METHODS AND COMPOSITIONS FOR INHIBITION OF CRISPR RE-CLEAVAGE EVENTS

Abstract

The disclosure relates to methods and compositions that serve to reduce the potential for targeted nuclease activity for example, but not limited to, CRISPR enzymes such as Cas9 and/or Cas12a. The disclosure also relates to methods and compositions be used as an HDR substrate to prevent Cas9 re-cleavage, and as a result increase the frequency of perfect HDR.

Claims

1. A modified HDR donor comprising modifications selected from the group comprising LNA, 2′-MOE, or 2′-OMe.

2. The modified HDR donor of claim 1, wherein the modifications are placed upstream of a CRISPR PAM recognition sequence.

3. The modified HDR donor of claim 2, wherein the modifications are placed 2, 3, 4, or 5 bases upstream of the CRISPR PAM recognition sequence.

4. The modified HDR donor of claim 1, wherein the modified HDR donor is a single stranded HDR donor.

5. The single stranded HDR donor of claim 4, wherein the single stranded HDR donor is a targeting strand.

6. The single stranded HDR donor of claim 4, wherein the single stranded HDR donor is a non-targeting strand.

7. The modified HDR donor of claim 1, wherein the modified HDR donor further comprises phosphorthioate internucleotide linkages.

8. A method for reducing targeted nuclease re-editing events comprising: a. contacting a candidate editing target locus site with an active CRISPR endonuclease system having a CRISPR protein, guide RNA, and a modified HDR donor.

9. The method of claim 8, wherein the modified HDR donor further comprises modifications selected from the group comprising LNA, 2′-MOE, or 2′-OMe.

10. The method of claim 9, wherein the modifications are placed 2, 3, 4, or 5 bases upstream of the CRISPR PAM recognition sequence.

11. The method of claim 8, wherein the modified HDR donor is a single stranded HDR donor.

12. The method of claim 11, wherein the single stranded HDR donor is a targeting strand.

13. The method of claim 11, wherein the single stranded HDR donor is a non-targeting strand.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0015] FIG. 1 provides a description of oligonucleotides used in the in vitro assay and placement of modified nucleotides (at positions 3, 4 and 5 upstream from the PAM sequence, which is situated 3′ of the protospacer as shown in FIG. 1) and modified linkages (linking positions 2-3, 3-4, 4-5 with respect to the PAM). In this assay, Cas9 targets the dsDNA duplex of 132-bp and the cleavage products are resolved as 75-bp and 57-bp dsDNA species in order to determine the %-cleavage at time points from 0-30 minutes

[0016] FIG. 2 shows workflow for digestion through analysis of cleavage products to determine the percent of the dsDNA substrate that is digested and, by extension, the degree to which the internal modifications are inhibiting digestion via S.p. Cas9.

[0017] FIG. 3 shows internal modification on both strands of the duplexed DNA oligonucleotides results in varying degrees of inhibition of Cas9 cleavage. Compared to unmodified oligonucleotides, the following relationship shows the performance of internal modifications in Cas9 inhibition after 30 minutes: Unmodified=RNA<PS<2′-OMe<LNA=MOE.

[0018] FIG. 4 demonstrates Cas9 digestion of double-stranded DNA with single-strand modifications showing that the MOE modification and the LNA modification provide the greatest inhibition of Cas9 re-cleavage when the modifications are positioned on the targeting strand and paired with an unmodified non-targeting strand.

[0019] FIG. 5 shows Cas9 digestion of double-stranded DNA with variable number of MOE and MOE-PS modifications and change in positions on the targeting strand.

[0020] FIG. 6 shows Cas9 digestion of double-stranded DNA with variable number of LNA and LNA-PS modifications and change in positions on the targeting strand

[0021] FIG. 7 shows editing in HEK293 cells in which NHEJ is reduced and the native, wild-type sequence is preserved with modified HDR donor templates. Templates contain the native gDNA sequence and reduce NHEJ in a dose-dependent fashion with the presence of modified nucleotides that inhibit Cas9 re-cleavage.

