Ubiquitin variants with high affinity to bind 53BP1 reduce amount of AAV required to achieve high HDR rates

Abstract

The present invention relates to tagless CM1 polypeptides and methods of using the same to enhance homologous directed repair (HDR) in recipient cells. Preferred donor template delivery vectors include an adenovirus-related vector for delivery of a donor template, and a preferably tagless CM1 polypeptide, referred to as CM1tf. The invention also provides isolated nucleic acids that encode label-free CM1 polypeptides.

Description

Cross - Reference to Related Applications

[0001] This application claims priority under 35 U.S.C. 119 based on U.S. Provisional Patent Application No. 63 / 399,452, filed on August 19, 2022, entitled "A UBIQUITIN VARIANT WITH HIGH AFFINITY FOR BINDING 53BP1 REDUCES THE AMOUNT OF AAV NEEDED TO ACHIEVE HIGH RATES OF HDR", the content of which is hereby incorporated by reference in its entirety. Sequence Listing

[0002] This application contains a sequence listing that has been submitted in XML format by the Patent Center and is hereby incorporated by reference in its entirety. The XML copy was created on August 15, 2023, and has the name IDT01 - 023 - PCT_ST26.xml. Technical Field

[0003] The present invention relates to the ability of ubiquitin variants to bind 53BP1 and bias the repair of double - strand breaks (DSBs) towards homologous - directed repair (HDR). Background Art

[0004] Double-strand breaks (DSBs) are repaired mainly by two mechanisms: non-homologous end joining (NHEJ), in which the broken ends are rejoined, but usually not precisely; or homology-directed repair (HDR), which typically involves using the sister chromatid or homologous chromosome as a repair template. The presence of sister chromatids promotes HDR, and there are cellular mechanisms that bias repair towards NHEJ during the G1 phase of the cell cycle [1]. A key determinant of repair pathway choice is 53BP1. 53BP1 was initially described as a binding partner of the tumor suppressor gene p53 and was later shown to be a key protein in NHEJ [2]. 53BP1 rapidly accumulates at double-strand breaks. In G1, 53BP1 recruits RIF1 and inhibits end resection [3,4]. End resection is a key step in repair pathway choice because it is required for HDR and inhibits NHEJ [1]. By inhibiting end resection, 53BP1 biases repair towards NHEJ, so the absence of 53BP1 leads to increased HDR [5]. Targeted nucleases and a DNA repair template homologous to the targeted cut site can be introduced into cells to promote precise genome editing via HDR [6]. Thus, potent inhibitors of 53BP1 are beneficial for precise genome editing.

[0005] The recruitment of 53BP1 to DSB sites depends on H4K20 methylation and H2AK15 ubiquitination. 53BP1 has tandem Tudor domains that have been shown to specifically bind monomethylated and demethylated H4K20, and methylation of H4K20 has been shown to be important for 53BP1 recruitment to double-strand breaks [7, 8]. D1521R is a mutation that disrupts the activity of the Tudor domain and its introduction impairs the ability of 53BP1 to form radiation-induced foci [9]. The minimal focus-forming region of 53BP1 consists of the Tudor domain flanked by an N-terminal oligomerization region and a C-terminal extension region. Notably, the accumulation of 53BP1 at DNA double-strand breaks (DSBs) requires the E3 ubiquitin ligase RNF168, which is responsible for catalyzing the ubiquitination of H2AK13 and H2AK15

[10] . The C-terminal extension region has been shown to contain a ubiquitination-dependent recruitment motif (UDR) that specifically binds H2AK15ub and is necessary for the recruitment of 53BP1 to DSB sites [9].

