Modified duplex donor template

Abstract

To provide a double-stranded DNA donor for improving donor efficacy and efficiency of homology-directed repair after introduction of a double-stranded break with a programmable nuclease.SOLUTION: Provided is 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 HDR donor comprises one or more modifications, wherein the one or more modifications comprise one or more hexaethylene glycol spacer 18 (Sp18) modifications at or near the 5 ' end and / or the 3 ' end.SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 62 / 925,366, filed October 24, 2019, which is incorporated herein by reference in its entirety.

[0002] Reference to sequence listing This application is filed with a sequence listing in computer-readable form in accordance with 37 C.F.R. § 1.821(c). The text file submitted by EFS, "013670-9060-US02_sequence_listing_22-OCT-2020_ST25.txt," was created on October 22, 2020, contains 235 sequences, has a file size of 86.3 Kbytes, and is incorporated by reference in its entirety.

[0003] Technical Field This paper describes compositions and methods for improving homology-directed repair (HDR) efficiency and reducing the homology-independent integration after introducing double-strand break by genetic engineering nuclease.Furthermore, the modification of double-stranded DNA donor improves the donor efficacy and efficiency of homology-directed repair after introducing double-strand break by programmable nuclease. [Background technology]

[0004] Genome editing using programmable nucleases allows for site-specific introduction of DNA into a target genome of interest. Several systems are capable of targeted genome editing, including transcription activator-like effector nucleases (TALENs), zinc finger (ZFNs), or clustered regularly interspaced short palindromic repeats (CRISPRs).

[0005] The CRISPR-Cas9 system has been widely used to perform 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, creating a double-strand break (DSB). This DSB is then corrected by the cell's innate DNA damage repair pathways. The two main 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- or double-stranded DNA molecules with homology on either side of the DSB as repair templates to generate the desired mutation in the target DNA [1].

[0006] Various forms of DNA, such as plasmid DNA, double-stranded linear DNA (dsDNA), and single-stranded DNA (ssDNA), can be used as repair templates in HDR experiments. Both dsDNA and ssDNA donors can induce innate immune responses in mammalian tissue culture cells. For short insertions (generally ≦120 bp) or mutations, chemically synthesized oligonucleotides, such as IDT® Ultramer™ ssDNA, can be used as single-stranded oligo donors (ssODNs) for HDR experiments. Using synthetic ssDNA allows for chemical modifications to the molecule to improve HDR efficiency. Templates for larger insertions (generally >120 bp) are limited due to the increased complexity of synthesis. While generating long ssDNA can be a laborious and costly process, linear dsDNA can generally be generated quickly and in large quantities. Because the more common NHEJ repair pathway promotes blunt-end ligation, the linear dsDNA donor can repair any DSBs present in the cell (including on-target Cas9 cleavage sites, any Cas9 off-target sites, and There is an increased risk of homology-independent integration into the target site (including any endogenous DSBs) [2, 3]. If homology-independent integration occurs at the target site, the entire donor, including the homology arms, is integrated, resulting in duplication of one or both homology arm regions.

[0007] It has been reported that 5'-biotin modification of linear dsDNA donors can reduce concatemer formation and integration via the NHEJ pathway [4]. Similarly, another group reported that 5'-end biotinylation or ssDNA overhangs can reduce blunt insertion [5]. Another group suggested that TEG and 2'-OMe ribonucleotide adapters at the 5' end of dsDNA donors could potentially increase HDR rates by restricting the access of the NHEJ machinery to the free end of the donor, but did not demonstrate a reduction in blunt integration [6].

[0008] A need exists for compositions and methods of modified dsDNA templates for HDR that increase the efficiency of HDR and reduce the unwanted homology-independent integration (both at the target site and at potential off-target or endogenous DSBs) typically associated with linear dsDNA donors. Summary of the Invention

[0009] One embodiment described herein is 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.In one aspect, the modifications comprise modifications to the 2' position of one or more nucleotides at or near the 5' end of the first homology arm region, and modifications to the 2' position of one or more nucleotides at or near the 5' end of the second homology arm region.In another aspect, the modifications comprise modifications to the 2' position of the 5'-end nucleotide, the second nucleotide from the 5' end, the third nucleotide from the 5' end, or a combination of nucleotides at or near the 5' end of the first homology arm region and the second homology arm region. In another embodiment, the modification at or near the 5' end of the duplex DNA HDR donor comprises one or more of 2'-O-methyl (2'-OMe), 2'-fluoro (2'-F), or 2'-O-methoxylethyl (2'-MOE). In another embodiment, the modification at or near the 5' end of the duplex DNA HDR donor comprises 2'-MOE. In another embodiment, the modification at or near the 5' end is a non-template mismatch to the target DNA. In another embodiment, the first and second homology arm regions are 40 to 150 nucleotides in length. In another embodiment, the first and second homology arm regions are at least 100 nucleotides in length. In another embodiment, the duplex DNA HDR donor further comprises a universal primer sequence. In another embodiment, the insertion region is greater than 100 bp. In one embodiment, 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 embodiment, the double-stranded HDR donor comprises a hairpin at either the 5'-end or the 3'-end. In another embodiment, the double-stranded HDR donor comprises a hairpin at both the 5'-end and the 3'-end.In another embodiment, the double-stranded DNA HDR donor improves homology-directed repair efficiency and reduces homology-independent integration in a programmable nuclease system.

[0010] Another embodiment described herein is a programmable nuclease system comprising a modified double-stranded DNA homology-directed repair (HDR) donor, a programmable nuclease enzyme, and a gRNA, wherein the gRNA molecule is capable of targeting the programmable nuclease molecule to a target nucleic acid. 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 embodiment, the modified double-stranded DNA HDR donor comprises modifications to the 2' position of the 5'-terminal nucleotide, the second nucleotide from the 5' end, the third nucleotide from the 5' end, or a combination of nucleotides at or near the 5' end of the first homology arm region and the second homology arm region. In another embodiment, the modified double-stranded DNA HDR donor comprises at least one 2'-OME, 2'-F, or 2'-MOE modification to 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 modified double-stranded DNA HDR donor improves homology-directed repair efficiency and reduces homology-independent integration in the programmable nuclease system.In another embodiment, the programmable nuclease system comprises one or more transcription activator-like effector nucleases (TALENs), zinc finger (ZFNs), or clustered regularly interspaced short palindromic repeats (CRISPRs).In another embodiment, the programmable nuclease system is CRISPR.In another embodiment, the programmable nuclease enzyme is CRISPR-associated-9 (Cas9).In another embodiment, the programmable nuclease system further comprises one or more HDR enhancers.

[0011] Another embodiment described herein is a method for increasing the homology-directed repair (HDR) rate and reducing homology-independent integration in a programmable nuclease system, comprising targeting a candidate editing target site locus with an active programmable nuclease system and a modified double-stranded DNA HDR donor. In one aspect, the modified double-stranded DNA HDR donor comprises a first homology arm region, an insertion region, and a second homology arm region, and 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 aspect, the modified double-stranded DNA HDR donor comprises modifications to the 2' position of the 5'-terminal nucleotide, the second nucleotide from the 5' end, the third nucleotide from the 5' end, or a combination of nucleotides at or near the 5' end of the first homology arm region and the second homology arm region. In another embodiment, the modified double-stranded DNA HDR donor comprises at least one 2'-OME, 2'-F, or 2'-MOE modification on 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 programmable nuclease systems.

[0012] Another embodiment described herein is the use of a modified double-stranded DNA HDR donor to increase the homology-directed repair (HDR) rate and reduce 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 priming sequences, and 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 aspect, the modifications comprise at least one 2'-OME, 2'-F, or 2'-MOE modification to one or more nucleotides at or near the 5' end of the double-stranded DNA HDR donor. Another embodiment described herein is a method for producing a modified double-stranded DNA HDR donor. The method comprises synthesizing an oligonucleotide comprising a first homology arm region, an insertion region, a second homology arm region, and optionally one or more universal priming 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 embodiment, the modifications comprise at least one 2'-OME, 2'-F, or 2'-MOE modification to one or more nucleotides at or near the 5' end of a duplex DNA HDR donor.