DETAILED DESCRIPTION

[0022] The disclosure relates to methods and compositions which reduce the potential for targeted nuclease activity for example, but not limited to, CRISPR enzymes such as Cas9 and/or Cas12a. The disclosure also relates to methods and compositions be used as an HDR substrate to prevent Cas9 re-cleavage, and as a result increase the frequency of perfect HDR.

[0023] Reference will now be made in detail to exemplary embodiments of the claimed invention. While the claimed invention will be described in conjunction with the exemplary embodiments, it will be understood that it is not intended to limit the claimed invention to those embodiments. To the contrary, it is intended to cover alternatives, modifications, and equivalents, as may be included within the spirit and scope of the claimed invention, as defined by the appended claims.

[0024] Those of ordinary skill in the art may make modifications and variations to the embodiments described herein without departing from the spirit or scope of the claimed invention. In addition, although certain methods and materials are described herein, other methods and materials that are similar or equivalent to those described herein can also be used to practice the claimed invention.

[0025] In addition, any of the compositions or methods provided, disclosed, or described herein can be combined with one or more of any of the other compositions and methods provided, disclosed, or described herein.

[0026] Definitions

[0027] Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by a person skilled in the art to which the claimed invention belongs. The terminology used herein is for describing particular embodiments only and is not intended to be limiting of the claimed invention. All technical and scientific terms used herein have the same meaning.

[0028] The following terms may have meanings ascribed to them below, unless specified otherwise. However, it should be understood that other meanings known or understood by those having ordinary skill in the art are also possible, and within the scope of the claimed invention. All publications, patent applications, patents, and other references mentioned or discussed herein are expressly incorporated by reference in their entireties. In the case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

[0029] As used herein, the singular forms “a,” “and,” and “the” include plural references, unless the context clearly dictates otherwise.

[0030] As used herein, the term “or” means, and is used interchangeably with, the term “and/or,” unless context clearly indicates otherwise.

[0031] As used herein, the term “including” means, and is used interchangeably with, the phrase “including but not limited to.”

[0032] As used herein, the term “such as” means, and is used interchangeably with, the phrase “such as, for example” or “such as but not limited.”

[0033] Unless specifically stated or obvious from context, as used herein, the term “about” is understood as within a range of normal tolerance in the art, for example, within two standard deviations of the mean. About can be understood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clear from context, all numerical values provided herein can be modified by the term about.

[0034] Ranges provided herein are understood to be shorthand for all of the values within the range. For example, a range of 1 to 50 is understood to include any number, combination of numbers, or sub-range from the group consisting 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50.

[0035] The term “decreased,” as used herein with regard to the re-cleavage of target genomic regions, refers to the level of cutting by guided endonuclease systems, such as CRISPR system after incorporation of the modified HDR donor. A guided endonuclease system has “decreased” re-cleavage activity if the level of its cleavage activity, as measured by the cleavage assay or as measured by other methods known in the art is less than the base cleavage activity before introduction of the modified HDR donor. For example, the re-cleavage activity of the guided endonuclease system is decreased if the re-cleavage activity is at least 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5% less than, or at least 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, or more than 10-fold less than the base cleavage activity before introduction of the modified HDR donor.

[0036] As used herein the term “imperfect HDR” are those HDR events which occur when the DSB is repaired, as intended, by adding the desired change in the single stranded donor oligo. However, the intended sequence is subsequently re-cut (re-cleaved) by the CRISPR system and then repaired by a NHEJ mechanism at the cut site. In the context of double-stranded HDR donors imperfect HDR refers to the blunt integration, reverse integration, or concatameric ligation events.

[0037] As used herein the term “perfect HDR” can be described as a repair outcome that reflects only the intended change to the target genomic sequence.

EXAMPLES

[0038] The claimed invention is further illustrated by the following Examples, which should not be construed as limiting. Those of skill in the art will recognize that the claimed invention may be practiced with variations of the disclosed structures, materials, compositions, and methods, and such variations are regarded as within the scope of the claimed invention.

Example 1

Methods to Digest and Resolve the CRISPR Products on a Fragment Analyzer (Agilent Technologies)

[0039] FIG. 2 shows a typical workflow for visualizing CRIPSR editing experiments. The typical workflow is as follows:

[0040] 1. DNA substrate duplexes from 132-nt oligos were combined in equal-mole amounts in IDT duplex buffer, heated to 95 C and cooled to room temperature at 10 uM final concentration.

