Modified double-stranded donor templates

Abstract

The present invention relates to modified double-stranded donor templates. In particular, compositions and methods are described herein for improving homologous directed repair (HDR) efficiency and reducing homologous independent integration following introduction of a double-stranded break using an engineered nuclease. Further, a double-stranded DNA donor is modified to improve donor potency and efficiency of homologous directed repair following introduction of a double-stranded break using a programmable nuclease.

Description

[0001] This application is a divisional application with an international application date of October 23, 2020, international application number PCT / US2020 / 057105, which entered the Chinese national phase on April 24, 2022, application number 202080074468.9, and invention name “Modified double-stranded donor template”.

[0002] CROSS REFERENCE TO RELATED APPLICATIONS

[0003] This application claims priority to U.S. Provisional Patent Application No. 62 / 925,366, filed on October 24, 2019, which is incorporated herein by reference in its entirety.

[0004] Reference to a sequence listing

[0005] This application is filed in accordance with 37 CFR § 1.821(c) with a sequence listing in computer readable form. The text file "013670-9060-US02_sequence_listing_10-DEC-2020_ST25.txt" submitted via EFS was generated on December 10, 2020, contains 235 sequences, has a file size of 86.4 kilobytes, and is incorporated herein by reference in its entirety. Technical Field

[0006] Described herein are compositions and methods for increasing the efficiency of homology-directed repair (HDR) and reducing homology-independent integration after double-strand breaks are introduced using engineered nucleases. In addition, double-stranded DNA donors are modified to increase donor potency and the efficiency of homology-directed repair after double-strand breaks are introduced using programmable nucleases. Background Art

[0007] Genome editing using programmable nucleases allows site-specific introduction of DNA into a target genome of interest. Many systems allow targeted genome editing, and these include transcription activator-like effector nucleases (TALENs), zinc fingers (ZFNs), or clustered regularly interspaced short palindromic repeats (CRISPRs).

[0008] The CRISPR-Cas9 system has been widely used for site-specific genome editing in eukaryotic cells. A sequence-specific guide RNA is required to recruit the Cas9 protein to the target site, after which the Cas9 endonuclease cleaves both strands of the target DNA, generating a double-strand break (DSB). This DSB is corrected by the cell’s innate DNA damage repair pathways. The two major pathways for DSB repair are the error-prone non-homologous end joining (NHEJ) pathway, which can result in random insertions or deletions (indels) in the target DNA, and the homology-directed repair (HDR) pathway, which uses single-stranded or double-stranded DNA molecules with homology to either side of the DSB as repair templates to generate desired mutations in the target DNA [1].

[0009] Various forms of DNA can be used as repair templates for HDR experiments, such as plasmid DNA, double-stranded linear DNA (dsDNA) or single-stranded DNA (ssDNA). Both dsDNA and ssDNA donors can induce innate immune responses in mammalian tissue culture cells. For short insertions (usually ≤120 bp) or mutations, chemically synthesized oligonucleotides such as IDT® Ultramer™ ssDNA can be used as single-stranded oligonucleotide donors (ssODN) for HDR experiments. The use of synthetic ssDNA allows chemical modifications to be placed in the molecule to potentially improve HDR efficiency. Due to the increased complexity of synthesis, templates for larger insertions (usually>120 bp) are limited. The production of long ssDNA may be a labor-intensive and costly process, while linear dsDNA can be produced quickly and in large quantities. Because the more prevalent NHEJ repair pathway facilitates blunt-end ligation, linear dsDNA donors have a higher risk of homology-independent integration into any DSB present in the cell, including the on-target Cas9 cleavage site, any Cas9 off-target sites, and any endogenous DSBs [2, 3]. When homology-independent integration occurs at the on-target site, the entire donor, including the homology arms, is incorporated, resulting in duplication of one or both homology arm regions.

[0010] It has been reported that the addition of a 5′-biotin modification to a linear dsDNA donor can reduce concatemer formation and integration via the NHEJ pathway [4]. Similarly, another group reported that biotin or ssDNA overhangs on the 5′-end can reduce blunt-end insertions [5]. Another group proposed that TEG and 2′-OMe ribonucleotide linkers on the 5′-end of a dsDNA donor could potentially increase HDR rates by limiting access of the NHEJ machinery to the free end of the donor, but did not demonstrate any reduction in blunt-end integration [6].

[0011] What is needed are compositions and methods of modified dsDNA templates for HDR that improve the efficiency of HDR and reduce unwanted homology-independent integration (at both the target site and potential off-target or endogenous DSBs) that is typically associated with linear dsDNA donors. Summary of the invention

[0012] One embodiment described herein is a double-stranded DNA homology-directed repair (HDR) donor, which comprises a first homology arm region, an insertion region, and a second homology arm region, wherein the first homology arm region and the second homology arm region comprise modifications to one or more nucleotides at or near the 5'-end. In one case, the modification comprises: modification of the 2'-position of one or more nucleotides at or near the 5'-end of the first homology arm region, and modification of the 2'-position of one or more nucleotides at or near the 5'-end of the second homology arm region. In another case, the modification comprises modification of the 2'-position of the 5'-terminal nucleotide, the 5'-penultimate nucleotide, the 5'-penultimate third (third) nucleotide, or a combination of the nucleotides at or near the 5'-end of the first homology arm region and the second homology arm region. In another case, the modification at or near the 5'-end of the double-stranded DNA HDR donor comprises one or more of 2'-O-methyl (2'-OMe), 2'-fluoro (2'-F), or 2'-O-methoxyethyl (2'-MOE). In another case, the modification at or near the 5′-end of the double-stranded DNA HDR donor includes 2′-MOE. In another case, the modification at or near the 5′-end is a non-template mismatch relative to the target DNA. In another case, the length of the first homology arm region and the second homology arm region is 40 to 150 nucleotides. In another case, the length of the first homology arm region and the second homology arm region is at least 100 nucleotides. In another case, the double-stranded DNA HDR donor also includes a universal primer sequence. In another case, the insertion region is greater than 100 bp. In one case, the insertion region is greater than 0.25 kb, greater than 0.5 kb, greater than 1 kb, greater than 2 kb, greater than 3 kb, greater than 4 kb, greater than 5 kb, greater than 6 kb, greater than 7 kb, greater than 8 kb, greater than 9 kb or greater than 10 kb. In another case, the double-stranded HDR donor includes a hairpin at either the 5′-end or the 3′-end. In another instance, the double-stranded HDR donor comprises a hairpin at both the 5′-end and the 3′-end. In another instance, the double-stranded DNA HDR donor improves homology-directed repair efficiency and reduces homology-independent integration in a programmable nuclease system.

[0013] Another embodiment described herein is a programmable nuclease system comprising a modified double-stranded DNA homology-directed repair (HDR) donor, a programmable nuclease, and a gRNA, wherein the gRNA molecule is capable of targeting the programmable nuclease molecule to a target nucleic acid. In one case, the modified double-stranded DNA HDR donor comprises a first homology arm region, an insertion region, and a second homology arm region; wherein the first homology arm region and the second homology arm region comprise modifications to one or more nucleotides at or near the 5'-end. In another case, the modified double-stranded DNA HDR donor comprises a modification of the 2'-position of the 5'-terminal nucleotide, the 5'-penultimate nucleotide, the 5'-penultimate third (third) nucleotide, or a combination of the nucleotides at or near the 5'-end of the first homology arm region and the second homology arm region. In another case, the modified double-stranded DNA HDR donor comprises at least one 2'-OME, 2'-F, or 2'-MOE modification of one or more nucleotides at or near the 5'-end. In another case, the modified double-stranded DNA HDR donor comprises one or more 2′-MOE modifications at or near the 5′-end. In another case, the modified double-stranded DNA HDR donor comprises a universal primer sequence. In another case, the modified double-stranded DNA HDR donor improves homology-directed repair efficiency and reduces homology-independent integration in a programmable nuclease system. In another case, the programmable nuclease system comprises one or more of a transcription activator-like effector nuclease (TALEN), a zinc finger (ZFN), or a clustered regularly interspaced short palindromic repeat sequence (CRISPR). In another case, the programmable nuclease system is CRISPR. In another case, the programmable nuclease is CRISPR-associated-9 (Cas9). In another case, the programmable nuclease system further comprises one or more HDR enhancers.

[0014] Another embodiment described herein is a method for increasing homology-directed repair (HDR) rates and reducing homology-independent integration in a programmable nuclease system, the method comprising targeting a candidate editing target site locus using an active programmable nuclease system and a modified double-stranded DNA HDR donor. In one instance, the modified double-stranded DNA HDR donor comprises a first homology arm region, an insertion region, and a second homology arm region; wherein the first homology arm region and the second homology arm region comprise modifications to one or more nucleotides at or near the 5'-end. In another instance, the modified double-stranded DNA HDR donor comprises a modification of the 2'-position of the 5'-terminal nucleotide, the 5'-penultimate nucleotide, the 5'-penultimate third (third) nucleotide, or a combination of the nucleotides at or near the 5'-end of the first homology arm region and the second homology arm region. In another instance, the modified double-stranded DNA HDR donor comprises at least one 2'-OME, 2'-F, or 2'-MOE modification of one or more nucleotides at or near the 5'-end. In another embodiment, the modified double-stranded DNA HDR donor comprises one or more 2'-MOE modifications at or near the 5'-end. In another embodiment, the modified double-stranded DNA HDR donor comprises a universal primer sequence. In another embodiment, the method further comprises one or more HDR enhancers. In another embodiment, the modified double-stranded DNA HDR donor improves homology-directed repair efficiency and reduces homology-independent integration in a programmable nuclease system.