[0006] Due to the affinity of 53BP1 for ubiquitinated H2A, Canny et al. recently screened for ubiquitin variants that interact with 53BP1. They discovered and engineered a ubiquitin variant that selectively binds 53BP1 and named it i53 (53BP1 inhibitor)

[11] . The top five matches from the ubiquitin variant screen were A10, A11, C08, G08, and H04, with G08 having the highest affinity. Contrary to expectations, the interaction of 53BP1 with G08 did not require the UDR, and this interaction was shown to occur between the Tudor domains of G08 and 53BP1. To generate i53, G08 was engineered by introducing the I44A mutation, which disrupts the solvent-exposed hydrophobic patch on ubiquitin, with which most ubiquitin-binding proteins interact [9,12]. Notably, this mutation in the context of H2AKC15ub(I44A) interfered with the interaction of 53BP1 with ubiquitinated H2A but did not interfere with the ability of i53 to enhance HDR, consistent with the mechanism by which i53 enhances HDR by interacting with the Tudor domain of 53BP1 rather than the UDR domain [9,11]. Additionally, i53 was engineered relative to G08 by removing the C-terminal di-glycine motif. Introduction of i53 into cells, but not the 53BP1-binding defective i53 variant DM (i53P69L+L70V), inhibited ionizing radiation-induced 53BP1 foci formation. Introduction of i53 by plasmid delivery, adeno-associated virus-mediated gene delivery, or mRNA delivery was shown to increase HDR efficiency. HDR efficiency was increased by introduction of i53 using both double-stranded DNA donors and single-stranded DNA donors, which have been shown to use different HDR mechanisms [11,13,14].

[0007] A previous application (U.S. Provisional Patent Application No. 63 / 321,384, filed Mar. 18, 2022, by Vakulskas et al., entitled "UBIQUITIN VARIANTS WITH IMPROVED AFFINITY FOR 53BP1" (Attorney Docket No.: IDT01-021-PRO3)) described an invention of a ubiquitin variant that contains nine amino acid substitutions relative to i53 and has a significantly increased (50-100-fold) affinity for binding 53BP1, which we call CM1. We demonstrated that this ubiquitin variant was able to enhance HDR to a greater extent at lower doses compared to i53 when using short ssDNA Alt-R donor oligonucleotides and long dsDNA Alt-R HDR donor modules as donor templates.

[0008] The present disclosure relates to an improved method for enhancing HDR in recipient cells by introducing CM1 into cells when using adeno-associated virus (AAV) for template delivery. Summary of the Invention

[0009] In a first aspect, there is provided a nucleic acid sequence encoding a tag-free ubiquitin polypeptide variant CM1.

[0010] In a second aspect, there is provided a protein sequence of a tag-free CM1 polypeptide.

[0011] In a third aspect, there is provided a method for enhancing homologous directed repair (HDR) in recipient cells. The method includes the steps of introducing a nucleic acid donor template and an isolated tag-free CM1 polypeptide into recipient cells. Brief Description of the Drawings

[0012] Figure 1A An exemplary heat map showing the HDR percentage of HPRT1 in HEK293 cells with and without 25 μM CM1tf is depicted.

[0013] Figure 1B An exemplary heat map showing the HDR percentage of SERPINC1 in HEK293 cells with and without 25 μM CM1tf is depicted.

[0014] Figure 1C An exemplary heat map showing the HDR percentage of SERPINC1 in K562 cells with and without 50 μM CM1tf is depicted. In Figure 1A -C, when using an AAV vector to deliver the donor template, the ubiquitin variant CM1tf enhances HDR when co-delivered with Cas9RNP. The heat map shows the HDR percentage measured by the EcoR1 cleavage assay with and without CM1tf. Using 4 μM Alt-R Cas9 electroporation enhancer, Cas9RNP (2 μM) with and without CM1tf was delivered into cells by Lonza nucleofection. After RNP delivery, an AAV donor containing an EcoR1 cleavage site inserted into 500 bp homology arms was added to the cells at a series of multiplicities of infection (MOI; multiplicity of infection) for 24 hours. The editing results are represented as the average HDR percentage ± standard deviation of three biological replicates.

[0015] Figure 2AExemplary heatmap depicting HDR percentages of HPRT1 in HEK293 cells under no enhancer, CM1tf, Alt-R HDR enhancer V2 (“V2”), or CM1tf+V2 conditions.

