of the apparatus and method of the present invention, what has been described herein is merely illustrative of the application of the principles of the present invention. Other arrangements, methods, modifications, and substitutions by one of ordinary skill in the art are therefore also considered to be within the scope of the present invention, which is not to be limited except by the claims.