[0013] Another embodiment described herein is a method for producing a modified double-stranded DNA HDR donor, comprising amplifying a target nucleic acid sequence comprising a first homology arm region, an insertion region, and a second homology arm region with one or more universal primers, wherein the universal priming sequence comprises a modification to one or more nucleotides at or near the 5' end. In one aspect, 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 explanation of the drawings]

[0014] [Figure 1A]

[0023] Figure 1A shows a schematic illustrating homology-independent and homology-dependent integration events when using a dsDNA HDR donor template for Cas9-directed cleavage (Figure 1A) or endogenous double-initiation cleavage or Cas9 off-target cleavage (Figure 1B). These homology-independent integration events result in the incorporation or duplication of homology arms at the double-strand break introduced by the programmable nuclease. [Figure 1B]

[0023] Figure 1A shows a schematic illustrating homology-independent and homology-dependent integration events when using a dsDNA HDR donor template for Cas9-directed cleavage (Figure 1A) or endogenous double-initiation cleavage or Cas9 off-target cleavage (Figure 1B). These homology-independent integration events result in the incorporation or duplication of homology arms at the double-strand break introduced by the programmable nuclease. [Figure 2] FIG. 1 shows evaluation of dsDNA donor integration via HDR or NHEJ pathways using a modified linear dsDNA donor containing a 1 kb insert. [Figure 3]

[0023] Figure 1 shows the evaluation of dsDNA donor integration via HDR or NHEJ pathways using modified linear dsDNA donors containing 42-bp insertions. The modifications were extended to multiple 2'-MOE ribonucleotides and internally placed 2'-MOE ribonucleotides. [Figure 4] Figure 1 shows evaluation of dsDNA donor integration via HDR or NHEJ pathways using a modified linear dsDNA donor containing a 42-bp insertion. Cas9 guides targeting non-homologous sites were used to mimic off-target Cas9 cleavage. [Figure 5] Figure 1 shows the evaluation of dsDNA donor integration via HDR or NHEJ pathways using a modified linear dsDNA donor containing a 42-bp insertion. The modifications were expanded to include an additional 2'-modification. Cas9 guides targeting non-homologous sites were used to mimic off-target Cas9 cleavage. [Figure 6]Figure 6 shows a method for synthesizing a hairpin-blocked dsDNA HDR template. Gray indicates a DNA hairpin composed of 2'-MOE ribonucleotides. Black indicates chemically synthesized unmodified DNA. White indicates the DNA template sequence with available primer binding sites. Figure 6A shows that for short HDR insertions, a hairpin-blocked dsDNA donor can be generated by annealing two chemically synthesized ssDNA oligos containing a 5'-MOE hairpin. Figure 6B shows that for longer HDR insertions, a hairpin-blocked dsDNA donor can be generated by PCR amplification. Primers with a 5'-MOE hairpin can be used to amplify the target HDR template. DNA polymerase should be unable to amplify through the MOE containing the hairpin. After several cycles, the final dsDNA product containing MOE hairpins at both 5' ends is generated. [Figure 7] This figure shows the evaluation of dsDNA donor integration via HDR or NHEJ pathways using donors with either a hairpin or 1xMOE-modified base at the 5' end. To introduce an EcoRI restriction site into the SERPCIN1 locus, the donor contained a 30-bp homology arm to mediate a 6-bp insertion. The hairpin consisted of a 3-bp stem with a "TTTT" loop and contained either unmodified DNA bases (DNA only) or 2'-MOE-modified bases (MOE-modified). The hairpin was not ligated for initial testing. [Figure 8A] Figure 8A shows the evaluation of dsDNA donor integration via the HDR (Figure 8A) or NHEJ (Figure 8B) pathways using modified linear dsDNA donors. The ratio of HDR to blunt integration is shown in Figure 8C. Donors were designed to mediate 42-bp insertions at four genomic loci and tested in two cell lines (n=8 per modification). Results are reported as fold change relative to the unmodified dsDNA donor for each site and cell line. [Figure 8B]Figure 8A shows the evaluation of dsDNA donor integration via the HDR (Figure 8A) or NHEJ (Figure 8B) pathways using modified linear dsDNA donors. The ratio of HDR to blunt integration is shown in Figure 8C. Donors were designed to mediate 42-bp insertions at four genomic loci and tested in two cell lines (n=8 per modification). Results are reported as fold change relative to the unmodified dsDNA donor for each site and cell line. [Figure 8C] Figure 8A shows the evaluation of dsDNA donor integration via the HDR (Figure 8A) or NHEJ (Figure 8B) pathways using modified linear dsDNA donors. The ratio of HDR to blunt integration is shown in Figure 8C. Donors were designed to mediate 42-bp insertions at four genomic loci and tested in two cell lines (n=8 per modification). Results are reported as fold change relative to the unmodified dsDNA donor for each site and cell line. [Figure 9A] Figure 9A shows the evaluation of dsDNA donor integration via HDR or NHEJ pathways using modified linear dsDNA donors mediating 300 bp, 500 bp, or 1 kb insertions at two genomic loci. For comparison, a long ssDNA donor targeting the SERPINC1 locus was provided. Figure 9B shows a comparative orthogonal analysis method for evaluating insertions at the SERPINC1 locus. Long-read sequencing using the MinION™ system from Oxford Nanopore Technologies (ONT) was compared with amplicon length analysis, in which PCR amplicons from genomic DNA samples were performed and quantified on a Fragment Analyzer. [Figure 9B]Figure 9A shows the evaluation of dsDNA donor integration via HDR or NHEJ pathways using modified linear dsDNA donors mediating 300 bp, 500 bp, or 1 kb insertions at two genomic loci. For comparison, a long ssDNA donor targeting the SERPINC1 locus was provided. Figure 9B shows a comparative orthogonal analysis method for evaluating insertions at the SERPINC1 locus. Long-read sequencing using the MinION™ system from Oxford Nanopore Technologies (ONT) was compared with amplicon length analysis, in which PCR amplicons from genomic DNA samples were performed and quantified on a Fragment Analyzer. [Figure 10] Figure 1 shows a schematic of a dsDNA HDR donor template design containing a universal priming sequence. 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 homologous to the universal priming sequence. [Figure 11]

[0023] Figure 1 shows 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. The donors were synthesized with or without a terminal universal priming sequence. [Figure 12]Figure 12. Visual assessment of HDR reads from 1xMOE donors using IGV. HDR reads from EMX1 and SERPINC1 1xMOE dsDNA donors produced with universal priming sequences were aligned to a reference containing either the correct HDR sequence (Figure 12A) or the HDR sequence with the universal sequence (i.e., incorrect HDR) (Figure 12B). For comparison, HDR reads from 1xMOE dsDNA donors lacking the universal sequence were aligned to the correct HDR reference (Figure 12C). Within the IGV plot, individual reads are represented as thin horizontal lines. Individual nucleotides that do not align correctly to the reference (i.e., insertions, gaps, or mutations) are marked in black. The background error rate from MinION™ sequencing can be assessed in Figure 12C. A representation of the HDR reference is shown above each IGV panel. Black represents the desired 500-bp insertion. The dashed region represents sequences homologous to the 100-bp donor homology arms. The dotted region represents the 30 bp universal priming sequence. The region of interest is indicated by an arrow. Misalignment to the incorrect HDR reference (Figure 12B) was evident in all HDR reads, indicating the absence of the 30 bp universal sequence after repair. The panel of EMX1 donors represents approximately 500 reads. The panel of SERPINC1 donors represents approximately 3700 reads. [Figure 13] Figure 1 shows the evaluation of HDR rates using either unmodified or 1xMOE-modified dsDNA donor templates. The donors were designed to insert GFP at the N- or C-terminus of the target gene and contained 200 bp homology arms. The donors were generated with a universal priming sequence. HDR rates were assessed by flow cytometry (reported as % GFP-positive cells). [Figure 14]Figure 14 shows an evaluation of yield when dsDNA HDR templates were prepared using either universal or gene-specific primers. Twelve sequences >500 bp and twelve sequences <500 bp were prepared and PCR yields were evaluated. The overall yield for each group is shown in Figure 14A, and a comparison between templates with and without universal primers for each sequence is shown in Figure 14B. DETAILED DESCRIPTION OF THE INVENTION

[0015] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art.For example, any nomenclature and techniques used in connection 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 conflict, the present document, including definitions, shall prevail.Although methods and materials similar or equivalent to those described herein can be used to practice or test the present invention, the following describes preferred methods and materials.

[0016] As used herein, the terms "amino acid," "nucleotide," "polypeptide," "polynucleotide," and "vector" have their common meanings as understood by a biochemist 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.

[0017] As used herein, the terms "include," "including," "contain," "containing," "having," and the like mean "comprising." The present disclosure also contemplates other embodiments that "comprise," "consist," and "consist essentially of" the embodiments, aspects, or elements presented herein, whether explicitly stated or not.

[0018] As used herein, the terms "a," "an," "the," and similar terms used in the context of this disclosure (especially in the context of the claims) are intended to mean Unless otherwise indicated herein or clearly contradicted by context, the terms "a," "an," and "the" should be construed to cover both the singular and the plural, and unless otherwise specified, the words "a," "an," and "the" mean "one or more."

[0019] As used herein, the term "or" can be conjunctive or disjunctive.

[0020] As used herein, the term "substantially" means to a large extent or to a great extent, but not completely.

[0021] As used herein, the term "about" or "approximately" applied to one or more values ​​of interest refers to a value similar to a stated reference value or within an acceptable error range of a particular value as determined by one of ordinary skill in the art, which depends in part on how the value is measured or determined, such as the limitations of the measurement system. In one aspect, the term "about" refers to any value, including both integer and fractional components, that is within a maximum ±10% variation of the value modified by the term "about." Alternatively, "about" can mean within three or more standard deviations, in accordance with practice in the art. Alternatively, as with respect to biological systems or processes, the term "about" can mean within an order of magnitude, in some embodiments, within five-fold, and in some embodiments, within two-fold of a value. As used herein, the symbol "~" means "about" or "approximately."