[0016] Figure 2B Exemplary heatmap depicting HDR percentages of SERPINC1 in HEK293 cells under no enhancer, CM1tf, V2, or CM1tf+V2 conditions. In Figure 2A -B, the ubiquitin variant CM1tf enhances HDR and provides additional enhancement when used with Alt-R HDR enhancer V2. The heatmap shows HDR percentages measured by EcoR1 cleavage assay using CM1tf, Alt-R HDR enhancer V2 (V2), or both. Cas9 RNPs with and without CM1tf were co-delivered into cells by Lonza nucleofection using 2 μM Cas9 RNP, 4 μM Alt-R Cas9 electroporation enhancer, and 0 or 25 μM CM1tf. After RNP delivery, an AAV donor containing an EcoR1 cleavage site inserted into 500 bp homology arms was added to the cells at a series of multiplicities of infection (MOI) for 24 hours. After nucleofection, the V2 enhancer was added to the medium at a final concentration of 1 μM for 24 hours. Editing results are represented as mean HDR percentage ± standard deviation of three biological replicates.

[0017] Figure 3 Exemplary heatmap summarizing that the IDT ubiquitin variant CM1tf enhances HDR when used with AAV donors having different homology arm lengths. The heatmap shows HDR percentages of STAT3 measured by EcoR1 cleavage assay with or without CM1tf. Cas9 RNPs with and without CM1tf were co-delivered into cells by Lonza nucleofection using 2 μM Cas9 RNP, 4 μM Alt-R Cas9 electroporation enhancer, and 0 or 25 μM CM1tf. After RNP delivery, AAV donors containing 500 base pair (bp), 300 bp, or 100 bp homology arms were added to the cells at a series of multiplicities of infection (MOI) for 24 hours. Editing results are represented as mean HDR percentage ± standard deviation of three replicates (single nucleofection, separate AAV delivery, and downstream processing).

[0018] Figure 4It is demonstrated that CM1tf is superior to i53 in its ability to enhance HDR using an AAV donor. The graph shows the percentage of HDR measured by the EcoR1 cleavage assay using CM1tf. Cas9 RNP was co-delivered and not co-delivered with CM1tf into cells by Lonza nucleofection using 2 μM Cas9 RNP, 4 μM Alt-R Cas9 electroporation enhancer, and a CM1tf concentration ranging from 200 μM to 6.25 μM. After RNP delivery, an AAV donor containing an EcoR1 cleavage site inserted into 500 bp homology arms was added to the cells at an MOI of 20,000 for 24 hours. The editing results are shown as the average HDR percentage, and the error bars represent the standard deviation of three biological replicates. Detailed Description

[0019] The present invention determines that CM1 is capable of enhancing HDR when using adeno-associated virus (AAV) to deliver templates into cells. When dealing with primary cells, it is particularly important to consider which donor to use, as different donors (including plasmid DNA, linear dsDNA, ssODN) cause different amounts of cytotoxicity, which can greatly affect the total cell yield [15-17]. Using AAV is generally the preferred method for delivering DNA templates into cells because it can introduce large sequences while avoiding the high toxicity associated with naked double-stranded DNA [18-21]. However, there is still a trade-off: a higher multiplicity of infection (MOI) can provide a higher editing level at the cost of increased toxicity, resulting in a lower cell yield. The production of AAV is also very time-consuming and expensive. Therefore, any product that can reduce the amount of AAV required to achieve a high editing level can increase cell yield and enhance the HDR rate while reducing manufacturing costs. Applications

[0020] In a first aspect, there is provided an isolated nucleic acid sequence encoding a tagless CM1 polypeptide. In a first aspect, the isolated nucleic acid sequence encodes a CM1tf polypeptide.

[0021] In a second aspect, there is provided an isolated protein sequence of a tagless CM1 polypeptide. In a first aspect, the tagless CM1 polypeptide includes a CM1tf polypeptide.

[0022] In a third aspect, there is provided a method for enhancing homologous directed repair (HDR) in recipient cells. The method includes the step of introducing a nucleic acid donor template and an isolated tagless CM1 polypeptide into the recipient cells. In a first aspect, the nucleic acid donor template includes an adeno-associated vector. In a second aspect, the isolated tagless CM1 polypeptide comprises a CM1tf polypeptide. Examples Example 1. Tagless CM1 (CM1tf) can enhance HDR when AAV is used for repair template delivery.