[0015] Another embodiment described herein is the use of a modified double-stranded DNA HDR donor for increasing homology-directed repair (HDR) rates and reducing homology-independent integration in a programmable nuclease system, wherein the modified double-stranded DNA HDR donor comprises a first homology arm region, an insertion region, a second homology arm region, and optionally one or more universal primer sequences; wherein the first homology arm region and the second homology arm region comprise modifications to one or more nucleotides at or near the 5'-end. In one case, the modification comprises at least one 2'-OME, 2'-F, or 2'-MOE modification of one or more nucleotides at or near the 5'-end of the double-stranded DNA HDR donor.

[0016] Another embodiment described herein is a method for making a modified double-stranded DNA HDR donor, the method comprising synthesizing an oligonucleotide comprising a first homology arm region, an insertion region, a second homology arm region, and optionally one or more universal primer sequences; wherein the first homology arm region and the second homology arm region comprise modifications to one or more nucleotides at or near the 5'-end. In one case, the modification comprises at least one 2'-OME, 2'-F or 2'-MOE modification of one or more nucleotides at or near the 5'-end of the double-stranded DNA HDR donor.

[0017] Another embodiment described herein is a method for making a modified double-stranded DNA HDR donor, the method comprising amplifying a target nucleic acid sequence comprising a first homology arm region, an insertion region, and a second homology arm region using one or more universal primers, wherein the universal primer sequence comprises a modification of one or more nucleotides at or near the 5'-end. In one case, the modification comprises at least one 2'-OME, 2'-F, or 2'-MOE modification at one or more nucleotides at or near the 5'-end of the universal primer. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1A –B shows a schematic diagram showing the effects of using a dsDNA HDR donor template for Cas9-directed cleavage ( Figure 1A ) or endogenous double-strand breaks or off-target cleavage by Cas9 ( Figure 1B ) These homology-independent integration events result in the incorporation or duplication of homology arms at the double-strand breaks introduced by programmable nucleases.

[0019] Figure 2 Shown is the evaluation of dsDNA donor integration using a modified linear dsDNA donor containing a 1 kb insert by either the HDR or NHEJ pathway.

[0020] Figure 3 Evaluation of dsDNA donor integration by HDR or NHEJ pathways using a modified linear dsDNA donor containing a 42 bp insert is shown. The modifications were extended to multiple 2′-MOE ribonucleotides and internally placed 2′-MOE ribonucleotides.

[0021] Figure 4 Evaluation of dsDNA donor integration by HDR or NHEJ pathways using a modified linear dsDNA donor containing a 42 bp insert is shown. Cas9 guides targeting non-homologous sites were used to mimic off-target Cas9 cleavage.

[0022] Figure 5Evaluation of dsDNA donor integration by HDR or NHEJ pathways using modified linear dsDNA donors containing a 42 bp insert is shown. Modifications were extended to include additional 2′-modifications. Cas9 guides targeting non-homologous sites were used to mimic off-target Cas9 cleavage.

[0023] Fig. 6A -B shows the synthesis method of hairpin-blocked dsDNA HDR template. Gray indicates DNA hairpin composed of 2′-MOE ribonucleotides. Black indicates chemically synthesized unmodified DNA. White indicates DNA template sequence with available primer binding sites. Fig. 6A It was shown that for short HDR inserts, a hairpin-blocked dsDNA donor can be generated by annealing two chemically synthesized ssDNA oligonucleotides containing a 5′-MOE hairpin. Figure 6B It is shown that for longer HDR insert sequences, hairpin-blocked dsDNA donors can be generated by PCR amplification. Primers with 5'-MOE hairpins can be used to amplify the target HDR template. The DNA polymerase should not be able to amplify past the MOE-containing hairpin. After several cycles, a final dsDNA product containing MOE hairpins on both 5'-ends should be generated.

[0024] Figure 7 Evaluation of dsDNA donor integration by HDR or NHEJ pathways using donors with hairpins or 1×MOE modified bases at the 5′-end is shown. The donor contains 30 bp homology arms and mediates a 6 bp insertion to introduce an EcoRI restriction site into the SERPCIN1 locus. The hairpin consists of a 3 bp stem and a "TTTT" loop and contains either unmodified DNA bases (DNA only) or 2′-MOE modified bases (MOE modified). No hairpin was attached for initial testing.

[0025] Fig. 8A –C shows the use of a modified linear dsDNA donor via HDR ( Fig. 8A ) or NHEJ ( Figure 8B ) pathway for the assessment of dsDNA donor integration. The ratio of HDR compared to blunt-end integration is shown in Figure 8C Donors were designed to mediate 42 bp insertions at 4 genomic locations and tested in 2 cell lines (n = 8 per modification). Results are reported as fold change relative to the unmodified dsDNA donor for each site and cell line.

[0026] Fig. 9AEvaluation of dsDNA donor integration by HDR or NHEJ pathways using modified linear dsDNA donors mediating 300 bp, 500 bp or 1 kb insertions at two genomic locations is shown. A long ssDNA donor targeting the SERPINC1 locus is provided for comparison. Fig. 9B A comparative orthogonal analysis method for assessing insertions at the SERPINC1 locus is shown. Long read sequencing using the MinION™ system from Oxford Nanopore Technologies (ONT) was compared to amplicon length analysis in which PCR amplicons from genomic DNA samples were run on a fragment analyzer and quantified.

[0027] Fig.10 A schematic diagram of the design of a dsDNA HDR donor template containing a universal primer sequence is shown. Hashed black indicates DNA sequences that are homologous between the genomic DNA target and the HDR donor (i.e., homology arms). Black indicates the desired insert DNA sequence. White indicates DNA sequences that are homologous to the universal primer sequence.

[0028] Fig.11 Evaluation of dsDNA donor integration via HDR or NHEJ pathways using modified linear dsDNA donors consisting of a 500 bp insert flanked by 100 bp homology arms is shown. Donors were synthesized with or without terminal universal primer sequences.

[0029] Fig. 12A C shows the visual assessment of HDR readouts from 1×MOE donors using IGV. HDR readouts from EMX1 and SERPINC1 1×MOE dsDNA donors made using universal primer sequences were aligned to a primer set containing the correct HDR sequence ( Fig. 12A ) or HDR sequences with generic sequences (i.e. incorrect HDR) ( Fig. 12B For comparison, HDR reads from a 1×MOE dsDNA donor lacking the universal sequence were aligned to the correct HDR reference sequence ( Fig. 12C ). In the IGV plot, each read sequence is represented by a thin horizontal line. Each nucleotide that does not align correctly with the reference sequence (i.e., insertion, gap, or mutation) is marked in black. The background error rate from MinION™ sequencing can be found in Fig. 12CSchematic representation of the HDR reference sequence is shown above each IGV plot. Solid black represents the desired 500 bp insert. The dashed area represents the sequence homologous to the 100 bp donor homology arm. The dotted area represents the 30 bp universal primer sequence. The region of interest is indicated by an arrow. Misalignment against the incorrect HDR reference sequence is evident in each HDR read sequence ( Fig. 12B ), indicating a 30 bp lack of universal sequence after repair. The plot for the EMX1 donor represents approximately 500 reads. The plot for the SERPINC1 donor represents approximately 3700 reads.

[0030] Fig.13 Evaluation of HDR rates when using unmodified or 1xMOE modified dsDNA donor templates is shown. Donors are designed to insert GFP at the N- or C-terminus of the target gene and contain 200 bp of homology arms. Donors were generated with universal primer sequences. HDR rates were evaluated by flow cytometry (reported as % of GFP positive cells).

[0031] Fig.14A -B shows the evaluation of the yield when making dsDNA HDR templates with universal primers or gene-specific primers. 12 sequences >500 bp and 12 sequences <500 bp were made and the PCR yield was evaluated. The total yield of each group is shown in Fig.14A , and for each sequence, the comparison between templates with and without universal primers is shown in Fig. 14B middle. DETAILED DESCRIPTION

[0032] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those of ordinary skill in the art are generally understood. For example, any nomenclature and techniques used in conjunction with cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization described herein are well known and commonly used in the art. In the event of a conflict, this document including definitions shall prevail. Preferred methods and materials are described below, although methods and materials similar or equivalent to those described herein may be used in the practice or testing of the present invention.

[0033] When used herein, the terms "amino acid", "nucleotide", "polypeptide", "polynucleotide" and "vector" have their common meanings as understood by biochemists of ordinary skill in the art. Standard single-letter nucleotides (A, C, G, T, U) and standard single-letter amino acids (A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W or R) are used herein.

[0034] When used herein, terms such as "including," "comprising," "having," and the like mean "comprising." The present disclosure also contemplates other embodiments comprising, "consisting of," and "consisting essentially of" the embodiments, aspects, or elements presented herein, whether explicitly stated or not.

[0035] As used herein, terms without a specific number used in the context of the present disclosure (especially in the context of the claims) should be interpreted to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. In addition, unless otherwise specified, references without a specific number mean "one or more".

[0036] As used herein, the term "or" can be conjunctive or disjunctive.

[0037] As used herein, the term "substantially" means to a great or significant extent, but not completely.

[0038] When used in this article, the term "about" or "approximately" applied to one or more values ​​of interest refers to a value close to the stated reference value or within an acceptable error range of the specific value, which is determined by a person of ordinary skill in the art and depends in part on how to measure or determine the value, such as the limitations of the measurement system. In one case, the term "about" refers to any value within a range of variation of up to ±10% of the value modified by the term "about", including both integers and decimal parts. Alternatively, according to the practice of this area, "about" can mean within 3 or more standard deviations. Alternatively, for example, for a biological system or process, the term "about" can mean within an order of magnitude of a value, within 5 times in some embodiments, and within 2 times in some embodiments. When used in this article, the symbol "~" means "about" or "approximately".