[0022] All ranges disclosed herein include both discrete endpoints and all integers and fractions specified within the range. For example, the range 0.1 to 2.0 includes 0.1, 0.2, 0.3, 0.4, ... 2.0. When an endpoint is modified by the term "about," the specified range is extended by up to ±10% variation of any value within the range or within three or more standard deviations that includes the endpoint.

[0023] As used herein, the terms "control" or "reference" are used interchangeably herein. A "reference" or "control" level may be a predetermined value or range used as a baseline or benchmark for evaluating measurement results. "Control" also refers to a control experiment or control cell.

[0024] As used herein, the phrase "effective amount" of a compound described herein refers to an amount of a compound described herein that elicits a biological response, such as a reduction or inhibition of enzyme or protein activity, or that ameliorates symptoms, relieves symptoms, slows or retards the progression of a disease, or prevents a disease.

[0025] As used herein, the terms "inhibit," "inhibition," or "inhibiting" refer to the 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 treatment.

[0026] As used herein, the term "universal primer" refers to a sequence that has no known alignment to the target sequence. Universal primers allow for sequence-independent amplification of the target sequence.

[0027] Disclosed herein are methods and compositions for improving HDR efficiency and reducing blunt integration events using dsDNA donor templates. In various embodiments, the disclosed methods and compositions are directed to homology-independent integration after genome editing using programmable nucleases. In some embodiments, the bulky modification is placed at the 5' end of the linear dsDNA donor. In further embodiments, the bulky modification is placed at or near the 5' end of the linear dsDNA donor. Additionally, modifications may be placed at the 2' position of the DNA at or near the 5' nucleotide or nucleotides of the dsDNA donor (e.g., 2'-MOE, 2'-OME, or 2'-F nucleotide). These modifications demonstrate improved effectiveness in reducing homology-independent integration. Furthermore, this reduction does not appear to be mediated by increased donor stability, as other modifications with established ability to block nuclease degradation (e.g., PS) do not reduce blunt integration rates to the same extent as other 2' modifications.

[0028] When homology-independent integration occurs at the on-target site, the entire donor, including the homology arms (Figure 1), is integrated, resulting in duplicated homology arms. Figure 1 shows a schematic diagram of homology-independent (duplicated homology arms) and homology-dependent integration events when using a dsDNA HDR donor template. Light gray bars indicate the target genomic DNA sequence, while white indicates the nonhomologous genomic DNA sequence (either an endogenous DSB or a Cas9 off-target site). 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 inserted DNA sequence. Figure 1A shows insertion of a dsDNA donor via HDR or NHEJ repair pathways at the on-target Cas9 cleavage site. Insertion via the NHEJ pathway results in duplication of the donor homology arms. Figure 1B shows insertion of dsDNA via the NHEJ repair pathway at an endogenous DSB or off-target Cas9 cleavage site.

[0029] In some embodiments, chemical modifications are introduced at the 5' end of a linear dsDNA donor. These chemical modifications are used to reduce the risk of NHEJ integration and improve its usefulness as a repair template in HDR experiments. In some embodiments, bulky or large modifications are introduced at the 5' end of a dsDNA donor. In additional embodiments, modifications may be introduced at the end or 5'-DNA nucleotide of a dsDNA oligonucleotide. In some embodiments, modifications may be introduced at or near the 5' end of a dsDNA oligonucleotide. In some embodiments, a DNA nucleotide or nucleotides at or near the 5' end of a dsDNA oligonucleotide may be modified. In some embodiments, the modification includes biotin, phosphorothioate (PS), triethylene glycol (TEG), locked nucleic acid (LNA, 2'-oxygen-4'-carbon methylene linkage), hexaethylene glycol (Sp18), 1,3-propanediol (SpC3), 2'-O-methoxyethyl (MOE) ribonucleotide, 2'-O-methyl ribonucleotide (2'-OMe), 2'-fluoro (2'-F) nucleotide, or ribonucleotide. In some embodiments, the modification is located at the 5'-terminal nucleotide, the second nucleotide from the 5'-terminus, the third nucleotide from the 5'-terminus, or a combination of nucleotides at or near the 5'-terminus of the dsDNA donor. In additional embodiments, the modification is located at the 2'-position of the 5'-terminal nucleotide, the second nucleotide from the 5'-terminus, the third nucleotide from the 5'-terminus, or a combination of nucleotides at or near the 5'-terminus of the dsDNA donor. In yet additional embodiments, the modification is located at the 2' position of the 5'-terminal nucleotide, the second nucleotide from the 5'-terminus, the third (tertiary) nucleotide from the 5'-terminus, or a combination of nucleotides at or near the 5'-terminus of the dsDNA donor.

[0030] The use of 2'-modified ribonucleotides, particularly 2'-O-methoxyethyl (MOE), was found to provide the best improvement when compared to biotin or other modifications. Additional experiments establishing the use of these modifications with donors that mediate large insertions are described herein.

[0031] Further improvements to the dsDNA donor manufacturing process were evaluated. Universal priming sequences were selected to lack homology with common genomes (human, mouse, rat, and zebrafish). Previous work by our group established the utility of these priming sequences in cloning applications (i.e., highly efficient and reliable amplification). (1) Successful amplification of a wide variety of sequences and (2) significant improvements in overall amplification yields can be achieved by incorporating these universal sequences into the donor manufacturing process. Testing of these universal sequences when placed adjacent to the complete HDR donor sequence is described herein. Because the risk of homology-independent integration is reduced when using 5'-dsDNA modifications, these sequences do not adversely affect the correct HDR rate using modified donors and are rarely incorporated during blunt integration.

[0032] The methods and compositions disclosed herein are for use in improving HDR efficiency and reducing homology-independent events (blunt integration events or multimerization events) of dsDNA donor templates. In various embodiments, the disclosed methods and compositions enable a reduction in homology-independent integration or an increase in homology-dependent integration after genome editing using programmable nucleases. In some embodiments, bulky nucleotide modifications are placed at the 5' end of a linear dsDNA donor. In additional embodiments, modifications placed at the 2'-position of the 5'-terminal nucleotide or a nucleotide near the 5'-end of the dsDNA (e.g., 2'-MOE, 2'-OMe) demonstrate improved efficacy in reducing homology-independent integration. Furthermore, this reduction does not appear to be mediated by increased donor stability, as other modifications with established ability to inhibit nuclease degradation (such as PS) also do not reduce blunt integration rates to the same extent as other 2'-modifications.

[0033] In some embodiments, chemical modifications are introduced at the 5' end of the linear dsDNA donor. These chemical modifications are used to reduce the risk of NHEJ integration and improve its utility as a repair template in HDR experiments. In some embodiments, bulky or large modifications are introduced. In additional embodiments, modifications may be introduced near the 5' end of the dsDNA oligonucleotide donor. In some embodiments, modifications may be introduced at or near the 5' end of the dsDNA oligonucleotide donor. In some embodiments, the nucleotide at or near the 5' end of the dsDNA oligonucleotide may be modified. In additional embodiments, modifications include: biotin (B); phosphorothioate (PS); *); triethylene glycol (TEG); locked nucleic acids, such as 2'-oxygen-4'-carbon methylene linkages (LNA); hexaethylene glycol (Sp18); 1,3-propanediol (SpC3); 2'-O-methoxyethyl (MOE) ribonucleotides, 2'-O-methyl ribonucleotides (2'-OMe), 2'-fluoro (2'-F) nucleotides, and ribonucleotides, but are not limited to these.

[0034] In a further embodiment, the use of hairpin structures at the ends of the dsDNA donor similarly reduces blunt integration.

[0035] In one embodiment, the end-modified dsDNA donor template will be suitable for subsequent use in introducing double-strand breaks by programmable nuclease.In a further embodiment, the programmable nuclease comprises transcription activator-like effector nuclease (TALEN), zinc finger (ZFN), or clustered regularly interspaced short palindromic repeats (CRISPR).In one embodiment, the programmable nuclease system is CRISPR.In one aspect, the programmable nuclease enzyme is CRISPR-associated 9 (Cas9).

[0036] In one embodiment, the 5'-end-modified dsDNA donor is generated by PCR amplification. Primers modified with biotin, phosphorothioate (PS) linkages, TEG, LNA, spacer 18 (SP18), C3 spacer (SpC3), or MOE are used to amplify the insertion region and generate a terminally modified dsDNA donor. In some embodiments, the insertion region is greater than 120 bp. In some embodiments, the insertion region is at least 1 kb long. In some 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.

[0037] In additional embodiments, modifications at or near the 5' end include biotin, phosphorothioate (PS), triethylene glycol (TEG), locked nucleic acids such as 2'-oxygen-4'-carbon methylene 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.

[0038] In further 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 duplex DNA donor. In some embodiments, the 2'-modification is 2'-MOE, 2'-OMe, or 2'-fluoro, and the nucleotide modification occurs at or near the 5' end of the duplex DNA donor. In some embodiments, the 5'-end modification is on the 5'-end nucleotide of the duplex DNA donor. In additional embodiments, the 5'-end modification is located at the 5'-end nucleotide, the second nucleotide from the 5' end, the third nucleotide from the 5' end, or a combination of nucleotides at or near the 5' end of the dsDNA donor. In other embodiments, the 5'-end modification is located at the 5' end, and the modification is located at the 5'-end nucleotide, the second nucleotide from the 5' end, or the third nucleotide from the 5' end. In yet another embodiment, the 5'-terminal modification is a 2'-MOE modified ribonucleotide located at the terminal 5' position, the second nucleotide position from the 5' end, the third nucleotide position from the 5' end, or a combination thereof. In yet another embodiment, the 5'-terminal modification is a 2'-MOE ribonucleotide located at the terminal 5' position, the second nucleotide position from the 5' end, the third nucleotide position from the 5' end, or a combination thereof.