[0023] We developed a tagless version of CM1 (termed CM1tf) and examined its ability to enhance HDR in cell lines using AAV donors (Table 1). AAV-DJ, a synthetic AAV serotype most closely related to AAV-2 and a chimera of types 2 / 8 / and 9, was chosen for examination because of its high transduction efficiency in a variety of cell types in vitro

[22] . The dose of CM1tf was kept constant while a range of MOIs were used for the AAV donors. The AAV donors were constructed such that a 6-base pair insertion fragment consisting of an EcoR1 cleavage site (GAATTC) was flanked by 500-base pair homology arms that matched the genomic sequences flanking the target cleavage site. Briefly, Cas9 V3 protein (IDT) and Alt-R sgRNA (IDT) were mixed at a ratio of 1:1.2, incubated for 10 minutes, and then Alt-R Cas9 Electroporation Enhancer (EE) (IDT), 1X PBS (Gibco), and CM1tf diluted in 1X PBS were added. HEK293 or K562 cells were washed and resuspended in SF buffer for Lonza nucleofection and added to the RNP+DNA mixture such that the final concentration of Cas9 RNP was 2 μM, EE was 4 μM, and CM1tf was 25 μM (for HEK293 cells) or 50 μM (for K562 cells). Using the Lonza nucleofection system, cells and RNP+DNA were electroporated using program DS-150 (for HEK293 cells) or FF-120 (for K562 cells). Cells were then seeded in 96-well plates at a rate of 20,000 cells / well, and AAV was added to the wells at MOIs ranging from 0 (no virus) to 160,000. The MOI was calculated as viral genomes (vg) per cell, with vg determined using qPCR. Cells were seeded into serum-free medium after recovery culture, followed by addition of the AAV virus, and supplemented with serum-containing medium 4 hours later. Genomic DNA was isolated using QuickExtract (Lucigen) 48 hours after RNP delivery. The results are shown in Figure 1. The use of CM1tf significantly enhanced HDR efficiency, such that approximately four-fold less AAV was required to achieve the same editing level in HEK293 cells without the CM1tf HDR enhancer. Example 2. When using AAV donors, CM1tf can be used in combination with Alt-R HDR Enhancer V2 to further enhance HDR.

[0024] To optimize HDR, it is often necessary to use a combination of multiple HDR boosters that act through different mechanisms to further increase HDR efficiency beyond what any single booster can achieve

[23] . Since CM1tf acts by promoting end resection and thus promotes HDR, it is possible to combine it with IDT Alt-R HDR Booster V2, a non-homologous end joining (NHEJ) inhibitor, to further increase HDR efficiency. To test this, Cas9 RNP with or without CM1tf was delivered into HEK293 cells as described in Example 1, and then the cells were seeded in medium with or without 1 μM V2 booster, and different MOI ranges of AAV were added to each well as described above. The results of the test are shown in Figure 2. Using CM1tf or V2 booster alone resulted in roughly the same increase in HDR efficiency, but when used together they provided an additional boost to HDR that exceeded what either booster achieved alone. Example 3. HDR can be enhanced when CM1tf is used with AAV-packaged donor DNA templates with different homologous arm lengths.

[0025] To test the compatibility of CM1tf with AAV donors of different homologous arm lengths and to confirm the optimal HA length for short inserts, we tested the effect of combining CM1tf with AAV donors containing 100, 300, or 500 bp homologous arms. RNP and CM1tf were delivered into HEk293 cells as described in Example 1. The results are as Figure 3 shown. The 300 bp homologous arm donor provided the highest HDR efficiency without a booster and maintained this trend with the addition of CM1tf. The use of CM1tf resulted in a similar level of enhanced HDR rate regardless of the homologous arm length. Example 4. CM1tf is superior to i53 in its ability to enhance HDR when using AAV donors.