[0039] All ranges disclosed herein include both endpoints as discrete values ​​and all integers and fractions specified within the range. For example, the range of 0.1-2.0 includes 0.1, 0.2, 0.3, 0.4 ... 2.0. If the endpoint is modified by the term "about", the specified range is expanded by a change of up to ±10% of any value (including the endpoint) within the range or within 3 or more standard deviations.

[0040] As used herein, the terms "control" or "reference" are used interchangeably. A "reference" or "control" level can be a predetermined value or range that is used as a baseline or benchmark against which measurements are evaluated. "Control" also refers to a control experiment or a control cell.

[0041] As used herein, the phrase "effective amount" of a compound described herein refers to an amount of a compound described herein that will elicit a biological response, such as a reduction or inhibition of enzyme or protein activity, or alleviate symptoms, alleviate symptoms, slow or delay disease progression, or prevent disease, etc.

[0042] As used herein, the term "inhibit" refers to a reduction or suppression of a given condition, symptom or disorder or disease, or a significant decrease in the baseline activity of a biological activity or process.

[0043] As used herein, the term "universal primer" refers to a sequence that has no known alignment to a target sequence. Universal primers allow for sequence-independent amplification of a target sequence.

[0044] Methods and compositions of dsDNA donor templates for improving HDR efficiency and reducing blunt-end integration events disclosed herein. In various embodiments, the methods and compositions disclosed herein allow for reduction of homology-independent integration after genome editing with programmable nucleases. In certain embodiments, bulky modifications are placed at the 5′-end of a linear dsDNA donor. In other embodiments, bulky modifications are placed at or near the 5′ end of a linear dsDNA donor. In addition, modifications may be placed at the 2′-position of the DNA of a nucleotide at or near one or more 5′-nucleotides of a dsDNA donor (e.g., 2′-MOE, 2′-OME, or 2′-F nucleotides). These modifications show improved efficacy in reducing homology-independent integration. In addition, this reduction does not appear to be mediated by increasing donor stability, because other modifications (PS, etc.) with a determined ability to block nuclease degradation do not reduce the blunt-end integration rate to the same extent as other 2′-modifications.

[0045] When homology-independent integration occurs at the on-target site, the entire donor, including the homology arms, is incorporated (Figure 1), which produces duplicate homology arms. Figure 1 is a schematic diagram of homology-independent (duplicate homology arms) and homology-dependent integration events when using a dsDNA HDR donor template. The light gray bars indicate the target genomic DNA sequence, while the white indicates the non-homologous genomic DNA sequence (at an endogenous DSB or Cas9 off-target site). The hashed black indicates the DNA sequence (i.e., homology arms) that is homologous between the genomic DNA target and the HDR donor. The black indicates the desired insert DNA sequence. Figure 1A The dsDNA donor is shown inserted into the target at the Cas9 cleavage site via HDR or NHEJ repair pathways. Insertion via the NHEJ pathway results in duplication of the donor homology arms. Figure 1B The insertion of dsDNA at endogenous DSBs or off-target Cas9 cleavage sites via the NHEJ repair pathway is shown.

[0046] In some embodiments, chemical modifications are introduced into the 5′-end of the linear dsDNA donor. These chemical modifications are used to reduce the risk of NHEJ integration and improve their function as repair templates in HDR experiments. In some embodiments, bulky or large modifications are introduced into the 5′-end of the dsDNA donor. In other embodiments, the modifications can be introduced into the end or 5′-DNA nucleotide of the dsDNA oligonucleotide. In some embodiments, the modifications can be introduced into or near the 5′-end of the dsDNA oligonucleotide. In some embodiments, one or more DNA nucleotides at or near the 5′-end of the dsDNA oligonucleotide can be modified. In certain embodiments, the modification comprises biotin, phosphorothioate (PS), triethylene glycol (TEG), locked nucleic acid (LNA, 2′-oxy-4′-carbon methylene linkage), hexaethylene glycol (Sp18), 1,3-propylene glycol (SpC3), 2′-O-methoxyethyl (MOE) ribonucleotide, 2′-O-methyl ribonucleotide (2′-OMe), 2′-fluoro (2′-F) nucleotide, or ribonucleotide. In certain embodiments, the modification is placed on the 5′-terminal nucleotide, the 5′-penultimate nucleotide, the 5′-third-to-last (third) nucleotide, or a combination of nucleotides at or near the 5′-end of the dsDNA donor. In other embodiments, the modification is placed at the 2′-position of the 5′-terminal nucleotide, the 5′-penultimate nucleotide, the 5′-third-to-last (third) nucleotide, or a combination of nucleotides at or near the 5′-end of the dsDNA donor. In yet another embodiment, the modification is placed at the 2'-position of the 5'-terminal nucleotide, the 5'-penultimate nucleotide, the 5'-third-to-last (third) nucleotide, or a combination of said nucleotides at or near the 5'-end of the dsDNA donor.

[0047] It has been found that the use of 2'-modified ribonucleotides, particularly 2'-O-methoxyethyl (MOE), provides the best improvement when compared to biotin or other modifications. Additional experiments using donors mediating large inserts to establish the use of these modifications are described herein.

[0048] Further improvements to the dsDNA donor manufacturing process were evaluated. Universal primer sequences with no homology to common genomes (human, mouse, rat, zebrafish) were selected. Previous work from our group established the utility of these primer sequences in cloning applications (i.e., efficient, reliable amplification). Significant improvements in (1) amplification success using a large number of different sequences and (2) overall amplification yield could be achieved by incorporating these universal sequences into the donor manufacturing process. This article describes the testing of these universal sequences when placed on either side of a complete HDR donor sequence. Due to the reduced risk of homology-independent integration when using 5′-dsDNA modifications, these sequences have no adverse effect on the correct HDR rate using modified donors and are rarely incorporated during blunt-end integration.

[0049] The methods and compositions disclosed herein relate to dsDNA donor templates for improving HDR efficiency and reducing homology-independent events (blunt-end integration events or multimerization events). In various embodiments, the methods and compositions disclosed herein allow for reducing homology-independent integration or increasing homology-dependent integration after genome editing using programmable nucleases. In certain embodiments, bulky nucleotide modifications are placed at the 5′-end of a linear dsDNA donor. In other embodiments, modifications (e.g., 2′-MOE, 2′-OMe) at the 2′-position of the nucleotide of one or more 5′-terminal nucleotides placed near the 5′-end of the dsDNA show improved efficacy in reducing homology-independent integration. In addition, this reduction does not appear to be mediated by increasing donor stability, as other modifications (PS, etc.) with a determined ability to inhibit acidase degradation do not reduce the blunt-end integration rate to the same extent as other 2′-modifications.

[0050] In certain embodiments, chemical modifications are introduced to the 5′-end of the linear dsDNA donor. These chemical modifications are used to reduce the risk of NHEJ integration and improve their utility as repair templates in HDR experiments. In certain embodiments, bulky or large modifications are introduced. In other embodiments, modifications can be introduced near the 5′-end of the dsDNA oligonucleotide donor. In certain embodiments, modifications can be introduced at or near the 5′-end of the dsDNA oligonucleotide donor. In certain embodiments, nucleotides at or near the 5′-end of the dsDNA oligonucleotide can be modified. In other embodiments, modifications include, but are not limited to, biotin (B), phosphorothioates (PS, ), triethylene glycol (TEG), locked nucleic acids such as 2′-oxy-4′-carbomethylene linkage (LNA), hexaethylene glycol (Sp18), 1,3-propylene glycol (SpC3), 2′-O-methoxyethyl (MOE) ribonucleotides, 2′-O-methyl ribonucleotides (2′-OMe), 2′-fluoro (2′-F) nucleotides and ribonucleotides.

[0051] In other embodiments, the use of hairpin structures on the ends of the dsDNA donor also reduces blunt-end integration.

[0052] In one embodiment, the end-modified dsDNA donor template is suitable for use after the introduction of a double-strand break by a programmable nuclease. In other embodiments, the programmable nuclease comprises a transcription activator-like effector nuclease (TALEN), a zinc finger (ZFN), or a clustered regularly interspaced short palindromic repeat (CRISPR). In one embodiment, the programmable nuclease system is CRISPR. In one case, the programmable nuclease is CRISPR associated-9 (Cas9).

[0053] In one embodiment, the 5′-end modified dsDNA donor is generated by PCR amplification. Primers modified with biotin, phosphorothioate (PS) linkage, TEG, LNA, spacer 18 (SP18), C3 spacer (SpC3), or MOE are used to amplify the insertion region and generate the end-modified dsDNA donor. In certain embodiments, the insertion region is greater than 120 bp. In certain embodiments, the insertion region is an insertion region of at least 1 kb. In certain embodiments, the insertion region is greater than 1 kb, greater than 2 kb, greater than 3 kb, greater than 4 kb, greater than 5 kb, greater than 6 kb, greater than 7 kb, greater than 8 kb, greater than 9 kb, or greater than 10 kb.

[0054] In other embodiments, modifications at or near the 5′-end include biotin, phosphorothioate (PS), triethylene glycol (TEG), locked nucleic acids such as 2′-oxy-4′-carbomethylene linkages (LNA), hexaethylene glycol (Sp18), 1,3 propanediol (SpC3), 2′-O-methoxyethyl ribonucleotides (MOE), 2′-O-methyl ribonucleotides (2′-OMe), 2′-fluoro (2′-F) nucleotides, and ribonucleotides.