[0039] In additional embodiments, the HDR donor comprises a homology arm on either side of the insertion. The homology arm is complementary to the sequence adjacent to the double-strand break introduced by the programmable nuclease. In some embodiments, the homology arm varies in length from at least 20 nucleotides to 500 nucleotides. In some embodiments, the homology arm is at least 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, or 500 nucleotides in length. In some embodiments, the length of the homology arm may be longer than 500 nucleotides. In additional embodiments, the homology arm is preferably at least 40 nucleotides in length, more preferably at least 100 nucleotides in length.

[0040] In some embodiments, the insertion is located between the homology arms. In some embodiments, the insertion is greater than 20 nucleotides in length. In some embodiments, the insertion is at least 1 nucleotide and up to 4 kb in length. In some embodiments, the insertion is in the range of 1-2 kb in length. In some embodiments, the insertion is 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, It may be 800bp, 900bp, 1kb, 2kb, 3kb, 4kb, 5kb, 6kb, 7kb, 8kb, 9kb in length, In yet further embodiments, the insert may be 10kb or more in length.

[0041] In additional embodiments, the HDR donor includes homology arms on either side of the insertion, and the insertion may include a SNP, MNP, or deletion. In some embodiments, the insertion is at least 1 nucleotide and up to 4 kb in length. In some embodiments, the insertion is in the range of 1-2 kb in length. In some embodiments, the insertion may 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, or 9 kb in length. In yet additional embodiments, the insertion may be 10 kb or longer in length.

[0042] The polynucleotides described herein include variants with substitutions, deletions, and / or additions, which may involve one or more nucleotides. The variants can be altered in the coding region, non-coding region, or both. The alterations in the coding region can result in conservative or non-conservative amino acid substitutions, deletions, or additions. Particularly preferred among these are silent substitutions, additions, and deletions that do not alter the binding properties and activity.

[0043] Further 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) the nucleotide sequence, or a degenerate, homologous, or codon-optimized variant thereof; or (b) a nucleotide sequence that can hybridize to the complement of any of the nucleotide sequences in (a).

[0044] By a polynucleotide having a nucleotide sequence that is at least, e.g., 90-99% "identical" to a reference nucleotide sequence, it is intended that the nucleotide sequence of the polynucleotide is identical to the reference sequence, except that the polynucleotide sequence may contain up to about 10-1 point mutations, additions, or deletions per 100 nucleotides of the reference nucleotide sequence.

[0045] In other words, to obtain a polynucleotide having a nucleotide sequence at least about 90-99% identical to a reference nucleotide sequence, up to 10% of the nucleotides in the reference sequence can be deleted, added, or substituted with other nucleotides, or up to 10% of the total nucleotides in the reference sequence can be inserted into the reference sequence. These mutations in the reference sequence can occur at the 5' or 3' terminal position of the reference nucleotide sequence, or anywhere between these terminal positions, and can be individually interspersed among nucleotides in the reference sequence or within one or more contiguous groups within the reference sequence. The same applies to polypeptide sequences at least about 90-99% identical to a reference polypeptide sequence.

[0046] In some embodiments, programmable nucleases (e.g., CRISPR enzymes) or components (e.g., gRNAs) can be introduced into cells using various approaches, including plasmid or viral expression vectors (leading to endogenous expression of either the enzyme, the gRNA, or both), delivery of the enzyme by separate gRNA / crRNA transfection, or delivery of the enzyme with the gRNA or crRNA as a ribonucleoprotein (RNP) complex.

[0047] It will be apparent to those skilled in the relevant art that suitable modifications and adaptations can be made to the compositions, formulations, methods, processes, devices, assemblies, and uses described herein without departing from the scope of any embodiment or aspect thereof. The compositions, devices, assemblies, and methods provided are exemplary and are not intended to limit the scope of any disclosed embodiment. All various embodiments, aspects, and options disclosed herein can be combined in any variation or iteration. The scope of the compositions, formulations, methods, devices, assemblies, and processes described herein includes all actual or potential combinations of the embodiments, aspects, options, examples, and preferences described herein. The compositions, formulations, devices, assemblies, or methods described herein may omit any component or step, substitute any component or step disclosed herein, or include any component or step disclosed elsewhere herein. The ratio of the mass of any component of any composition or formulation disclosed herein to the mass of the other components in the formulation or the total mass of the other components in the formulation is disclosed herein as if expressly disclosed. If the meaning of any term in a patent or publication incorporated by reference conflicts with the meaning of the term used in this disclosure, the meaning of the term or phrase in this disclosure will control. All patents and publications cited herein are incorporated by reference for their specific teachings.

[0048] reference 1. Chang et al., “Non-homologous DNA end joining and alternative pathways to double-strand break repair.” Nature Reviews Molecular Cell Biology18:495-506 (2017). 2. Roth et al., “Reprogramming human T cell function and specificity with non-viral genome targeting,” Nature 559 (7714): 405-409 (2018). 3. Li et al., “Design and specificity of long ssDNA donors for CRISPR-based knock-in,” bioRxiv doi: 10.1101 / 178905 (2017). 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). 5. Canaj et al., “Deep profiling reveals substantial heterogeneity of integration outcomes in CRISPR knock-in experiments,” bioRxiv doi: 10.1101 / 841098 (2019). 6. Ghanta et al., “5’Modifications Improve Potency and Efficacy of DNA Donors for Precision Genome Editing,” bioRxiv doi: 10.1101 / 354480 (2018). 7. Robinson et al., “Integrative Genomics Viewer,” Nature Biotechnology 29: 24-26 (2011).

[0049] Embodiments 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. A2. A duplex DNA HDR donor according to A1, wherein the modifications include a modification to the 2' position of one or more nucleotides at or near the 5' end of the first homology arm region, and a modification to the 2' position of one or more nucleotides at or near the 5' end of the second homology arm region. A3. The modification is at the 2' position of the 5'-terminal nucleotide, the second nucleotide from the 5'-terminal, the third nucleotide from the 5'-terminal, or a combination of nucleotides at or near the 5'-terminal of the first homology arm region and the second homology arm region. A double-stranded DNA HDR donor according to A1 to A2, comprising: A4. A duplex DNA HDR donor according to A1, wherein the modification at or near the 5' end of the duplex DNA HDR donor comprises one or more of 2'-OME, 2'-F, or 2'-MOE. A5. A double-stranded DNA HDR donor according to A1 to A4, wherein the modification at or near the 5' end of the double-stranded DNA HDR donor includes 2'-MOE. A6. A duplex DNA HDR donor according to A1 to A5, wherein the modification at or near the 5' end is a non-template mismatch to the target DNA. A7. A double-stranded DNA HDR donor according to A1 to A6, wherein the first homology arm region and the second homology arm region are 40 to 150 nucleotides in length. A8. A duplex DNA HDR donor according to A1 to A7, wherein the first homology arm region and the second homology arm region are at least 100 nucleotides in length. A9. A double-stranded DNA HDR donor according to any one of A1 to A8, further comprising a universal primer sequence. A10. A double-stranded DNA HDR donor according to A1 to A9, wherein the insertion region is greater than 100 bp. A11. A duplex DNA HDR donor according to A1 to A10, wherein 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. A12. A duplex DNA HDR donor according to A1 to A11, wherein the duplex HDR donor comprises a hairpin at either the 5' end or the 3' end. A13. A double-stranded DNA HDR donor according to A1 to A12, wherein the double-stranded HDR donor comprises hairpins at both the 5' and 3' ends. A14. A double-stranded DNA HDR donor according to A1 to A13, which improves homology-directed repair efficiency and reduces homology-independent integration in a programmable nuclease system.

[0050] B1. A programmable nuclease system comprising a modified double-stranded DNA homology-directed repair (HDR) donor, a programmable nuclease enzyme, and a gRNA, wherein the gRNA molecule is capable of targeting the programmable nuclease molecule to a target nucleic acid. B2. The programmable nuclease system of B1, wherein the modified double-stranded DNA HDR donor comprises a first homology arm region, an insertion region, and a second homology arm region, and the first homology arm region and the second homology arm region comprise modifications to one or more nucleotides at or near the 5' end. B3. The programmable nuclease system described in B1-B2, wherein the modified double-stranded DNA HDR donor comprises a modification to the 2' position of the 5'-terminal nucleotide, the second nucleotide from the 5'-terminal end, the third nucleotide from the 5'-terminal end, or a combination of nucleotides at or near the 5'-terminal end of the first homology arm region and the second homology arm region. 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 to one or more nucleotides at or near the 5' end. 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. B6. The programmable nuclease system of B1 to B5, wherein the modified double-stranded DNA HDR donor comprises a universal primer sequence. B7. The programmable nuclease system of B1 to B6, wherein the modified double-stranded DNA HDR donor improves homology-directed repair efficiency and reduces homology-independent integration in the programmable nuclease system. B8. The programmable nuclease system of B1-B7, comprising one or more of a transcription activator-like effector nuclease (TALEN), a zinc finger (ZFN), or a clustered regularly interspaced short palindromic repeats (CRISPR). B9. The programmable nuclease system of B1 to B8, which is a CRISPR. B10. The programmable nuclease system of B1 to B9, wherein the programmable nuclease enzyme is CRISPR-associated-9 (Cas9). B11. The programmable nuclease system of B1-B10, further comprising one or more HDR enhancers.