[0041] 2. The Cas9 digest reactions were carried out in BioRad 96-well, full-skirted plates at 37 C. Components in the 10 ul reaction include 1× Cas9 reaction buffer (from 5× stock), 2 pmol of dsDNA substrate, and 5 pmol RNP complex (1:1 mol ratio of v3 Cas9:gRNA formed as discussed previously in Jacobi, et al).

[0042] 3. Reactions were quenched at 0, 0.5, 5 and 30 minutes with 2 ul of 300 mM EDTA (50 mM final concentration). Cas9 was digested with proteinase K (1 ul of 20 mg/ml solution) for 10 minutes at 56 C. Samples were further diluted with 60.85 ul of nuclease-free water and vortexed.

[0043] 4. Reaction products were resolved via capillary electrophoresis on the Fragment Analyzer via CRP-910-33 CRISPR Discovery method, dsDNA 810 gel with 180 s vacuum injection time.

[0044] 5. Calculations for % cleavage from band intensities were calculated as follows: 100×((Digested product 1+Digested product 2)/(Digested product 1+Digested product+Full length product)).

Example 2

Dual Strand Internal Modifications and the Effect on the Reduction of CRISPR Re-Cleavage Events

[0045] In initial experiments, both of the duplexed oligonucleotides, comprising the targeting and non-targeting strands, contained the modified nucleic acids at the locations indicated in FIG. 1. Unmodified oligonucleotides as well as the RNA (ribonucleic acid)-modified oligonucleotides showed very similar cleavage kinetics and gave >70% digestion after 30 minutes under the previously discussed reaction conditions. The dsDNA substrates containing the phosporothioate (PS) modified linkages reduced cleavage activity by about 50%. The internal 2′-OMe (2′-O-methyl-RNA) modifications reduced the cleavage activity to 14.5%. Both the LNA (locked nucleic acid)-modified oligonucleotides and the MOE (2′-O-methoxyethly RNA)-modified oligonucleotides gave <2% digestion (FIG. 3).

Example 3

Single Strand Internal Modifications and the Effect on the Reduction of CRISPR Re-Cleavage Events

[0046] After examining the inhibitory activity of the dual-strand modifications the impact of duplexed DNA comprised of one modified and one unmodified strand on Cas9 cleavage inhibition was assessed. In this example, the modifications with dual-strand inhibition, 2′-OMe, LNA and 2′-MOE, were again compared to an unmodified dsDNA substrate; however, the modifications were placed on either the targeting strand or the non-targeting strand of the gRNA in order to understand the impact of the single-stranded modifications on in vitro cleavage. In FIG. 4, the MOE and the LNA modifications placed on the targeting strand confer almost 100% resistance to cleavage with WT Cas9; whereas, the same modifications on the non-targeting strand (cleaved by the RuvC nuclease domain) only give about 50% inhibition compared to the percent digestion observed for the unmodified duplex. Although the inhibitory effect from the 2′-OMe modifications is reduced, it is notable that the modified nucleotides on the non-targeting strand lead to a reduction in Cas9 digestion which is greater than the reduction in digestion mediated by the targeting strand, giving 60% digestion and 71% digestion, respectively.

Example 4

Single Strand and Single Internal Modifications and the Effect on the Reduction of CRISPR Re-Cleavage Events

[0047] As shown in Example 3 the MOE and LNA modifications on the targeting strand provided an almost 100% inhibition of Cas9 cleavage out to 30 minutes (FIG. 4). In Example 4 modified oligonucleotides with a varied number of internal modifications were designed. Example 4 further demonstrates the minimal requirement for modifications that inhibit Cas9 cleavage. In both cases of a single MOE modification along with a single phosphorothioate modification (where “MOEPS upstream” has a PS-linkage between nucleotide positions 3 and 4 from the PAM sequence and a MOE modification at nucleotide position 3 and “MOEPS downstream” has a PS linkage at the same location but a MOE modification of the nucleotiede at position 4), it is apparent that a single nucleotide modification at a nucleotide adjacent to the cut-site paired with a PS-linkage at the canonical Cas9 cleavage site represent the minimally sufficient modifications to fully inhibit Cas9 cleavage in vitro. (FIG. 5).