[0055] In other embodiments, the modification at or near the 5′-end includes a modification of the 2′-position of a DNA nucleotide at or near the 5′-end of the double-stranded DNA donor. In certain embodiments, the 2′-modification is 2′-MOE, 2′-OMe or 2′-fluorine, and the modification of the nucleotide occurs at or near the 5′-end of the double-stranded DNA donor. In certain embodiments, the 5′-end modification is on the 5′-terminal nucleotide of the double-stranded DNA donor. In other embodiments, the 5′-end modification is located at the 5′-terminal nucleotide, the 5′-penultimate nucleotide, the 5′-penultimate third (third) nucleotide, or a combination of the nucleotides at or near the 5′-end of the dsDNA donor. In other embodiments, the 5′-end modification is located at the 5′-terminal nucleotide, the 5′-penultimate nucleotide, or the 5′-penultimate third (third) nucleotide. In another embodiment, the 5'-end modification is a 2'-MOE modified ribonucleotide located at the terminal 5'-position, the second to last nucleotide position from the 5'-end, the third to last (third) nucleotide position from the 5'-end, or a combination thereof. In another embodiment, the 5'-end modification is a 2'-MOE ribonucleotide located at the terminal 5'-position, the second to last nucleotide position from the 5'-end, the third to last (third) nucleotide position from the 5'-end, or a combination thereof.

[0056] In other embodiments, the HDR donor comprises homology arms on either side of the insertion sequence. The homology arms are complementary to the sequences on both sides of the double-strand break introduced by the programmable nuclease. In some embodiments, the length of the homology arms varies from at least 20 nucleotides to 500 nucleotides. In some embodiments, the length of the homology arms is at least 40, 50, 60 70, 80, 90, 100, 150, 200, 300, 400 or 500 nucleotides. In some embodiments, the length of the homology arms may be greater than 500 nucleotides. In other embodiments, the homology arms are preferably at least 40 nucleotides long, more preferably at least 100 nucleotides long.

[0057] In some embodiments, the insert sequence is placed between homology arms. In some embodiments, the length of the insert sequence is greater than 20 nucleotides. In some embodiments, the insert sequence is at least 1 nucleotide long to 4 kb long. In some embodiments, the length of the insert sequence is in the range of 1–2 kb. In some embodiments, the length of the insert sequence can be at least 1 bp, 2 bp, 3 bp, 4 bp, 5 bp, 6 bp, 7 bp, 8 bp, 9 bp, 10bp, 20 bp, 30 bp, 40 bp, 50 bp, 60 bp, 70 bp, 80 bp, 90 bp, 100 bp, 200 bp, 300 bp, 400bp, 500 bp, 600 bp, 700 bp, 800 bp, 900 bp, 1 kb, 2 kb, 3 kb, 4 kb, 5 kb, 6 kb, 7 kb, 8kb, 9 kb. In yet another embodiment, the insert sequence may be 10 kb or longer in length.

[0058] In other embodiments, the HDR donor comprises homology arms on either side of the insert, wherein the insert may include a SNP, MNP, or deletion. In certain embodiments, the insert may be at least 1 nucleotide long up to 4 kb long. In certain embodiments, the insert may be in the range of 1–2 kb in length. In certain embodiments, the length of the insert sequence can be at least 1 bp, 2 bp, 3 bp, 4 bp, 5 bp, 6 bp, 7 bp, 8 bp, 9 bp, 10 bp, 20 bp, 30 bp, 40 bp, 50 bp, 60 bp, 70 bp, 80 bp, 90 bp, 100 bp, 200 bp, 300 bp, 400 bp, 500 bp, 600 bp, 700 bp, 800 bp, 900 bp, 1 kb, 2 kb, 3 kb, 4 kb, 5 kb, 6 kb, 7 kb, 8 kb, 9 kb. In yet another embodiment, the length of the insert fragment can be 10 kb or longer.

[0059] The polynucleotides described herein include variants with substitutions, deletions and / or additions that may involve one or more nucleotides. The variants may be altered in the coding region, the noncoding region, or both. The changes in the coding region may produce conservative or non-conservative amino acid substitutions, deletions or additions. Particularly preferred are silent substitutions, additions and deletions that do not change the performance and activity of the binding.

[0060] Other embodiments described herein include nucleic acid molecules comprising a polynucleotide having a nucleotide sequence that is about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical, more preferably at least about 90-99% identical to (a) a nucleotide sequence or a degenerate, homologous or codon-optimized variant thereof, or (b) a nucleotide sequence that is capable of hybridizing to the complement of any of the nucleotide sequences in (a).

[0061] A polynucleotide having a nucleotide sequence that is, for example, at least 90-99% "identical" to a reference nucleotide sequence means that the nucleotide sequence of the polynucleotide is identical to the reference sequence except that the polynucleotide sequence may include up to about 10 to 1 point mutation, addition, or deletion per every 100 nucleotides of the reference nucleotide sequence.

[0062] In other words, in order to obtain a polynucleotide having a nucleotide sequence with about at least 90-99% identity to a reference nucleotide sequence, up to 10% of the nucleotides in the reference sequence may be deleted, added or replaced by another nucleotide, or a plurality of nucleotides up to 10% of the total nucleotides in the reference sequence may be inserted into the reference sequence. These mutations of the reference sequence may occur at the 5'- or 3'-terminal position of the reference nucleotide sequence or at any position between those terminal positions, interspersed individually between the nucleotides in the reference sequence or in one or more consecutive groups in the reference sequence. The same applies to polypeptide sequences having about at least 90-99% identity to a reference polypeptide sequence.

[0063] In certain embodiments, the programmable nuclease (e.g., CRISPR enzyme) or component (e.g., gRNA) can be introduced into cells using a variety of different approaches. Examples include plasmid or viral expression vectors (which cause endogenous expression of the enzyme, gRNA, or both), delivery of the enzyme and gRNA / crRNA transfection alone, or delivery of the enzyme with gRNA or crRNA as a ribonucleoprotein (RNP) complex.

[0064] It is obvious to those of ordinary skill in the relevant art that suitable modifications and adaptations can be made to the compositions, formulations, methods, processes, devices, components and applications described herein without departing from the scope of any embodiment or aspect thereof. The compositions, devices, components and methods provided are exemplary and are not intended to limit the scope of any disclosed embodiment. All various different embodiments, aspects and options disclosed herein can be combined in any variation or iteration form. The scope of the compositions, formulations, methods, devices, components and processes described herein includes all actual or potential combinations of the embodiments, aspects, options, examples and preferences described herein. The compositions, formulations, devices, components or methods described herein may omit any component or step, replace any component or step disclosed herein, or include any component or step disclosed elsewhere herein. The mass of any component of any composition or formulation disclosed herein and the mass of any other component in the formulation or the ratio of the total mass of other components in the formulation are disclosed herein as if they were explicitly disclosed. If the meaning of any term in any patent or publication incorporated by reference conflicts with the meaning of the term used in the present disclosure, the meaning of the term or phrase in the present disclosure shall prevail. All patents and publications cited herein are incorporated herein by reference with their specific teachings.

[0065] References

[0066] 1. Chang et al., “Non-homologous DNA end joining and alternative pathways to double-strand break repair,” Nature Reviews Molecular Cell Biology 18:495–506 (2017).

[0067] 2. Roth et al., “Reprogramming human T cell function and specificity with non-viral genome targeting,” Nature 559 (7714): 405–409 (2018).

[0068] 3. Li et al., “Design and specificity of long ssDNA donors for CRISPR-based knock-in,” bioRxiv doi:10.1101 / 178905 (2017).

[0069] 4. Gutierrez-Triann et al., “Efficient single-copy HDR by 5′ modified long dsDNA donors,” eLife 2018;7:e39468; DOI: 10.7554 / eLife.39468 (2018).

[0070] 5. Canaj et al., “Deep profiling reveals substantial heterogeneity of integration outcomes in CRISPR knock-in experiments,” bioRxiv doi: 10.1101 / 841098 (2019).

[0071] 6. Ghanta et al., “5′ modifications Improve Potency and Efficacy of DNA Donors for Precision Genome Editing,” bioRxiv doi: 10.1101 / 354480 (2018).

[0072] 7. Robinson et al., “Integrative Genomics Viewer,” Nature Biotechnology 29: 24–26 (2011).

[0073] Implementation

[0074] A1. A double-stranded DNA homology-directed repair (HDR) donor, comprising a first homology arm region, an insertion region, and a second homology arm region; wherein the first homology arm region and the second homology arm region comprise modifications to one or more nucleotides at or near the 5'-end.

[0075] A2. The double-stranded DNA HDR donor described in A1, wherein the modifications include: modification of the 2′-position of one or more nucleotides at or near the 5′-end of the first homology arm region, and modification of the 2′-position of one or more nucleotides at or near the 5′-end of the second homology arm region.

[0076] A3. The double-stranded DNA HDR donor described in A1-A2, wherein the modification includes modification of the 2′-position of the 5′-terminal nucleotide, the 5′-second-to-last nucleotide, the 5′-third-to-last (third) nucleotide, or a combination of said nucleotides at or near the 5′-end of the first homology arm region and the second homology arm region.

[0077] A4. The double-stranded DNA HDR donor described in A1, wherein the modification at or near the 5′-end of the double-stranded DNA HDR donor comprises one or more of 2′-OME, 2′-F or 2′-MOE.

[0078] A5. The double-stranded DNA HDR donor of A1-A4, wherein the modification at or near the 5′-end of the double-stranded DNA HDR donor comprises 2′-MOE.

[0079] A6. The double-stranded DNA HDR donor of A1-A5, wherein the modification at or near the 5′-end is a non-template mismatch relative to the target DNA.