[0051] C1. A method for increasing homology-directed repair (HDR) rates and decreasing homology-independent integration in a programmable nuclease system, comprising targeting a candidate editing target site locus with an active programmable nuclease system and a modified double-stranded DNA HDR donor. C1. The method of C1, wherein the modified double-stranded DNA HDR donor comprises a first homology arm region, an insertion region, and a second homology arm region, and the first homology arm region and the second homology arm region comprise modifications to one or more nucleotides at or near the 5' end. C2. The method of C1, wherein the modified double-stranded DNA HDR donor comprises a modification to the 2' position of the 5'-terminal nucleotide, the second nucleotide from the 5'-terminus, the third nucleotide from the 5'-terminus, or a combination of nucleotides at or near the 5'-terminus of the first homology arm region and the second homology arm region. C3. The method of C1 to C2, wherein the modified double-stranded DNA HDR donor comprises at least one 2'-OME, 2'-F, or 2'-MOE modification to one or more nucleotides at or near the 5' end. C4. The method of any one of C1 to C3, wherein the modified double-stranded DNA HDR donor comprises one or more 2'-MOE modifications at or near the 5' end. C5. The method according to any one of C1 to C4, wherein the modified double-stranded DNA HDR donor comprises a universal primer sequence. C6. The method of C1 to C5, further comprising one or more HDR enhancers. C7. The method of any one of C1 to C6, wherein the modified double-stranded DNA HDR donor improves homology-directed repair efficiency and reduces homology-independent integration in the programmable nuclease system.

[0052] D1. Use of a modified double-stranded DNA HDR donor to increase the homology-directed repair (HDR) rate and reduce 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 priming sequences; and the first homology arm region and the second homology arm region comprise modifications to one or more nucleotides at or near the 5' end. D2. The use of D1, wherein the modification comprises at least one 2'-OME, 2'-F, or 2'-MOE modification to one or more nucleotides at or near the 5' end of the duplex DNA HDR donor.

[0053] E1. A method for producing a modified double-stranded DNA HDR donor, comprising: and optionally one or more universal priming 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 a modification to one or more nucleotides at or near the 5' end. E2. The method of E1, wherein the modification comprises at least one 2'-OME, 2'-F, or 2'-MOE modification to one or more nucleotides at or near the 5' end of the duplex DNA HDR donor.

[0054] F1. A method for producing a modified double-stranded DNA HDR donor, comprising amplifying a target nucleic acid sequence comprising a first homology arm region, an insert region, and a second homology arm region with one or more universal primers, wherein the universal priming sequence comprises a modification to one or more nucleotides at or near the 5' end. 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. [Example]

[0055] Example 1 Modified dsDNA donors increase HDR rates and decrease NHEJ insertions. Initial studies were performed to compare the HDR insertion rate for unmodified linear dsDNA, donors containing a 5'-biotin modification, or donors with alternative modifications at or near the 5' end, relative to homology-independent (i.e., blunt) integration. The dsDNA donor was generated by PCR amplification of a plasmid containing a 1 kb insert with 100 bp flanking homology arms targeting the human SERPINC1 gene (100-1000-100; SEQ ID NO: 1; see Table 1 for amplification primer sequences; SEQ ID NOs: 2-21). Amplification primers were designed with either the unmodified sequence or the indicated modifications. Purified dsDNA donors were transfected at 100 nM (1 μg) in a final volume of 28 μL nucleofection buffer with 2 μM Cas9 V3™ RNP (IDT, Coralville, Iowa) targeting SERPINC1 using Lonza nucleofection (Lonza, Basel, Switzerland) to yield 3.5 × 10 nucleotides. 5 The SC1 (SERPINC1) protospacer sequence used can be found in Table 1 (SEQ ID NO: 22). Cells were lysed 48 hours later using QuickExtract™ DNA extraction solution (Lucigen, Madison, WI). HDR and blunt integration rates were assessed by digital droplet PCR (ddPCR) (Bio-Rad, Hercules, CA) using PCR assays with primers flanking the junction between the target DNA and the insert (Table 1; SEQ ID NOs: 23-27). Both the HDR and blunt junction assays included one primer outside the homology arm sequence to avoid amplification from non-integrated donors. The HDR assay probe (SEQ ID NO: 25) covered the junction between the target site and the insert sequence. The blunt assay probe (SEQ ID NO: 27) covered the junction between the target site and the integration homology arm sequence.

[0056] [Table 1-1]

[0057] [Table 1-2]

[0058] dsDNA donors containing known nuclease-resistant modifications, such as phosphorothioate linkages (2xPS, 3xPS, or 6xPS) or LNA nucleotides on the 5'-end, did not improve the HDR:blunt ratio over unmodified donors, as the modifications increased the rates of both HDR and blunt integration (Figure 2). Donor 5'-modifications (biotin, TEG, Sp18, and SpC3) increased the HDR rate with varying degrees of reduction in blunt integration. Of these donors, TEG, biotin, and Sp18 showed increased HDR:blunt ratios (1.8-, 2.0-, and 2.5-fold improvements over unmodified, respectively). See Figure 2. Donors containing 2'-O-methoxyethyl (2'-MOE)-modified ribonucleotides at both 5'-ends showed the greatest increase in HDR:blunt ratio (5.0-fold improvement over unmodified donors). See Figure 2. The HDR rate was similar between 2'-MOE and other modified donors, suggesting that increased stability alone was not the cause of the increased HDR:blunt ratio. Furthermore, as previously mentioned, LNA- and PS-modified donors did not increase the HDR:blunt ratio, indicating that the decrease in blunt integration is likely not due to increased nuclease resistance of the template. We also demonstrate that the use of 2'-modified ribonucleotides near the 5' end of the donor is insufficient to reduce blunt integration, and that the 2'-MOE-modified template is the most suitable for this activity among the modifications tested here.

[0059] Example 2 5'-modifications demonstrate lower off-target integration when short donor templates are used. The rate of homology-independent integration depends on the total length of the donor, with blunt insertions increasing as the donor size decreases. To determine whether 5'-end modifications would reduce the rate of blunt insertions using a smaller 42-bp insert (compared to the 1 kb insert tested in Example 1), a modified dsDNA donor targeting the SERPINC1 locus described in Example 1 was generated (SC1-166S; SEQ ID NO: 22). The donor consisted of a 42-bp insert containing an EcoRI restriction site with 40-bp homology arms (SC1 40-42-40; SEQ ID NO: 28), generated by PCR amplification of a plasmid containing the 42-bp insert with 100-bp flanking homology arms (see Table 2 for amplification primer sequences; SEQ ID NOs: 29-44).

[0060] Three modifications from Example 1 (biotin, Sp18, and MOE) were selected for further testing, while the 6xPS modification was included as a moderate performance control. To determine whether additional modified residues could further reduce blunt incorporation, three modifications were added to the 5' end. Donors with 3.5 × 10 2'-MOE ribonucleotides (3 × MOE) were also included. 2'-MOE ribonucleotides were also tested at various distances from the 5' end (Int MOE-3 and -5, with 2'-MOEs located 3 or 5 nucleotides from the 5' end; SEQ ID NOs: 41-44). Modified and unmodified donors were transfected with 3.5 × 10 2'-MOE ribonucleotides in a final volume of 28 μL using Lonza nucleofection (Lonza, Basel, Switzerland). 5HEK-293 cells were delivered at 500 nM (1.1 μg) with 2 μM Cas9 V3™ (IDT, Coralville, IA) RNP targeting the SERPINC1 locus. After 48 hours, DNA was extracted using QuickExtract™ DNA extraction solution (Lucigen, Madison, WI). Integration rates were assessed by RFLP using EcoRI digestion with a Fragment Analyzer™ machine (Advanced Analytical, Ames, IA) for band quantification. HDR and blunt integration events were distinguishable by a 40-bp size difference due to overlapping homology arms.

[0061] [Table 2]

[0062] The 1x MOE and 3x MOE modified donors provided the greatest improvement in the HDR:blunt ratio (4.1-fold and 4.6-fold improvement over unmodified, respectively). See Figure 3. As previously observed, 6x PS, biotin, and Sp18 provided some improvement (1.9-fold, 2.7-fold, and 2.8-fold improvement over unmodified, respectively), but did not reduce blunt integration to the same extent as MOE-modified donors. See Figure 3. Interestingly, the position of the 2'-MOE ribonucleotide within the donor slightly affected its utility in reducing blunt integration. Shifting the MOE ribonucleotide 3 or 5 nucleotides from the 5' end of the donor only improved the HDR:blunt ratio by 3.1-fold or 2.4-fold compared to unmodified donors. See Figure 3. Thus, users of the art should consider using a 2'-MOE ribonucleotide positioned within 2-3 nucleotides of the 5' end of the donor template. One would predict that the addition of 2'-MOE ribonucleotides would result in a significant reduction in NHEJ-mediated insertions.