[0026] Compared to using ssDNA donors, achieving the best possible HDR efficiency with AAV donors may require different optimal doses of CM1tf. Additionally, the nature of the donor may affect the gain achieved with CM1tf compared to i53. To verify this, Cas9 RNP was co-delivered with CM1tf or i53 in a range of doses into HEK293 cells as described in Example 1, and then AAV was added to the cells at a set multiplicity of infection (MOI) of 20,000 24 hours later. The results are as Figure 4As shown previously, detection of CM1tf using an ssDNA donor showed that CM1tf had roughly the same gain for HDR in the dose range of 6.25 to 50 μM, while the optimal concentration of i53 was approximately 100 - 150 μM, with a reduced gain at 200 μM. Here, we show that in the case of using an AAV donor, although CM1tf consistently outperforms i53, higher doses of CM1tf up to 200 μM do show a slightly larger gain for HDR as the dose increases. This is even more pronounced for i53, whose efficacy increases significantly from 100 μM to 200 μM, although this is not the trend observed with the ssDNA donor. These results suggest that the nature of the donor used is an important consideration for CM1tf dose. However, compared to i53, the gain of CM1tf for HDR is much less affected by dose, and CM1tf achieves a generally higher level of editing compared to i53. Example 5. Sequences

[0027] Table 1. Amino Acid and DNA Sequences The first SEQ ID NO listed in a corresponds to the amino acid sequence; the second SEQ ID NO listed corresponds to the nucleotide sequence.

[0028] Table 2. Guidelines

[0029] Table 3. AAV Donor

[0030] Table 4: Primers for EcoR1 Cleavage Assay Definitions

[0031] To aid in the understanding of the present invention, several terms are defined below.

[0032] Unless the context clearly requires otherwise or is clearly contrary to the meaning herein, the terms "a", "an", "the", and similar terms used in the context of describing the present invention (especially in the context of the following claims) shall be construed to cover both the singular and the plural. Unless otherwise specified, the terms "comprising", "having", "including", and "containing" and the like shall be construed as open-ended terms (i.e., "including but not limited to"). Unless otherwise specified herein, the numerical ranges recited herein are merely intended to be a shorthand method of indicating each individual numerical value falling within the range, and each individual numerical value is considered to be individually disclosed as if it were separately recited herein. Unless the context clearly requires otherwise or is clearly contrary to the meaning herein, all methods described herein can be performed in any suitable order. Unless otherwise asserted, all examples or exemplary statements used herein (e.g., "such as") are merely intended to more clearly illustrate the present invention and do not constitute a limitation on the scope of the invention. No statement in the specification should be construed as indicating that any non-claimed element is essential for the practice of the present invention.

[0033] The term "CRISPR" refers to the clustered regularly interspaced short palindromic repeat bacterial adaptive immune system.

[0034] The terms "Cas" and "Cas endonuclease" generally refer to CRISPR-associated endonucleases.

[0035] The term "Cas protein" generally refers to the wild-type protein of CRISPR-associated endonucleases (including the interchangeable terms Cas and Cas endonuclease), including variants thereof.

[0036] The term "Cas nucleic acid" generally refers to the nucleic acid of CRISPR-associated endonucleases, including guide RNA, single guide RNA (sgRNA), CRISPR RNA (crRNA), or trans-activating crRNA (tracrRNA).

[0037] The terms "Cas9" and "CRISPR / Cas9" refer to the CRISPR-associated bacterial adaptive immune system of Streptococcus pyogenes. Examples of this system are disclosed in U.S. Patent Application No. 15 / 729,491, filed October 10, 2017, and U.S. Patent Application No. 15 / 964,041, filed April 26, 2018 (Attorney Docket Nos. IDT01-009-US and IDT01-009-US-CIP, respectively), the contents of which are incorporated herein by reference.

[0038] When the term "variant" modifies a protein (e.g., ubiquitin), the term "variant" refers to a protein that contains at least one amino acid substitution, an additional amino acid (e.g., such as an affinity tag or a nuclear localization signal), or a combination thereof, of a reference protein (usually the amino acid sequence of the wild-type protein).

[0039] The term "polypeptide" refers to any linear or branched peptide that contains multiple amino acids. Polypeptides include proteins or fragments or fusions thereof, provided that such proteins, fragments, or fusions retain useful biochemical or biological activity.