[0080] A7. The double-stranded DNA HDR donor described in A1–A6, wherein the length of the first homology arm region and the second homology arm region is 40 to 150 nucleotides.

[0081] A8. The double-stranded DNA HDR donor described in A1-A7, wherein the length of the first homology arm region and the second homology arm region is at least 100 nucleotides.

[0082] A9. The double-stranded DNA HDR donor described in A1–A8, wherein the double-stranded DNA HDR donor further comprises a universal primer sequence.

[0083] A10. The double-stranded DNA HDR donor described in A1-A9, wherein the inserted region is larger than 100 bp.

[0084] A11. The double-stranded DNA HDR donor described in A1-A10, wherein the inserted region is greater than 0.25 kb, greater than 0.5 kb, greater than 1 kb, greater than 2 kb, greater than 3 kb, greater than 4 kb, greater than 5 kb, greater than 6 kb, greater than 7 kb, greater than 8 kb, greater than 9 kb or greater than 10 kb.

[0085] A12. The double-stranded DNA HDR donor of A1-A11, wherein the double-stranded HDR donor comprises a hairpin at either the 5′-end or the 3′-end.

[0086] A13. The double-stranded DNA HDR donor of A1-A12, wherein the double-stranded HDR donor comprises hairpins at both the 5′-end and the 3′-end.

[0087] A14. The double-stranded DNA HDR donor described in A1–A13, wherein the double-stranded DNA HDR donor improves homology-directed repair efficiency and reduces homology-independent integration in a programmable nuclease system.

[0088] B1. A programmable nuclease system comprising a modified double-stranded DNA homology-directed repair (HDR) donor, a programmable nuclease and a gRNA, wherein the gRNA molecule is capable of targeting the programmable nuclease molecule to a target nucleic acid.

[0089] B2. The programmable nuclease system described in B1, wherein the modified double-stranded DNA HDR donor comprises a first homology arm region, an insertion region, and a second homology arm region; wherein the first homology arm region and the second homology arm region comprise modifications to one or more nucleotides at or near the 5'-end.

[0090] B3. The programmable nuclease system described in B1–B2, wherein the modified double-stranded DNA HDR donor comprises a modification of the 2′-position of the 5′-terminal nucleotide, the 5′-second-to-last nucleotide, the 5′-third-to-last (third) nucleotide, or a combination of said nucleotides at or near the 5′-end of the first homology arm region and the second homology arm region.

[0091] B4. The programmable nuclease system of B1-B3, wherein the modified double-stranded DNA HDR donor comprises at least one 2′-OME, 2′-F or 2′-MOE modification of one or more nucleotides at or near the 5′-end.

[0092] B5. The programmable nuclease system of B1-B4, wherein the modified double-stranded DNA HDR donor comprises one or more 2′-MOE modifications at or near the 5′-end.

[0093] B6. The programmable nuclease system of B1-B5, wherein the modified double-stranded DNA HDR donor comprises a universal primer sequence.

[0094] B7. The programmable nuclease system described in B1–B6, wherein the modified double-stranded DNA HDR donor improves homology-directed repair efficiency and reduces homology-independent integration in the programmable nuclease system.

[0095] B8. The programmable nuclease system described in B1–B7, wherein the programmable nuclease system comprises one or more of a transcription activator-like effector nuclease (TALEN), a zinc finger (ZFN), or a clustered regularly interspaced short palindromic repeat (CRISPR).

[0096] B9. The programmable nuclease system described in B1–B8, wherein the programmable nuclease system is CRISPR.

[0097] B10. The programmable nuclease system of B1–B9, wherein the programmable nuclease is CRISPR-associated-9 (Cas9).

[0098] B11. The programmable nuclease system described in B1-B10, wherein the programmable nuclease system further comprises one or more HDR enhancers.

[0099] C1. A method for increasing homology-directed repair (HDR) rates and reducing homology-independent integration in a programmable nuclease system, the method comprising targeting a candidate editing target site locus with an active programmable nuclease system and a modified double-stranded DNA HDR donor.

[0100] C1. The method described in C1, wherein the modified double-stranded DNA HDR donor comprises a first homology arm region, an insertion region, and a second homology arm region; wherein the first homology arm region and the second homology arm region comprise modifications to one or more nucleotides at or near the 5'-end.

[0101] C2. The method described in C1, wherein the modified double-stranded DNA HDR donor comprises a modification of the 2′-position of the 5′-terminal nucleotide, the 5′-second-to-last nucleotide, the 5′-third-to-last (third) nucleotide, or a combination of the nucleotides at or near the 5′-end of the first homology arm region and the second homology arm region.

[0102] C3. The method of C1–C2, wherein the modified double-stranded DNA HDR donor comprises at least one 2′-OME, 2′-F or 2′-MOE modification of one or more nucleotides at or near the 5′-end.

[0103] C4. The method of C1–C3, wherein the modified double-stranded DNA HDR donor comprises one or more 2′-MOE modifications at or near the 5′-end.

[0104] C5. The method described in C1–C4, wherein the modified double-stranded DNA HDR donor comprises a universal primer sequence.

[0105] C6. The method described in C1–C5, wherein the method further comprises one or more HDR enhancers.

[0106] C7. The method described in C1–C6, wherein the modified double-stranded DNA HDR donor improves homology-directed repair efficiency and reduces homology-independent integration in a programmable nuclease system.

[0107] D1. A modified double-stranded DNA HDR donor for use in a programmable nuclease system to increase the homology-directed repair (HDR) rate and reduce homology-independent integration, wherein the modified double-stranded DNA HDR donor comprises a first homology arm region, an insertion region, a second homology arm region and optionally one or more universal primer sequences, wherein the first homology arm region and the second homology arm region comprise modifications to one or more nucleotides at or near the 5'-end.

[0108] D2. The use described in D1, wherein the modification comprises at least one 2′-OME, 2′-F or 2′-MOE modification of one or more nucleotides at or near the 5′-end of the double-stranded DNA HDR donor.

[0109] E1. A method for making a modified double-stranded DNA HDR donor, the method comprising synthesizing a first oligonucleotide comprising a first homology arm region, an insertion region, a second homology arm region and optionally one or more universal primer sequences, synthesizing a second complementary oligonucleotide sequence, and hybridizing the first oligonucleotide and the second oligonucleotide sequence, wherein the first homology arm region and the second homology arm region comprise modifications to one or more nucleotides at or near the 5'-end.

[0110] E2. The method of E1, wherein the modification comprises at least one 2′-OME, 2′-F or 2′-MOE modification of one or more nucleotides at or near the 5′-end of the double-stranded DNA HDR donor.

[0111] F1. A method for making a modified double-stranded DNA HDR donor, the method comprising amplifying a target nucleic acid sequence comprising a first homology arm region, an insertion region, and a second homology arm region using one or more universal primers, wherein the universal primer sequence comprises a modification of one or more nucleotides at or near the 5′-end.

[0112] F2. The method of F1, wherein the modification comprises at least one 2'-OME, 2'-F or 2'-MOE modification at one or more nucleotides at or near the 5'-end of the universal primer.

[0113] Example

[0114] Example 1

[0115] Improved HDR rates and reduced NHEJ insertions when using modified dsDNA donors

[0116] Initial experiments were performed to compare the insertion rates of homology-independent (i.e., blunt-end) integration relative to HDR of unmodified linear dsDNA, donors containing a 5′-biotin modification, or donors with optional modifications at or near the 5′-end. dsDNA donors were generated by PCR amplification of a plasmid containing a 1 kb insert flanked by 100 bp homology arms targeting the human SERPINC1 gene (100-1000-100; SEQ ID NO: 1; for amplification primer sequences, see Table 1; SEQ ID NOs: 2–21). Amplification primers were designed to have either unmodified sequence or the modifications indicated. Purified dsDNA donor was delivered to 3.5 × 10 cells / mL at a concentration of 100 nM (1 µg) in a final volume of 28 µL nucleofection buffer containing 2 µM Cas9V3™ RNP targeting SERPINC1 (IDT, Coralville, Iowa) using Lonza nucleofection reagent (Lonza, Basel, Switzerland). 5 HEK-293 cells. The SC1 (SERPINC1) protospacer sequence used can be found in Table 1 (SEQ ID NO: 22). After 48 hours, cells were lysed using QuickExtract™ DNA extraction solution (Lucigen, Madison, WI). HDR and blunt-end integration rates were assessed using PCR assays by digital droplet PCR (ddPCR) (Bio-Rad, Hercules, CA) using primers flanking the junction between the target DNA and the insert sequence (Table 1; SEQ ID NOs: 23–27). Both HDR and blunt-end junction assays contain a primer outside the homology arm sequence to avoid amplification from non-integrated donors. The HDR assay probe (SEQ ID NO: 25) covers the junction between the target site and the insert sequence. The blunt-end assay probe (SEQ ID NO: 27) covers the junction between the target site and the integrated homology arm sequence.

[0117]

[0118]

[0119] dsDNA donors containing known nuclease resistance modifications such as phosphorothioate linkages (2×PS, 3×PS, or 6×PS) or LNA nucleotides at the 5′-end do not increase the HDR:blunt end ratio compared to unmodified (unmod) donors because such modifications increase both HDR and blunt end integration rates ( Figure 2 ). 5′-Modifications on the donors (biotin, TEG, Sp18, and SpC3) resulted in increased HDR rates and reduced blunt-end integration to varying degrees. Among these donors, TEG, biotin, and Sp18 showed an increase in HDR:blunt ratios (1.8, 2.0, and 2.5-fold, respectively, compared to unmodified). See Figure 2 The donor containing 2′-O-methoxy-ethyl (2′-MOE) modified ribonucleotides at both 5′-ends gave the greatest improvement in the HDR:blunt end ratio (5.0-fold improvement compared to the unmodified donor). Figure 2 . HDR rates were similar between 2′-MOE modified and other modified donors, indicating that improved stability alone does not result in an increase in HDR:blunt end ratios. Furthermore, as stated earlier, LNA-modified donors and PS-modified donors did not increase HDR:blunt end ratios, indicating that the reduction in blunt end integration is most likely not derived from increased template nuclease resistance. It was also demonstrated that the use of any 2′-modified ribonucleotide near the 5′-end of the donor was not sufficient to reduce blunt end integration, and that this activity was greatest for 2′-MOE modified templates among the modifications tested here.