[0063] Example 3 2'-MOE modification reduces incorporation at non-homologous double-strand breaks In addition to blunt integration at the target 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 cleavage of dsDNA. To evaluate the impact of 2'-MOE modification on donor integration at potential non-homologous DSBs, dsDNA donors with unmodified or modified 5' ends (unmodified, 1x MOE, or 6x 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 targeting gRNA (SC1-166S; SEQ ID NOS: 22) or mock "off-target" gRNAs with no homology to the donor (AAVS1-670AS; SEQ ID NOS: 54; HPRT 38087; SEQ ID NOS: 55).

[0064] 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 transfected with 3 x 10 nucleotides at a 100 nM dose (0.3 μg) with 2 μM Cas9 V3™ RNP (IDT, Coralville, IA) and 2 μM Alt-R™ Cas9 Electroporation Enhancer™ in a final volume of 28 μL by Lonza nucleofection (Lonza, Basel, Switzerland). 5 The vector was delivered into 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 using a Fragment Analyzer™ machine (Advanced Analytical, Ames, IA). HDR and blunt integration events can be distinguished by the 50-bp size difference due to the overlap of the homology arms (Figure 4).

[0065] [Table 3]

[0066] Blunt integration rates >9% were observed for unmodified dsDNA at the on-target Cas9 site and both "off-target" Cas9 sites. See Figure 4. A decrease in blunt integration (<1%) was observed with 2'-MOE-modified donors, demonstrating that 2'-MOE modification also reduces NHEJ-mediated insertion at non-homologous DSBs. See Figure 4. As previously observed, 6xPS modification resulted in a modest decrease in blunt integration.

[0067] Example 4 Reduced off-target integration is not a result of increased nuclease protection; specific 2' modifications are required for efficient reduction To determine whether the ability of the 2'-MOE modification to reduce blunt integration was specific or a general feature of modifications at the 2' position of the approximately 5'-most nucleotide, additional 2' modifications and 5'-terminal non-templated 2'-MOE ribonucleotides (SEQ ID NOS: 70-71) were tested (RNA, LNA, 2'-OMe, or 2'-F). (Non-templated ribonucleotides are defined as non-homologous to the target DNA sequence.) The donor consisted of the sequence previously described in Example 2 (SC1 40-42-40; SEQ ID NOS: 28), 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 specific to Example 4 listed in Table 4; SEQ ID NOS: 60-71).

[0068] [Table 4]

[0069] The dsDNA donor was co-delivered with Cas9 complexed with either the target gRNA (SC1-166S; SEQ ID NO: 22) or a gRNA with no homology to the donor (TNPO3; SEQ ID NO: 72). The donor was delivered into HEK-293 cells at a 250 nM dose (0.6 μg) containing 2 μM Cas9 V3 RNP by Lonza nucleofection (Lonza, Basel, Switzerland) in a final volume of 28 μL. After 48 hours, DNA was extracted using QuickExtract™ DNA extraction solution (Lucigen, Madison, WI). Integration rates were assessed using EcoRI. The DNA was assessed by RFLP and each band was quantified using a Fragment Analyzer™ machine (Advanced Analytical, Ames, IA).

[0070] Although HDR was unaffected or slightly boosted by various modifications, blunt integration at both on-target and off-target DSBs was reduced whenever 2'-MOE modifications were present (Figure 5). In contrast, most additional 2' modifications did not affect or increase the blunt integration rate. 2'-OMe modifications reduced the blunt integration rate at on-target DSBs, but not to the same extent as 2'-MOE modifications. See Figure 5. 2'-OMe modifications did not significantly reduce the blunt integration rate at off-target DSBs.

[0071] Taken together, these data suggest that the ability of the 2'-MOE modification to reduce homology-independent integration is (a) not a function of increased stability by promoting nuclease resistance, since other stabilizing modifications do not produce similar results, and (b) not a generalized function of 2'-modifications of most 5'-ribonucleotides, since other 2'-modifications do not produce similar results.

[0072] Example 5 Use of a hairpin as a blocking group to reduce homology-independent integration. In addition to chemical modification, the use of DNA hairpins at the ends of dsDNA donors can be used to reduce homology-independent integration. The generation of these hairpin-blocked donors is achieved in several ways (Figure 6A-B). For small HDR events (typically insertions of ≤120 bp with 40 bp homology arms), both DNA strands were chemically synthesized with a 5'-MOE hairpin sequence. These MOE adapters contain complementary sequences that allow the formation of a hairpin structure. The DNA strands were annealed to form the dsDNA HDR donor. For larger HDR events, a DNA primer containing a similar 5'-MOE hairpin was chemically synthesized and used to amplify the desired HDR donor. The use of an MOE ribonucleotide within the hairpin structure inhibits DNA polymerase progression through the hairpin. In both synthesis methods, the hairpin-blocked donor can be used as a nicked HDR template or ligated to generate a fully closed molecule.

[0073] The use of hairpins in chemically synthesized short oligos was functionally tested in cells. A 66-nt sequence was designed to mediate a 6-base GAATTC insertion into the SERPINC1 locus. This sequence and its reverse complement were synthesized as ssODNs that were either completely unmodified (Table 5; SEQ ID NOs: 75-76), contained an unmodified hairpin at the 5' end (SEQ ID NOs: 77-78), contained an MOE-modified hairpin at the 5' end (SEQ ID NOs: 79-80), or contained a non-template MOE-modified base at the 5' end (SEQ ID NOs: 81-82). Paired ssODNs were diluted to 100 μM and mixed at a 1:1 ratio to generate a final duplex of 50 μM. The oligo mixture was heated to 95°C for 1 minute and then slowly cooled to room temperature to allow the strands to anneal. The duplexed dsDNA donor was co-delivered with Cas9 complexed with the target gRNA (SEQ ID NO: 22). The donor was delivered into HEK-293 cells at a concentration of 2 μM using 2 μM Cas9 V3 RNP and 2 μM Alt-R® Cas9 electroporation enhancer by Lonza nucleofection (Lonza, Basel, Switzerland) in a final volume of 28 μL. After 48 h, DNA was extracted using QuickExtract™ DNA extraction solution (Lucigen, Madison, WI). Integration rates were assessed by RFLP using EcoRI, and individual bands were quantified using a Fragment Analyzer™ machine (Advanced Analytical, Ames, IA) (Figure 7).

[0074] [Table 5]

[0075] Using a shorter 66-bp unmodified dsDNA donor resulted in more efficient integration via the NHEJ pathway compared to the HDR pathway (55.5% blunt vs. 27.6% HDR). Introducing a DNA-only hairpin at the donor end improved the repair profile (42.4% blunt vs. 33.0% HDR). Inclusion of an MOE modification within the hairpin significantly improved this functionality to levels similar to those observed with a single 1x MOE at the 5' end (58.4% HDR vs. 8.9% blunt for the MOE-modified hairpin; 66.5% HDR vs. 6.9% blunt for the 1x MOE). Additional optimization of the modified hairpin (i.e., ligation, stem-loop length optimization, etc.) can be implemented to further improve this functionality.

[0076] Example 6 2'-MOE modification improves the desired repair outcome at multiple sites and in multiple cell lines. In the following experiments, 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 into cells of interest using Lonza nucleofection according to the recommended protocol.

[0077] To further validate the ability of 2'-MOE modification to result in correct repair with higher HDR rates and reduced blunt integration, unmodified and 1xMOE-modified dsDNA donors were tested at four additional genomic loci (HPRT, AAVS1 670, AAVS1 T2, and EMX1) in two cell lines (HEK293 and K562). The donors were designed to mediate a 42-bp insertion and had 40-bp homology arms (SEQ ID NOS: 83-86). The donors were generated by PCR amplification as previously described (primer sequences specific to Example 6, SEQ ID NOS: 95-142, listed in Table 6). Donors were delivered into the indicated cell lines in a final volume of 28 μL of nucleofection buffer containing 2 μM Cas9 V3™ RNP (IDT, Coralville, Iowa) and 2 μM Alt-R™ Cas9 Electroporation Enhancer™ at 250 nM using the recommended protocol for Lonza nucleofection (Lonza, Basel, Switzerland). are 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 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 ).