[0048] Interestingly, the single LNAPS (LNA modification with a single PS) modification (FIG. 6) demonstrates some inhibition of Cas9 cleavage in either the upstream or downstream position. However, the “2-LNA” substrate, with positions 3 and 4 modified by LNAs, is the modification that gives the greatest inhibition in this experiment.

Example 5

Testing of Modified ssDNA HDR in Cells

[0049] Finally, the modified ssDNA HDR donor templates were tested in cells. In this example, each donor template represented the WT sequence. In this example, if the donor template is incorporated, this should represent the most likely scenario for re-cleavage to occur following repair, that is, one in which the repaired product remains a perfect match for the co-delivered RNP complex. The cell culture and nucleofection methods follow.

[0050] The cell culture and nucleofection methods employed here are as follows. HEK293 cells were transfected with 2 μM S.p Cas9 v3 RNP targeting TNPO3 (guide RNA sequence: 5′-CCACGGAGAGCCTTCTGCCC-3′) along with 2 μM IDT Alt-R Electroporation Enhancer, and varying dose of ssDNA HDR donor templates (0.1, 0.5, 1.0, 2.0, 3.2 μM). Ribonucleoprotein complexes (RNP) and single-stranded oligodeoxynucleotide (ssODN) or modified ssDNA HDR donor templates were delivered to HEK293 cells using the Amaxa Nucleofector System by Lonza. Cells were counted and pelleted using centrifugation. The pelleted cells were washed with 10 mL 1× phosphate-buffered saline (PBS). The cells were pelleted again and resuspended in Nucleofection Solution. Alt-R CRISPR-Cas9 crRNA/tracrRNA targeting the TNPO3 site was assembled as an RNP complex as described in Jacobi, et al. For each electroporation, 5 μL of RNP complex was added to 20 μL of cells in Nucleofection Solution (3-5E5 cells/nucleofection). A single-stranded Ultramer DNA oligo consisting 40-nucleotide (nt) arms of homology flanking the genomic DNA cut site were designed to retain the WT genomic DNA sequence. Donor DNA and IDT Alt-R Cas9 Electroporation Enhancer were added to the reaction to the final concentrations noted above in a final volume of 28 μL PBS. The reaction was mixed by pipetting and 25 μL was transferred to an electroporation cuvette plate. The cells were electroporated according to the manufacturer's protocol for this cell line. After electroporation, the cells were resuspended in 75 μL pre-warmed culture media in the electroporation cuvette. Triplicate aliquots of 25 μL of resuspended cells were further cultured in 175 μL pre-warmed media. The cells were allowed to grow until 100% confluency, 48-72 hours in total, after which genomic DNA was isolated via Quick Extract Buffer (Epicentre). The percent wild-type sequence as well as the percent of NHEJ repair for each treatment was quantified by NGS amplicon sequencing on the Illumina MiSeq platform and data analysis done via IDT's in-house data analysis pipeline (rhAmpSeq, CRISP-Alt-Rations).

[0051] FIG. 7, shows the results graphed with “Percent NHEJ” as the readout on the y-axis. As can be seen, the “RNP-only” condition, which lack a ssDNA donor, as well as the “Unmod” donor condition result in 60-70% NHEJ, and the unmodified DNA donor does not confer much Cas9 cleavage inhibition. In contrast, we observe >50% inhibition of NHEJ in the 3.2 μM ssDNA delivery condition for both MOEPS and LNAPS variables tested. In a dose-response fashion, the results suggest that internally-modified ssDNA HDR donors are incorporated into the genome of HEK293 cells and confer a resistance to S.p. Cas9 re-cleavage

Bibliography

[0052] Jacobi, et al. Methods. May 15, 2017:121-122. Simplified CRISPR tools for efficient genome editing.

[0053] Skarnes, et al. Methods. Jul. 15, 2019: 164-165. Improving homology-directed repair efficiency in human stem cells.

[0054] Rose, et al. Nature Communications. 11:2697 (2020). Suppression of unwanted CRISPR-Cas9 editing by co-administration of catalytically inactivation truncated guide RNAs.