[0120] Example 2

[0121] 5′-modifications show lower off-target integration when using shorter donor templates

[0122] Homology-independent integration rates are dependent on overall donor length, with blunt-end insertions increasing as donor size decreases. To determine whether 5′-end modification reduces blunt-end insertion rates when using a smaller 42 bp insert (compared to the 1 kb insert tested in Example 1), a modified dsDNA donor (SC1-166S; SEQ ID NO: 22) targeting the SERPINC1 locus described in Example 1 was generated. The donor consisted of a 42 bp insert containing an EcoRI restriction site and 40 bp homology arms (SC1 40-42-40; SEQ ID NO: 28) and was generated by PCR amplification of a plasmid containing the 42 bp insert and 100 bp flanking homology arms (see Table 2 for amplification primer sequences; SEQ ID NOs: 29–44).

[0123] Three modifications from Example 1 (biotin, Sp18, and MOE) were selected for additional testing, with the 6×PS modification included as a moderately performing control. A donor with three 2′-MOE ribonucleotides on the 5′-end (3×MOE) was also included to determine if blunt-end integration could be further reduced by additional modified residues. 2′-MOE ribonucleotides at different distances from the 5′-end were also tested (Int MOE -3 and -5, where the 2′-MOE is located 3 or 5 nucleotides from the 5′-end; SEQ ID NOs: 41–44). Modified and unmodified donors were delivered at a concentration of 500 nM (1.1 µg) in a final volume of 28 µL with 2 µM Cas9V3™ (IDT, Coralville, IA) RNPs targeting the SERPINC1 locus using Lonza Nucleofection Reagent (Lonza, Basel Switzerland) to 3.5 × 10 5 HEK-293 cells. After 48 h, DNA was extracted using QuickExtract™ DNA Extraction Solution (Lucigen, Madison, WI). Integration rates were assessed by RFLP using EcoRI digestion, and a Fragment Analyzer™ instrument was used for band quantification (Advanced Analytical, Ames, IA). HDR and blunt-end integration events can be distinguished by a 40 bp size difference caused by the duplication of the homology arms.

[0124]

[0125]

[0126] 1×MOE and 3×MOE modified donors resulted in the greatest increase in HDR:blunt end ratio (4.1- and 4.6-fold, respectively, relative to unmod). Figure 3 As previously observed, 6×PS, biotin, and Sp18 produced some improvement (1.9-, 2.7-, and 2.8-fold, respectively, relative to unmod), but did not reduce blunt-end integration to the same extent as the MOE-modified donor. Figure 3 Interestingly, the position of the 2′-MOE ribonucleotide in the donor did slightly affect its utility in reducing blunt-end integration. Moving the MOE ribonucleotide 3 or 5 nucleotides from the 5′-end of the donor resulted in only a 3.1- or 2.4-fold increase in the HDR:blunt-end ratio compared to the unmodified donor. Figure 3 Therefore, one skilled in the art would predict that placing a 2′-MOE ribonucleotide within 2–3 nucleotides of the 5′-end of the donor template would result in a substantial reduction in NHEJ-mediated insertions.

[0127] Example 3

[0128] 2′-MOE modification reduces integration at non-homologous double-strand breaks

[0129] In addition to blunt-end integration at the targeted cleavage site, the double-stranded donor can potentially integrate at any other double-strand break in the genome, including off-target Cas9 cleavage sites and endogenous breaks in dsDNA. To evaluate the effect of 2′-MOE modification on donor integration at potential non-homologous DSBs, dsDNA donors with unmodified or modified 5′-ends (unmod, 1×MOE, or 6×PS) were generated by PCR amplification (see Table 3 for amplification primer sequences; SEQ ID NOs: 48–53) and co-delivered with Cas9 complexed with the target gRNA (SC1-166S; SEQ ID NO: 22) or a mock “off-target” gRNA that is not homologous to the donor (AAVS1-670AS; SEQ ID NO: 54; HPRT 38087; SEQ ID NO: 55).

[0130] The donor consisted of a 42 bp insert containing an EcoRI restriction site and 50 bp homology arms targeting the SERPINC1 locus (SC1 50-42-50; SEQ ID NO: 47). The donor was delivered at a dose of 100 nM (0.3 µg) in a final volume of 28 µL together with 2 µM Cas9 V3™ RNP (IDT, Coralville, IA) and 2 µM Alt-R™ Cas9 Electroporation Enhancer™ to 3 × 10 5 K562 cells. After 48 hours, DNA was extracted using QuickExtract™ DNA Extraction Solution (Lucigen, Madison, WI). Integration rates were assessed by RFLP using EcoRI digestion and individual bands were quantified on a Fragment Analyzer™ instrument (AdvancedAnalytical, Ames, IA). HDR and blunt-end integration events can be distinguished by a 50 bp size difference caused by the duplication of the homology arms ( Figure 4 ).

[0131]

[0132] For unmodified dsDNA, blunt-end integration rates >9% were observed at the on-target Cas9 site and two “off-target” Cas9 sites. Figure 4Reduced blunt-end integration (<1%) was observed using 2′-MOE modified donors, confirming that 2′-MOE modification can also reduce NHEJ-mediated insertions at non-homologous DSBs. Figure 4 As previously observed, 6×PS modification resulted in a moderate reduction in blunt-end integration.

[0133] Example 4

[0134] Reduction in off-target integration is not a result of improved nuclease protection, and efficient reduction requires specific 2′-modifications

[0135] To determine whether the ability of the 2′-MOE modification to reduce blunt-end integration is a specific or general function of modifications at the 2′-position closest to the 5′-terminal nucleotide, other 2′-modifications (RNA, LNA, 2′-OMe, or 2′-F) as well as non-templated 2′-MOE ribonucleotides (SEQ ID NOs: 70–71) were tested on the 5′-end. (Non-templated ribonucleotides are defined as not homologous to the target DNA sequence.) The donor consisted of the sequence described previously in Example 2 (SC1 40-42-40; SEQ ID NO: 28), i.e., a 42 bp insert containing an EcoRI restriction site and 40 bp homology arms targeting the SerpinC1 locus. The donor was generated by PCR amplification as previously described (primer sequences unique to Example 4 are listed in Table 4; SEQ ID NOs: 60–71).

[0136]

[0137] The dsDNA donor was co-delivered with Cas9 complexed with a target gRNA (SC1-166S; SEQ ID NO: 22) or a gRNA not homologous to the donor (TNPO3; SEQ ID NO: 72). The donor was delivered to HEK-293 cells at a dose of 250 nM (0.6 µg) in a final volume of 28 µL with 2 µM Cas9 V3 RNP using Lonza Nucleofection Reagent (Lonza, Basel, Switzerland). After 48 hours, DNA was extracted using QuickExtract™ DNA Extraction Solution (Lucigen, Madison, WI). Integration rates were assessed by RFLP using EcoRI and each band was quantified on a Fragment Analyzer™ instrument (Advanced Analytical, Ames, IA).

[0138] Although HDR was unaffected or slightly enhanced by the various modifications, blunt-end integration at both on-target and off-target DSBs was reduced as long as the 2′-MOE modification was present ( Figure 5 ). In contrast, most other 2′-modifications did not affect or increased blunt-end integration rates. 2′-OMe modification did reduce blunt-end integration rates at DSBs on the target, but not to the same extent as 2′-MOE modification. Figure 5 2′-OMe modification did not significantly reduce the blunt-end integration rate at off-target DSBs.

[0139] Together, these data suggest that the ability of the 2′-MOE modification to reduce homology-independent integration is (a) not a result of increased stability by promoting nuclease resistance, as other stabilizing modifications did not produce similar results, and (b) not a generalized result of 2′-modifications at the 5′-most ribonucleotide, as other 2′-modifications did not produce similar results.

[0140] Example 5

[0141] Using hairpins as blocking groups to reduce homology-independent integration

[0142] In addition to chemical modification, the use of DNA hairpins at the ends of dsDNA donors can also be used to reduce homology-independent integration. The generation of these hairpin-blocked donors has been achieved by several methods ( Fig. 6A –B). In the case of small HDR events (typically ≤ 120 bp of insert sequence and 40 bp homology arms), two DNA strands with 5′-MOE hairpin sequences are chemically synthesized. These MOE adapters contain complementary sequences, allowing the formation of a hairpin structure. The DNA strands are annealed to form a dsDNA HDR donor. In the case of larger HDR events, DNA primers with similar 5′-MOE hairpins are chemically synthesized and used to amplify the desired HDR donor. The use of MOE ribonucleotides within the hairpin structure blocks the DNA polymerase from progressing through the hairpin. For both synthesis methods, the hairpin-blocked donor can be used as a nicked HDR template or ligated to produce a fully closed molecule.