[0040] Fusion proteins typically include additional amino acid information that is not native to the protein to which the additional amino acid information is covalently linked. Such additional amino acid information can include tags that enable purification or identification of the fusion protein. Such additional amino acid information can include peptides that enable the fusion protein to be transported into cells and / or to specific intracellular locations. Examples of tags for these purposes include the following: AviTag, which is a peptide (GLNDIFEAQKIEWHE; SEQ ID NO: 21) that allows the enzyme BirA to biotinylate the protein such that the protein can be separated by streptavidin; Calmodulin-tag, which is a peptide (KRRWKKNFIAVSAANRFKKISSSGAL; SEQ ID NO: 22) that binds to the protein calmodulin; E-tag, which is a peptide (GAPVPYPDPLEPR; SEQ ID NO: 23) that is recognized by an antibody; FLAG-Tag, which is a peptide (DYKDDDDK; SEQ ID NO: 24) that is recognized by an antibody; HA-tag, which is a peptide (YPYDVPDYA; SEQ ID NO: 25) derived from hemagglutinin that is recognized by an antibody; His-tag, which typically consists of 5-10 histidine residues that bind to nickel or cobalt chelates (e.g., HHHHHH; SEQ ID NO: 26); Myc-tag, which is a peptide (EQKLISEEDL; SEQ ID NO: 27) derived from c-myc that is recognized by an antibody; NE-tag, which is a novel 18-amino acid synthetic peptide (TKENPRSNQEESYDDNES; SEQ ID NO: 28) that is recognized by a monoclonal IgG1 antibody and has utility in a wide range of applications including Western blot, enzyme-linked immunosorbent assay (ELISA), flow cytometry, immunocytochemistry, immunoprecipitation, and affinity purification of recombinant proteins; S-tag, which is a peptide (KETAAAKFERQHMDS; SEQ ID NO: 29) derived from ribonuclease A; SBP-tag, which is a peptide (MDEKTTGWRGGHVVEGLAGELEQLRARLEHHPQGQREP; SEQ ID NO: 30) that binds to streptavidin; Softag 1, which is designed for mammalian expression (SLAELLNAGLGGS;SEQ ID NO: 31); Softag 3, which is intended for prokaryotic expression (TQDPSRVG; SEQ ID NO: 32); Strep-tag, which is a peptide that binds to streptavidin or modified streptavidin (called Strepctactin) (Strep-tagII: WSHPQFEK; SEQ ID NO: 33); TC tag, which is a tetracysteine tag recognized by the FlAsH and ReAsH bisarsenical compounds, (CCPGCC; SEQ ID NO: 34); V5 tag, which is a peptide recognized by an antibody (GKPIPNPLLGLDST; SEQ ID NO: 35); VSV tag, which is a peptide recognized by an antibody (YTDIEMNRLGK; SEQ ID NO: 36); Xpress tag (DLYDDDDK; SEQ ID NO: 37); Isopep tag, which is a peptide that covalently binds to the pilin-C protein (TDKDMTITFTNKKDAE; SEQ ID NO: 38); SpyTag, which is a peptide that covalently binds to the SpyCatcher protein (AHIVMVDAYKPTK; SEQ ID NO: 39); SnoopTag, which is a peptide that covalently binds to the SnoopCatcher protein (KLGDIEFIKVNK; SEQ ID NO: 40); BCCP (biotin carboxyl carrier protein), a protein domain biotinylated by BirA to enable recognition by streptavidin; Glutathione-S-transferase-tag, which is a protein that binds to immobilized glutathione; Green fluorescent protein-tag, which is a self-fluorescent protein that can be bound by an antibody; HaloTag, which is a mutant bacterial haloalkane dehalogenase covalently linked to a reactive haloalkane substrate to allow attachment to a variety of substrates; Maltose binding protein-tag, which is a protein that binds to amylose agarose; Nus-tag; Thioredoxin-tag;and an Fc tag, which is derived from an immunoglobulin Fc domain that permits dimerization and solubility and can be used for purification on Protein A Sepharose. Nuclear localization signals (NLS), such as those obtained from SV40, permit a protein to be transported to the nucleus immediately upon entry into the cell. Given that the native Cas9 protein originates from bacteria and thus does not naturally contain an NLS motif, addition of one or more NLS motifs to a recombinant Cas9 protein is expected to show improved genome editing activity when used in eukaryotic cells where the target genomic DNA substrate is located in the nucleus. Those skilled in the art will appreciate these different fusion tag technologies, and how to make and use fusion proteins containing them.;

[0041] The term "tag-free" refers to a polypeptide that lacks additional amino acid information that is not native to the polypeptide.