[0078] [Table 6-1]

[0079] [Table 6-2]

[0080] As previously observed, all 5' modifications improved HDR rates relative to unmodified dsDNA donors (A). While the fold improvement in HDR relative to unmodified dsDNA varied across sites and cell lines, the average improvement in HDR rates was relatively similar across all modifications tested (improvements ranging from 1.2-1.3-fold). In contrast, MOE-modified donors showed a significant reduction in blunt integration compared to 2xPS and biotin donors (B). On average, the fold reduction in blunt integration compared to unmodified dsDNA was 1.6 (2xPS), 2.3 (biotin), 2.9 (1xMOE), 3.2 (1xMOE, 2xPS), and 3.3 (3xMOE). When HDR and blunt integration improvements were assessed together at each site (C, reported as the ratio of HDR:blunt repair events), MOE-modified dsDNA donors outperformed the other modifications. The average fold change relative to unmodified dsDNA was 2.3 (2xPS), 3.1 (biotin), 3.6 (1xMOE), 4.1 (1xMOE 2xPS), and 4.3 (3xMOE). Together, these results demonstrate that MOE modification is the most efficient at promoting correct repair events after CRISPR editing.

[0081] Example 7 HDR rates are increased and NHEJ insertions are reduced using modified dsDNA donors that mediate large insertions. As a follow-up to studies using short inserts, we performed experiments to compare HDR and blunt integration rates using dsDNA donors mediating 300-bp, 500-bp, or 1000-bp insertions at two genomic loci (SERPINC1 and EMX1; see Table 7 for donor sequences and amplification primers, SEQ ID NOS: 147-154). Donors were generated by PCR amplification of plasmids containing the desired inserts with 100-bp flanking homology arms. Amplification primers were designed with either the unmodified sequence or the indicated modifications. Long ssDNA (Megamers™) was ordered for comparison at the SERPINC1 locus. Donors were transfected with 3.5 × 10 nucleofection vectors in a final volume of 28 μL of nucleofection buffer containing 2 μM Cas9 V3™ RNP (IDT, Coralville, Iowa) targeting SERPINC1 or EMX1 at 100 nM using Lonza nucleofection (Lonza, Basel, Switzerland). 5 The protospacer sequences were delivered to HEK-293 cells. 24 hours after transfection, the cells were treated with IDT Alt-R™ HDR Enhancer V2 (1 μM). The protospacer sequences used are shown in Table 1 (SEQ ID NO: 22) and Table 7 (SEQ ID NO: 161).

[0082] After 48 hours, cells were lysed using QuickExtract™ DNA extraction solution (Lucigen, Madison, WI). HDR and blunt 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 a PCR barcoding extension and ligation sequencing kit according to the manufacturer's recommended protocol. Final data analysis was performed using IDT's in-house data analysis pipeline (CRISPAltRations) (Figure 9A). Insertion rates were assessed separately by amplicon length analysis of the SERPINC1 sample. Isolated gDNA was PCR amplified using SERPINC1 RFLP primers (SEQ ID NOs: 45-46). Amplicons were run on a Fragment Analyzer™ machine for band quantification. Insertion events were identified based on the expected amplicon size for the integration event (Figure 9B).

[0083] [Table 7-1]

[0084] [Table 7-2]

[0085] [Table 7-3]

[0086] Compared with unmodified dsDNA donors, 1xMOE-modified donors mediated large insertions at both loci, resulting in higher HDR rates (average 28.6% to 30.9% for EMX1 and 44.4% to 53.6% for SERPINC1) and lower blunt integration rates (average 3.2% to 1.1% for EMX1 and 8.4% to 2.5% for SERPINC1). Long ssDNA donors mediated the same insertions at the SERPINC1 locus resulted in very low blunt integration rates (<1%). Long ssDNA donors mediated the highest rates of HDR for 300-bp insertions (71.3% vs. 54.9% for modified dsDNA donors). However, modified dsDNA donors performed as well as or better than long ssDNA donors for HDR with large insertions (55.2% vs. 53.3% for 500-bp insertions and 50.8% vs. 29.4% for 1000-bp insertions). A similar trend was observed for SERPINC1 samples when using the orthologous evaluation method ( Figure 9B ).

[0087] Example 8 The utilization of a universal priming sequence to produce modified dsDNA donors does not adversely affect HDR repair. To evaluate the impact of incorporating a universal priming sequence into the donor template, dsDNA donors mediating 500-bp insertions in EMX1 and SERPINC1 (see Table 7, SEQ ID NOS: 148 and 151) were prepared with either locus-specific or universal primers (Table 7, SEQ ID NOS: 153-160; Table 8, SEQ ID NOS: 166-181). The placement of the universal priming sequence relative to the donor is shown in Figure 10. Modifications tested included 1x MOE, 3x MOE, and biotin with a phosphorothioate modification (biotin 5x PS as described in [5]). Donors were transfected with 2 μM Cas9 V3™ R targeting SERPINC1 or EMX1 using Lonza nucleofection (Lonza, Basel, Switzerland). 3.5 × 10 at 100 nM in nucleofection buffer in a final volume of 28 μL containing NP (IDT, Coralville, Iowa)5 The transfection was performed on HEK-293 cells. 24 hours after transfection, cells were treated with IDT Alt-R™ HDR Enhancer V2 (1 μM). After 48 hours, cells were lysed using QuickExtract™ DNA extraction solution (Lucigen, Madison, WI). HDR and blunt integration rates were measured using the MinION™ platform (Oxford The sequences were assessed by long-read sequencing using Nanopore Technologies (Oxford, UK) and analyzed as previously described (Figure 11).

[0088] [Table 8]

[0089] HDR and blunt integration rates were relatively similar for dsDNA donors generated with or without a universal priming sequence. For donors without a universal priming sequence, the improvement in HDR and decrease in blunt rate were similar for various modifications. The main exception to this trend was the 3xMOE modification of the SERPINC1 site, where blunt insertions were still reduced compared to unmodified dsDNA, but HDR was not improved to the same extent as with 1xMOE or Biotin5xPS (unmodified: 45.1% HDR, 9.0% blunt; 1xMOE: 55.2% HDR, 2.3% blunt; 3xMOE: 44.5% HDR, 1.2% blunt). In contrast, much larger differences in performance were observed for modified donors produced with a universal priming sequence. Interestingly, when the universal priming site was included in the donor sequence, the integration rate of unmodified dsDNA donors at both sites was lower (EMX1: 28.8% vs. 24.8% HDR, SERPINC1: 45.2% vs. 34.4% HDR). At both sites, 1x MOE modification resulted in the greatest improvement in HDR rate when integrated into donors containing the universal sequence. Further analysis of HDR reads from 1x MOE-modified donors was performed in Integrative Genomics Viewer [7] (IGV, Broad Institute, Cambridge, MA). HDR reads aligned to either a reference amplicon containing the correct HDR sequence (Figure 12A) or a reference amplicon containing both the desired insert and universal priming sequence (Figure 12B). No evidence of universal sequence incorporation was observed in HDR readouts, and therefore universal sequences can be incorporated into the manufacturing process of modified dsDNA donors without adversely affecting functional performance.

[0090] Example 9 Use of modified dsDNA donors manufactured with universal priming sequences to generate GFP fusions in human cell lines. 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 were produced using either unmodified or 1xMOE-modified primers with universal priming sequences as previously described. The guide and donor sequences used are listed in Table 9. Donors were transfected at 50 nM in a final volume of 28 μL nucleofection buffer containing 2 μM Cas9 V3™ RNP (IDT, Coralville, Iowa) targeting GAPDH, CLTA, or RAB11a using Lonza nucleofection (Lonza, Basel, Switzerland). 3.5 × 10 5 The antibody was delivered to 1000 K562 cells. After transfection, the cells were plated in duplicate wells. For one set of wells, the cells were treated with IDT Alt-R™ HDR Enhancer V2 (1 μM) for 24 hours after transfection. The cells were passaged for 7 days, at which time the HDR rate was assessed by flow cytometry. Briefly, the cells were washed with PBS and then plated in 1-2 × 10 6The cells were resuspended at 1000 cells / mL. Immediately before analysis for viability staining, Hoechst 33258 was added to the cell suspension at a final concentration of 4 μg / mL. Cells were analyzed using a Becton Dickinson LSR II cytometer (BD Bioscience, San Jose, CA) to assess GFP expression levels (Figure 12).

[0091] [Table 9-1]

[0092] [Table 9-2]

[0093] Overall HDR rates varied across the sites tested, with the maximum GFP-positive rates achieved under optimal conditions being 17.2% (GAPDH), 44.9% (CLTA), and 64% (RAB11a). No GFP signal was observed in cells receiving dsDNA donors without RNP (data not shown). HDR rates increased using modified dsDNA donor templates in both untreated conditions (1.6-, 1.3-, and 1.2-fold improvements over unmodified dsDNA for GAPDH, CLTA, and RAB11a, respectively) and HDR enhancer-treated conditions (1.4-, 1.4-, and 1.1-fold improvements over unmodified dsDNA, respectively). On average, using 1x MOE-modified dsDNA donors resulted in a 1.3-fold increase in HDR rates over unmodified dsDNA donors across all conditions. In comparison, Alt-R Use of HDR Enhancer V2 increased HDR rates by an average of 2.4-fold across all sites and conditions. Use of the modified donor and HDR enhancer in combination boosted HDR rates by an average of 3.2-fold across all sites. Collectively, this demonstrates the combinatorial utility of using optimal reagents (i.e., modified donors and small molecule enhancers) in HDR experiments.