[0143] The use of hairpins on chemically synthesized short oligonucleotides was functionally tested in cells. A 66-nt sequence was designed to mediate the insertion of the six-base GAATTC in the SERPINC1 locus. This sequence and its reverse complement were synthesized as ssODNs that were completely unmodified (Table 5; SEQ ID NOs: 75–76), contained an unmodified hairpin at the 5′-end (SEQ ID NOs: 77–78), contained a MOE-modified hairpin at the 5′ end (SEQ ID NOs: 79–80), or contained a non-templated MOE-modified base at the 5′-end (SEQ ID NOs: 81–82). Paired ssODNs were diluted to 100 µM and then mixed in a 1:1 ratio to produce 50 μM final duplexes. The oligonucleotide mixture was heated at 95°C for 1 min and then slowly cooled to room temperature to allow strand annealing. The duplex dsDNA donor was co-delivered with Cas9 complexed with target gRNA (SEQ ID NO: 22). Donors were delivered to HEK-293 cells at a concentration of 2 µM in a final volume of 28 µL using Lonza Nucleofection Reagent (Lonza, Basel Switzerland) along with 2 µM Cas9 V3 RNP and 2 µM Alt-R® Cas9 Electroporation Enhancer. DNA was extracted 48 hours later using QuickExtract™ DNA Extraction Solution (Lucigen, Madison, WI). Integration rates were assessed by RFLP using EcoRI and each band was quantified on a Fragment Analyzer™ instrument (Advanced Analytical, Ames, IA). Figure 7 ).

[0144]

[0145] Use of a shorter 66 bp unmodified dsDNA donor resulted in efficient integration via the NHEJ pathway relative to the HDR pathway (55.5% blunt end vs. 27.6% HDR). Introduction of a DNA-only hairpin to the end of the donor provided improvements in repair profiles (42.4% blunt end vs. 33.0% HDR). Inclusion of a MOE modification within the hairpin significantly improved this function to levels similar to those observed when a single 1×MOE was used on the 5′-end (58.4% HDR vs. 8.9% blunt end for MOE-modified hairpins; 66.5% HDR vs. 6.9% blunt end for 1×MOE). Additional optimizations (i.e., ligation, stem-loop length optimization, etc.) can be performed on the modified hairpins to further improve this function.

[0146] Example 6

[0147] 2′-MOE modification improves desired repair outcomes at multiple sites and in multiple cell lines

[0148] In the experiments described below, all guides were tested as Alt-R™ crRNA:tracrRNA complexed with Alt-R™ S. pyogenes Cas9 nuclease. The RNP complex and dsDNA donor were delivered to the cells of interest using the Lonza Nucleofection Reagent following the recommended protocol.

[0149] To further validate the ability of 2′-MOE modification to drive correct repair through higher HDR rates and reduced blunt-end integration, unmodified and 1×MOE-modified dsDNA donors were tested at four additional genomic loci (HPRT, AAVS1 670, AAVS1 T2, EMX1) in two cell lines (HEK293, K562). Donors were designed to mediate 42 bp insertions and have 40 bp homology arms (SEQ ID NOs: 83–86). Donors were generated by PCR amplification as previously described (primer sequences unique to Example 6 are listed in Table 6, SEQ ID NOs: 95–142). Donors were delivered to the indicated cell lines at a concentration of 250 nM with 2 µM Cas9 V3™ RNP (IDT, Coralville, Iowa) and 2 µM Alt-R™ Cas9 Electroporation Enhancer™ in a final volume of 28 μL of nucleofection buffer using the recommended protocol of Lonza Nucleofection Reagent (Lonza, Basel, Switzerland). The protospacer sequences used can be found in Table 6 (SEQ ID NOs: 135–138). After 48 hours, cells were lysed using QuickExtract™ DNA Extraction Solution (Lucigen, Madison, WI). Repair events were quantified by NGS amplicon sequencing (rhAmpSeq™) on the Illumina MiSeq platform (locus-specific sequencing primers are listed in Table 6, SEQ ID NOs: 139–146), and data analysis was performed using IDT's in-house data analysis pipeline (CRISPAltRations) described in U.S. Patent Application No. 16 / 919,577, the teachings of which are incorporated herein by reference ( FIG. 8 ).

[0150]

[0151]

[0152]

[0153] As previously observed, 5′-modifications all increased HDR rates compared to unmodified dsDNA donors (A). Although the fold increase in HDR compared to unmodified dsDNA varied by site and cell line, the average increase in HDR rates was relatively similar for all modifications tested (ranging from 1.2 to 1.3 fold increase). In contrast, MOE-modified donors showed a greater reduction in blunt-end integration compared to 2×PS and biotin donors (B). On average, the fold reduction in blunt-end integration relative to unmodified dsDNA was 1.6 (2×PS), 2.3 (biotin), 2.9 (1×MOE), 3.2 (1×MOE, 2×PS), and 3.3 (3×MOE). When the improvements in HDR and blunt-end integration were evaluated together for each site (C, reported as the ratio of HDR:Blunt repair events), MOE-modified dsDNA donors outperformed the other modifications. The average fold changes compared to unmodified dsDNA were 2.3 (2×PS), 3.1 (biotin), 3.6 (1×MOE), 4.1 (1×MOE 2×PS), and 4.3 (3×MOE). Altogether, these results confirm that MOE modification is most efficient in driving correct repair events after CRISPR editing.

[0154] Example 7

[0155] Improved HDR rates and reduced NHEJ insertions using modified dsDNA donors mediating large inserts

[0156] As a follow-up to the work using short insert sequences, experiments were performed to compare HDR and blunt-end integration rates when using dsDNA donors to mediate insertions of 300 bp, 500 bp, or 1000 bp at two genomic loci (SERPINC1 and EMX1; see Table 7, SEQ ID NOs: 147–154 for donor sequences and amplification primers). Donors were generated by PCR amplification of plasmids containing the desired insert sequence and 100 bp of flanking homology arms. Amplification primers were designed with either unmodified sequence or the indicated modifications. Long ssDNA (Megamers™) was ordered for comparison at the SERPINC1 locus. Donors were delivered to 3.5 × 10 cells at a concentration of 100 nM along with 2 µM Cas9 V3™ RNPs targeting SERPINC1 or EMX1 (IDT, Coralville, Iowa) in a final volume of 28 µL of nucleofection buffer using Lonza nucleofection reagent (Lonza, Basel, Switzerland). 5HEK-293 cells. After transfection, cells were treated with IDTAlt-R™ HDR Enhancer V2 (1 µM) for 24 hrs. The protospacer sequences used are shown in Table 1 (SEQ ID NO: 22) and Table 7 (SEQ ID NO: 161).

[0157] After 48 h, cells were lysed using QuickExtract™ DNA extraction solution (Lucigen, Madison, WI). HDR and blunt-end integration rates were assessed by long-read sequencing using the MinION™ platform (Oxford Nanopore Technologies, Oxford, UK). The locus-specific amplification primers used are listed (Table 7, SEQ ID NOs: 162–165). Final sequencing libraries were prepared using the PCR Barcode Amplification and Ligation Sequencing Kit following the manufacturer’s recommended protocol. Final data analysis was performed using IDT’s in-house data analysis pipeline (CRISPAltRations) ( Fig. 9A ). For SERPINC1 samples, insertion rate was independently assessed by amplicon length analysis. Isolated gDNA was PCR amplified using SERPINC1 RFLP primers (SEQ ID NO: 45–46). Amplicons were run on a Fragment Analyzer™ instrument for band quantification. Insertion events were identified based on the expected amplicon size for integration events ( Fig. 9B ).

[0158]

[0159]

[0160]

[0161]

[0162] When compared to unmodified dsDNA donors mediating large inserts at both genomic loci, 1×MOE modified donors resulted in higher HDR rates (mean values ​​of 28.6% vs. 30.9% at EMX1 and 44.4% vs. 53.6% at SERPINC1) and lower blunt-end integration rates (mean values ​​of 3.2% vs. 1.1% at EMX1 and 8.4% vs. 2.5% at SERPINC1). Long ssDNA donors mediating the same insertion at the SERPINC1 locus resulted in extremely low blunt-end integration rates (<1%). The long ssDNA donors mediated the highest HDR rates for 300 bp inserts (71.3% vs. 54.9% using modified dsDNA donors). However, for HDR using larger inserts, the modified dsDNA donors performed as well or better than the long ssDNA (55.2% vs. 53.3% for 500 bp inserts, 50.8% vs. 29.4% for 1000 bp inserts). Similar trends were observed for the SERPINC1 samples when using an orthogonal evaluation method ( Fig. 9B ).

[0163] Example 8

[0164] Making modified dsDNA donors using universal primer sequences has no adverse effects on HDR repair

[0165] To evaluate the impact of incorporating universal primer sequences into the donor templates, dsDNA donors were prepared that mediated 500 bp insertions at EMX1 and SERPINC1 (see Table 7, SEQ ID NOs: 148 and 151) with either locus-specific primers or universal primers (Table 7 SEQ ID NOs: 153–160; Table 8 SEQ ID NOs: 166–181). The placement of the universal primer sequences relative to the donors is shown in Fig.10 The modifications tested included 1×MOE, 3×MOE, and biotin with phosphorothioate modification (biotin 5×PS, as described in [5]). The donor was delivered to 3.5 × 10 cells at a concentration of 100 nM together with 2 μM Cas9 V3™ RNP targeting SERPINC1 or EMX1 (IDT, Coralville, Iowa) in a final volume of 28 μL of nucleofection buffer using Lonza nucleofection reagent (Lonza, Basel, Switzerland). 5HEK-293 cells. Following transfection, cells were treated with IDT Alt-R™ HDR Enhancer V2 (1 µM) for 24 hrs. After 48 h, cells were lysed using QuickExtract™ DNA Extraction Solution (Lucigen, Madison, WI). HDR and blunt-end integration rates were assessed by long-read sequencing using the MinION™ platform (Oxford Nanopore Technologies, Oxford, UK) and analyzed as previously described ( Fig.11 ).