[0042] "Ubiquitin" or "human ubiquitin (hμMan Ubiquitin)" refers to the wild-type ubiquitin polypeptide amino acid sequence.

[0043] The terms "i53", "i53 ubiquitin (i53 Ubiquitin)" or "ubiquitin i53 (Ubiquitin i53)" refer to a ubiquitin variant polypeptide amino acid sequence that lacks the carboxy-terminal diglycine of the wild-type ubiquitin polypeptide and includes several amino acid substitutions (Q2L, I44A, Q49S, Q62L, E64D, T66K, L69P and V70L) relative to the wild-type ubiquitin polypeptide. References 1. Chapman, J.R., Taylor, M.R. & Boulton, S.J. Playing the end game: DNA double-strand break repair pathway choice. Molecular cell 47, 497-510 (2012). 2.Iwabuchi,K.,Bartel,P.L.,Li,B.,Marraccino,R.&Fields,S.Two cellularproteins that bind to wild-type but not mutant p53.Proceedings of theNational Academy of Sciences ofthe United States ofAmerica 91,6098-6102(1994). 3.Escribano-Díaz,C.et al.A cell cycle-dependent regulatory circuitcomposed of 53BP1-RIF1 and BRCA1-CtIP controls DNA repair pathwaychoice.Molecular cell 49,872-883(2013). 4.Feng,L.,Fong,K.W.,Wang,J.,Wang,W.&Chen,J.RIF1 counteracts BRCA1-mediated end resection during DNA repair.The Journal of biologicalchemistry288,11135-11143(2013). 5.Xie,A.et al.Distinct roles of chromatin-associated proteins MDC1and 53BP1 in mammalian double-strand break repair.Molecular cell 28,1045-1057(2007). 6.Gaj,T.,Sirk,S.J.,Shui,S.L.&Liu,J.Genome-Editing Technologies:Principles and Applications.Cold Spring Harbor perspectivesin biology 8(2016). 7. Botuyan, M. V. et al. Structural basis for the methylation state-specific recognition of histone H4-K20 by 53BP1 and Crb2 in DNA repair. Cell 127, 1361-1373 (2006). 8. Charier, G. et al. The Tudor tandem of 53BP1: a new structural motif involved in DNA and RG-rich peptide binding. Structure (London, England: 1993) 12, 1551-1562 (2004). 9. Fradet-Turcotte, A. et al. 53BP1 is a reader of the DNA-damage-induced H2A Lys15 ubiquitin mark. Nature 499, 50-54 (2013). 10. Mattiroli, F. et al. RNF168 ubiquitinates K13-15 on H2A / H2AX to drive DNA damage signaling. Cell 150, 1182-1195 (2012). 11. Canny, M. D. et al. Inhibition of 53BP1 favors homology-dependent DNA repair and increases CRISPR-Cas9 genome-editing efficiency. Nature biotechnology 36, 95-102 (2018). 12. Dikic, I., Wakatsuki, S. & Walters, K. J. Ubiquitin-binding domains - from structures to functions. Nature reviews. Molecular cell biology 10, 659-671 (2009). 13. Davis, L. & Maizels, N. Two Distinct Pathways Support Gene Correction by Single-Stranded Donors at DNA Nicks. Cell reports 17, 1872 - 1881 (2016). 14. Verma, P. & Greenberg, R. A. Noncanonical views of homology-directed DNA repair. Genes & development 30, 1138 - 1154 (2016). 15. Roth, T. L. et al. Reprogramming human T cell function and specificity with non-viral genome targeting. Nature 559, 405 - 409 (2018). 16. Li, X. L. et al. Highly efficient genome editing via CRISPR-Cas9 in human pluripotent stem cells is achieved by transient BCL-XL overexpression. Nucleic Acids Res 46, 10195 - 10215 (2018). 17. Sun, L., Wu, J., Du, F., Chen, X. & Chen, Z. J. Cyclic GMP-AMP synthase is a cytosolic DNA sensor that activates the type I interferon pathway. Science 339, 786 - 791 (2013). 18. Dudek, A. M. & Porteus, M. H. Answered and Unanswered Questions in Early-Stage Viral Vector Transduction Biology and Innate Primary Cell Toxicity for Ex-Vivo Gene Editing. Front Immunol 12, 660302 (2021). 19. Yu, K. R., Natanson, H. & Dunbar, C. E. Gene Editing of Human Hematopoietic Stem and Progenitor Cells: Promise and Potential Hurdles. Hum Gene Ther 27, 729 - 740 (2016). 20. Eyquem, J. et al. Targeting a CAR to the TRAC locus with CRISPR / Cas9 enhances tumour rejection. Nature 543, 113 - 117 (2017). 21. Martin, R. M. et al. Highly Efficient and Marker - free Genome Editing of Human Pluripotent Stem Cells by CRISPR - Cas9 RNP and AAV6 Donor - Mediated Homologous Recombination. Cell Stem Cell 24, 821 - 828 e825 (2019). 22. Grimm, D. et al. In vitro and in vivo gene therapy vector evolution via multispecies interbreeding and retargeting of adeno - associated viruses. J Virol 82, 5887 - 5911 (2008). 23. Riesenberg, S. & Maricic, T. Targeting repair pathways with small molecules increases precise genome editing in pluripotent stem cells. Nat Commun 9, 2164 (2018). 24. Brault, J. et al. CRISPR-targeted MAGT1 insertion restores XMEN patient hematopoietic stem cells and lymphocytes. Blood 138, 2768-2780 (2021). 25. De Ravin, S. S. et al. Enhanced homology-directed repair for highly efficient gene editing in hematopoietic stem / progenitor cells. Blood 137, 2598-2608 (2021). 26. Sweeney, C. L. et al. Correction of X-CGD patient HSPCs by targeted CYBB cDNA insertion using CRISPR / Cas9 with 53BP1 inhibition for enhanced homology-directed repair. Gene Ther 28, 373-390 (2021). 27. Wienert, B. et al. Timed inhibition of CDC7 increases CRISPR-Cas9 mediated templated repair. Nat Commun 11, 2109 (2020).