[0094] Example 10 Use of universal priming sequences increases consistency and improves yield when producing dsDNA HDR templates To evaluate the impact of universal priming sequences on the dsDNA HDR template production process, 24 sequences were generated using either universal priming sequences (Table 8, SEQ ID NOS: 172-181) or gene-specific primers (Table 10, SEQ ID NOS: 188-235) with various modifications. As previously described, all donors were required to generate the sequences of interest. The fragments were generated by amplification from a plasmid containing the gene (pUCIDT Amp or pUCIDT Kan vector). PCR amplification was performed using KOD Hot Start DNA Polymerase (EMD) according to the manufacturer's recommendations, with 200 nM primers and 10 ng of plasmid DNA in a final reaction volume of 50 μL. Thermocycling was performed using a Bio-Rad S1000 thermal cycler with the following cycling conditions: 3 min incubation at 95°C, followed by 36 amplification cycles (95°C for 20 s; 65°C for 10 s; 70°C for 20–30 s / kb). The annealing temperature was adjusted according to the gene-specific primer melting temperature. Following SPRI bead cleanup, all products were analyzed using a Fragment Analyzer (Agilent), and the sequences were verified by next-generation sequencing using an Illumina-Nextera DNA library preparation kit. The overall amplification efficiency from the universal or gene-specific primers was assessed by measuring the final yield, reported as ng / μL (Figure 14A).

[0095] [Table 10-1]

[0096] [Table 10-2]

[0097] Due to the difference in yield between short (<500 bp) and long (>500 bp) amplicons, the overall yield after amplification using either universal or gene-specific primers was evaluated separately for the 12 short and 12 long HDR templates (Figure 14A). Overall, the use of universal primers for both short and long amplicons resulted in significantly higher yields. For the long amplicons, the use of universal primers resulted in a mean concentration of 138.3 ng / μL (±18.0 SD) after cleanup, while the use of gene-specific primers resulted in a mean concentration of 77.8 ng / μL (±32.6 SD). For the short amplicons, the use of universal primers resulted in a mean concentration of 40.9 ng / μL (±5.9 SD) after cleanup. Using gene-specific primers resulted in an average concentration of 15.9 ng / μL (±6.4 SD), while using universal primers resulted in an average concentration of 15.9 ng / μL (±6.4 SD). A direct comparison between sequences amplified with universal or gene-specific primers (Figure 14B) reveals a large variation in yield when gene-specific primers are used. In contrast, using universal primers resulted in higher yields (2.9- and 2.0-fold improvements over the average for short and long amplicons, respectively) and more consistent yields across sequences of similar length. Due to the higher yields and greater consistency, using universal primers better supports the development of high-throughput manufacturing processes.

Claims

1. 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 their 5′ ends.

2. 2. The duplex DNA HDR donor of claim 1, wherein the modifications comprise a modification to the 2' position of one or more nucleotides at or near the 5' end of the first homology arm region and a modification to the 2' position of one or more nucleotides at or near the 5' end of the second homology arm region.

3. 3. The duplex DNA HDR donor of claim 1 or 2, wherein the modifications comprise modifications to the 2' position of the 5'-terminal nucleotide, the second nucleotide from the 5'-terminus, the third nucleotide from the 5'-terminus, or a combination of nucleotides at or near the 5'-terminus of the first homology arm region and the second homology arm region.

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

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

6. 6. The duplex DNA HDR donor of any one of claims 1 to 5, wherein the modification at or near the 5' end is a non-templated mismatch to the target DNA.

7. 7. The double-stranded DNA HDR donor of any one of claims 1 to 6, wherein the first homology arm region and the second homology arm region are 40 to 150 nucleotides in length.

8. The double-stranded DNA HDR donor of any one of claims 1 to 7, wherein the first homology arm region and the second homology arm region are at least 100 nucleotides in length.

9. The double-stranded DNA HDR donor of any one of claims 1 to 8, wherein the double-stranded DNA HDR donor further comprises a universal primer sequence.

10. The double-stranded DNA HDR donor of any one of claims 1 to 9, wherein the insertion region is greater than 100 bp.

11. 11. The duplex DNA HDR donor of any one of claims 1 to 10, 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.

12. 12. The duplex DNA HDR donor of any one of claims 1 to 11, wherein the duplex HDR donor comprises a hairpin at either the 5' or 3' end.

13. 13. The duplex DNA HDR donor of any one of claims 1 to 12, wherein the duplex HDR donor comprises a hairpin at both the 5' and 3' ends.

14. 14. The double-stranded DNA HDR donor of any one of claims 1 to 13, wherein the double-stranded DNA HDR donor improves homology-directed repair efficiency and reduces homology-independent integration in a programmable nuclease system.

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

16. 16. The programmable nuclease system of claim 15, 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 a modification to one or more nucleotides at or near the 5' end.

17. 17. The programmable nuclease system of claim 15 or 16, wherein the modified duplex DNA HDR donor comprises a modification to the 2' position of the 5'-terminal nucleotide, the second nucleotide from the 5'-terminus, the third nucleotide from the 5'-terminus, or a combination of nucleotides at or near the 5'-terminus of the first homology arm region and the second homology arm region.

18. 18. The programmable nuclease system of any one of claims 15 to 17, wherein the modified duplex DNA HDR donor comprises at least one 2'-OME, 2'-F, or 2'-MOE modification to one or more nucleotides at or near the 5' end.

19. 19. The programmable nuclease system of any one of claims 15 to 18, wherein the modified double-stranded DNA HDR donor comprises one or more 2'-MOE modifications at or near the 5' end.

20. 20. The programmable nuclease system of any one of claims 15 to 19, wherein the modified double-stranded DNA HDR donor comprises a universal primer sequence.

21. 21. The programmable nuclease system of any one of claims 15 to 20, wherein the modified double-stranded DNA HDR donor improves homology-directed repair efficiency and reduces homology-independent integration in the programmable nuclease system.

22. 22. The programmable nuclease system of any one of claims 15 to 21, comprising one or more of transcription activator-like effector nucleases (TALENs), zinc fingers (ZFNs), or clustered regularly interspaced short palindromic repeats (CRISPRs).

23. The programmable nuclease system of any one of claims 15 to 22, which is CRISPR.

24. 24. The programmable nuclease system of any one of claims 15 to 23, wherein the programmable nuclease enzyme is CRISPR-associated-9 (Cas9).

25. 25. The programmable nuclease system of any one of claims 15 to 24, further comprising one or more HDR enhancers.

26. 1. A method for increasing homology-directed repair (HDR) rates and decreasing homology-independent integration in a programmable nuclease system, comprising targeting a candidate editing target site locus with an active programmable nuclease system and a modified duplex DNA HDR donor.

27. 27. The method of Claim 26, wherein the modified duplex DNA HDR donor comprises a first homology arm region, an insert region, and a second homology arm region, wherein the first homology arm region and the second homology arm region comprise a modification to one or more nucleotides at or near the 5' end.

28. 28. The method of claim 26 or 27, wherein the modified duplex DNA HDR donor comprises a modification to the 2' position of the 5' terminal nucleotide, the second nucleotide from the 5' terminal, the third nucleotide from the 5' terminal, or a combination of nucleotides at or near the 5' terminal of the first homology arm region and the second homology arm region.

29. 29. The method of any one of claims 26-28, wherein the modified duplex DNA HDR donor comprises at least one 2'-OME, 2'-F, or 2'-MOE modification to one or more nucleotides at or near the 5' end.

30. 30. The method of any one of claims 26-29, wherein the modified double-stranded DNA HDR donor comprises one or more 2'-MOE modifications at or near the 5' end.

31. 31. The method of any one of claims 26 to 30, wherein the modified double-stranded DNA HDR donor comprises a universal primer sequence.

32. 32. The method of any one of claims 26 to 31, further comprising one or more HDR enhancers.

33. 33. The method of any one of claims 26 to 32, wherein the modified double-stranded DNA HDR donor improves homology-directed repair efficiency and reduces homology-independent integration in a programmable nuclease system.

34. 1. Use of a modified double-stranded DNA HDR donor to increase homology-directed repair (HDR) rates and decrease 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 priming sequences; and the first homology arm region and the second homology arm region comprise modifications to one or more nucleotides at or near the 5' end.

35. 35. The use of claim 34, wherein the modification comprises at least one 2'-OME, 2'-F, or 2'-MOE modification to one or more nucleotides at or near the 5' end of the duplex DNA HDR donor.

36. 1. A method for producing a modified double-stranded DNA HDR donor, comprising synthesizing an oligonucleotide comprising a first homology arm region, an insert region, a second homology arm region; and optionally, one or more universal priming sequences, wherein the first homology arm region and the second homology arm region comprise a modification to one or more nucleotides at or near the 5′ end.

37. 37. The method of claim 36, wherein the modification comprises at least one 2'-OME, 2'-F, or 2'-MOE modification to one or more nucleotides at or near the 5' end of the duplex DNA HDR donor.

38. 1. A method for producing a modified double-stranded DNA HDR donor, comprising amplifying a target nucleic acid sequence comprising a first homology arm region, an insert region, and a second homology arm region with one or more universal primers, wherein the universal priming sequence comprises a modification to one or more nucleotides at or near the 5′ end.

39. 39. The method of claim 38, 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.

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