[0166]

[0167] HDR and blunt-end integration rates were relatively similar for dsDNA donors generated with or without universal primer sequences. For donors without universal primer sequences, increases in HDR rates and decreases in blunt-end rates were similar between the various modifications. The main exception to this trend was the 3×MOE modification at the SERPINC1 locus, where blunt-end insertions were still reduced relative to unmodified dsDNA, but HDR was not improved to the same extent as 1×MOE or biotin 5×PS (unmod: 45.1% HDR, 9.0% blunt ends; 1×MOE: 55.2% HDR, 2.3% blunt ends; 3×MOE: 44.5% HDR, 1.2% blunt ends). In contrast, much greater performance differences were observed for modified donors made using universal primer sequences. Interestingly, when the universal primer site was included in the donor sequence, the unmodified dsDNA donor had lower integration rates for both sites (EMX1: 28.8% vs. 24.8% HDR, SERPINC1: 45.2% vs. 34.4% HDR). For both sites, the 1×MOE modification provided the greatest increase in HDR rates when incorporated into donors containing the universal sequence.

[0168] HDR reads from 1×MOE-modified donors were further analyzed in the Integrative Genomics Viewer [7] (IGV, Broad Institute, Cambridge, MA). When the HDR reads were compared to a reference amplicon containing the correct HDR sequence ( Fig. 12A ) or a reference amplicon containing both the desired insert and universal primer sequences ( Fig. 12B ) alignment, no evidence of universal sequence incorporation was observed in the HDR readouts. Thus, universal sequences can be incorporated into the manufacturing process of modified dsDNA donors without adversely affecting functional performance.

[0169] Example 9

[0170] Generation of GFP fusions in human cell lines using modified dsDNA donors made with universal primer sequences

[0171] To evaluate the functional performance of modified dsDNA donor templates in applications such as protein tagging, donors were designed to generate GFP-tagged GAPDH (C-terminal fusion), CLTA (N-terminal fusion), and RAB11a (N-terminal fusion). Donors with universal primer sequences were made using unmodified or 1xMOE-modified primers as described previously. The guide and donor sequences used are listed in Table 9. Donors were delivered to 3.5 × 10 cells at a concentration of 50 nM with 2 μM Cas9V3™ RNPs targeting GAPDH, CLTA, or RAB11a (IDT, Coralville, Iowa) in a final volume of 28 μL of nucleofection buffer using Lonza nucleofection reagent (Lonza, Basel, Switzerland). 5 K562 cells. After transfection, cells were plated in duplicate wells. For one set of wells, cells were treated with IDT Alt-R™ HDR Enhancer V2 (1µM) 24 hr after transfection. Cells were passaged for 7 days, at which time point HDR rates were assessed by flow cytometry. Briefly, cells were washed in PBS and then plated at 1–2 × 10 6 The cells were resuspended at 100 cells / mL. Hoechst 33258 was added to the cell suspension at a final concentration of 4 µg / ml and immediately analyzed for viability staining. The cells were analyzed on a Becton Dickinson LSR II cytometer (BD Bioscience, San Jose, CA) to assess GFP expression levels (Figure 12).

[0172]

[0173]

[0174] Among the loci tested, overall HDR rates varied, and the highest GFP-positive rates obtained under optimal conditions were 17.2% (GAPDH), 44.9% (CLTA), and 64% (RAB11a). No GFP signal was observed in cells that received dsDNA donors without RNPs (data not shown). The use of modified dsDNA donor templates increased HDR rates under both untreated conditions (1.6-, 1.3-, and 1.2-fold increases relative to unmodified dsDNA for GAPDH, CLTA, and RAB11a, respectively) and HDR enhancer-treated conditions (1.4-, 1.4-, and 1.1-fold increases relative to unmodified dsDNA, respectively). On average, the use of 1×MOE-modified dsDNA donors increased HDR rates 1.3-fold relative to unmodified dsDNA donors across all conditions. In contrast, the use of Alt-R HDR enhancer V2 increased HDR rates by an average of 2.4-fold across all loci and conditions. Across all loci, the combined use of modified donors and HDR enhancers increased HDR rates by an average of 3.2-fold. Overall, this demonstrates the power of using a combination of optimal reagents (i.e., modified donors and small molecule enhancers) in HDR experiments.

[0175] Example 10

[0176] Use of universal primer sequences when making dsDNA HDR templates can result in greater consistency and improved yields

[0177] To evaluate the effect of universal primer sequences on the manufacturing process of dsDNA HDR templates, 24 sequences were generated using universal primer sequences with different modifications (Table 8, SEQ ID NOs: 172–181) or gene-specific primers (Table 10, SEQ ID NOs: 188–235). All donors were generated by amplification from plasmids containing the sequence of interest (pUCIDT Amp or pUCIDT Kan vectors) as described previously. PCR amplification was performed using KOD Hot Start DNA Polymerase (EMD) in a 50 μL final reaction volume using 200 nM primers and 10 ng of plasmid DNA as recommended by the manufacturer. Thermal cycling was performed using a Bio-Rad S1000 thermal cycler with the following cycling conditions: incubation at 95°C for 3 min, followed by 36 amplification cycles (95°C for 20 sec; 65°C for 10 sec; 70°C for 20–30 sec / kb). The annealing temperature was adjusted according to the gene-specific primer melting temperature. After SPRI bead cleanup, all products were analyzed using a Fragment Analyzer (Agilent) and sequence verified by NGS using an Illumina-Nextera DNA library preparation kit. The overall amplification efficiency from universal or gene-specific primers was assessed by measuring the final yield and reported in ng / µL ( Fig.14A ).

[0178]

[0179]

[0180]

[0181] Due to differences in yields of short (<500 bp) and long (>500 bp) amplicons, the overall yields after amplification with universal or gene-specific primers were evaluated separately for 12 short and 12 long HDR templates ( Fig.14A ). Overall, for both short and long amplicons, the yields were significantly higher when using universal primers. For long amplicons, use of universal primers resulted in a mean clean-up concentration of 138.3 ng / µL (± 18.0 SD), while use of gene-specific primers resulted in a mean concentration of 77.8 ng / µL (± 32.6 SD). For short amplicons, use of universal primers resulted in a mean clean-up concentration of 40.9 ng / µL (± 5.9 SD), while use of gene-specific primers resulted in a mean concentration of 15.9 ng / µL (± 6.4SD). Direct comparisons between each sequence amplified using universal or gene-specific primers ( Fig. 14B) revealed large variations in yield when using gene-specific primers. In contrast, among sequences of similar length, the use of universal primers resulted in higher yields (an average of 2.9 and 2.0-fold for short and long amplicons, respectively) and greater yield consistency. Due to the higher yields and greater consistency, the use of universal primers will better support the development of high-throughput manufacturing processes.

Claims

1. A double-stranded DNA homology-directed repair (HDR) donor, comprising a first homology arm region, an insertion region, and a second homology arm region; wherein the first homology arm region and the second homology arm region comprise modifications to one or more nucleotides at or near the 5'-end.

2. The double-stranded DNA HDR donor according to claim 1, wherein the modification comprises: A modification of the 2'-position of one or more nucleotides at or near the 5'-end of the first homology arm region, and a modification of the 2'-position of one or more nucleotides at or near the 5'-end of the second homology arm region.

3. The double-stranded DNA HDR donor according to claim 1 or 2, wherein the modification comprises modification of the 2′-position of the 5′-terminal nucleotide, the 5′-second-to-last nucleotide, the 5′-third-to-last (third) nucleotide, or a combination of said nucleotides at or near the 5′-end of the first homology arm region and the second homology arm region.

4. The double-stranded DNA HDR donor according to any one of claims 1 to 3, wherein the modification at or near the 5′-end of the double-stranded DNA HDR donor comprises one or more of 2′-OME, 2′-F or 2′-MOE.

5. The double-stranded DNA HDR donor according to any one of claims 1 to 4, wherein the modification at or near the 5'-end of the double-stranded DNA HDR donor comprises 2'-MOE.

6. A programmable nuclease system comprising a modified double-stranded DNA homology-directed repair (HDR) donor, a programmable nuclease and a gRNA, wherein the gRNA molecule is capable of targeting the programmable nuclease molecule to a target nucleic acid.

7. A method for increasing homology-directed repair (HDR) rates and reducing homology-independent integration in a programmable nuclease system, the method comprising targeting a candidate editing target site locus with an active programmable nuclease system and a modified double-stranded DNA HDR donor.

8. A use of a modified double-stranded DNA HDR donor for improving homology-directed repair (HDR) rates and reducing homology-independent integration in a programmable nuclease system, wherein the modified double-stranded DNA HDR donor comprises a first homology arm region, an insertion region, a second homology arm region, and optionally one or more universal primer sequences, wherein the first homology arm region and the second homology arm region comprise modifications to one or more nucleotides at or near the 5'-end.

9. A method for making a modified double-stranded DNA HDR donor, the method comprising synthesizing an oligonucleotide comprising a first homology arm region, an insertion region, a second homology arm region, and optionally one or more universal primer sequences, wherein the first homology arm region and the second homology arm region comprise modifications to one or more nucleotides at or near the 5'-end.

10. A method for making a modified double-stranded DNA HDR donor, the method comprising amplifying a target nucleic acid sequence comprising a first homology arm region, an insertion region, and a second homology arm region using one or more universal primers, wherein the universal primer sequence comprises a modification of one or more nucleotides at or near the 5′-end.

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