[0044] All references cited herein, including publications, patent applications, and patents, are incorporated herein by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in full herein.

[0045] Preferred embodiments of the invention have been described herein, including the best mode known to the inventors of practicing the invention. Variations of these preferred embodiments will be apparent to those of ordinary skill in the art upon reading the foregoing description.

[0046] The inventors expect that those skilled in the art will adopt these variations as needed, and the inventors do not intend to limit the present invention to the specific content described herein. Accordingly, the present invention includes all modifications and equivalent schemes within the scope permitted by applicable law, which modifications and equivalent schemes are covered by the technical scope described in the appended claims. In addition, unless otherwise expressly stated herein or clearly contrary to the context, all possible combinations of variations of the above-described components also fall within the scope of the present invention.

Claims

1. An isolated nucleic acid sequence, wherein, it encodes a tagless CM1 polypeptide.

2. The isolated nucleic acid sequence according to claim 1, wherein, the nucleic acid sequence of the ubiquitin polypeptide variant encodes a CM1tf polypeptide.

3. An isolated tagless CM1 polypeptide.

4. The isolated tagless CM1 polypeptide according to claim 3, wherein, the tagless CM1 polypeptide comprises a CM1tf polypeptide.

5. A method for enhancing homologous directed repair (HDR) in recipient cells, characterized by comprising: introducing a nucleic acid donor template and an isolated tagless CM1 polypeptide into the recipient cells.

6. The method according to claim 5, wherein, an adeno-associated vector is used to introduce the nucleic acid donor template.

7. The method according to claim 5, wherein, the isolated tagless CM1 polypeptide comprises a CM1tf polypeptide.

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