Ubiquitin mutants with high affinity for binding 53BP1 reduce the amount of AAV required to achieve high HDR rates

Abstract

The present invention relates to tag-free CM1 polypeptides and methods for improving homology-directed repair (HDR) in recipient cells using the same. A preferred donor template delivery vehicle includes an adenovirus-associated vector for delivering the donor template, and a preferred tag-free CM1 polypeptide, designated CM1tf. An isolated nucleic acid encoding the tag-free CM1 polypeptide is also provided.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority under 35 U.S.C. § 119 to U.S. Provisional Patent Application No. 63 / 399,452, filed August 19, 2022, entitled "UBIQUITIN VARIANT WITH HIGH AFFINITY FOR BINDING 53BP1 REDUCES THE AMOUNT OF AAV NEEDED THE HIGH RATES OF HDR," the contents of which are incorporated herein by reference in their entirety.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted via the Patent Center in XML format, and is incorporated herein by reference in its entirety. Said XML copy, created on August 15, 2023, is named IDT01-023-PCT_ST26.xml.

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

[0004] Double-strand breaks (DSBs) are repaired primarily by two mechanisms: nonhomologous end joining (NHEJ), in which the broken ends are often imprecisely rejoined, or homologous recombination repair (HDR), which typically involves using a sister chromatid or homologous chromosome as a repair template. HDR is promoted by the presence of sister chromatids, and cellular mechanisms exist that bias toward NHEJ repair during the G1 phase of the cell cycle [1]. A key determinant of repair pathway selection is 53BP1. 53BP1 was first described as a binding partner of the tumor suppressor gene p53 and later shown to be a critical protein in NHEJ [2]. 53BP1 rapidly accumulates at double-strand break sites. In G1, 53BP1 recruits RIF1, inhibiting end resection [3, 4]. End resection is required for HDR and inhibits NHEJ, making it a critical step in repair pathway selection [1]. By inhibiting end resection, 53BP1 biases repair toward NEHJ, and consequently, loss of 53BP1 leads to increased HDR [5]. Targeted nucleases can be introduced into cells along with DNA repair templates with homology to the target cleavage site, facilitating precise genome editing via HDR [6]. Therefore, potent inhibitors of 53BP1 are useful for precise genome editing.

[0005] Recruitment of 53BP1 to DSB sites depends on both H4K20 methylation and H2AK15 ubiquitination. 53BP1 contains tandem Tudor domains, which have been shown to specifically bind monomethylated and demethylated H4K20, suggesting that H4K20 methylation is important for 53BP1 recruitment to double-strand breaks [7,8]. Introduction of a mutation, D1521R, that inhibits Tudor domain activity impairs the ability of 53BP1 to form ionizing radiation-induced forsythesis [9]. The minimal forsythesis-forming region of 53BP1 consists of the Tudor domain flanked by the N-terminal oligomerization region and a C-terminal extension. Notably, 53BP1 accumulation at DSBs requires the E3 ubiquitin ligase RNF168, which mediates the ubiquitination of H2AK13 and H2AK15

[10] . The C-terminal extension was shown to contain a ubiquitination-dependent induction motif (UDR) that specifically binds H2AK15ub and is required for 53BP1 recruitment to DSB sites [9].

[0006] Due to the affinity of 53BP1 for ubiquitinated H2A, Canny et al. recently conducted a screen for ubiquitin mutants that interact with 53BP1 and discovered and engineered a ubiquitin mutant that selectively binds to 53BP1, which they named i53 (an inhibitor of 53BP1)

[11] . The top five hits from the ubiquitin mutant screen were A10, A11, C08, G08, and H04, with G08 showing the highest affinity. Contrary to expectations, the interaction between 53BP1 and G08 did not require the UDR, and the interaction was shown to occur between G08 and the 53BP1 Tudor domain. 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 H2AKc15ub(I44A)-associated mutation inhibits 53BP1 interaction with ubiquitinated H2A but does not inhibit i53's ability to enhance HDR, consistent with i53 enhancing HDR through interaction with the 53BP1 Tudor domain rather than the UDR domain [9,11]. Furthermore, i53 was obtained by modifying G08 by removing the C-terminal diglycine motif. Introduction of i53 into cells inhibited ionizing radiation-induced 53BP1 forsythesis formation, whereas the 53BP1-binding-deficient i53 mutant DM (i53P69L+L70V) did not. Introduction of i53 via plasmid delivery, adeno-associated virus-mediated gene delivery, or mRNA delivery was all shown to improve HDR rates. The introduction of i53 has improved HDR rates using both double-stranded and single-stranded DNA donors, which have been shown to use different HDR mechanisms [11,13,14].

[0007] In a previous application (U.S. Provisional Patent Application No. 63 / 321,384, entitled "UBIQUITIN VARIANTS WITH IMPROVED AFFINITY FOR 53BP1" (Attorney Docket No. IDT01-021-PRO3), filed March 18, 2022, to Vakulskas et al., the inventors described a ubiquitin variant containing nine amino acid substitutions compared to i53, which the inventors call CM1, with dramatically improved (50-100-fold) binding affinity for 53BP1. The inventors demonstrated that this ubiquitin variant was able to enhance HDR to a greater extent than i53 at lower doses when a short ssDNA Alt-R donor oligo and a long dsDNA Alt-R HDR donor block were used as donor templates.

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

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

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

[0011] In a third aspect, a method for improving homology-directed repair (HDR) in a recipient cell is provided, the method comprising introducing into the recipient cell a nucleic acid donor template and an isolated tag-free CM1 polypeptide. [Brief explanation of the drawings]

[0012] [Figure 1A]FIG. 1A shows an example heatmap showing the rate of HDR in HPRT1 in HEK293 cells with and without 25 μM CM1tf. [Figure 1B] Figure 1B shows an example heatmap showing the percentage of HDR in SERPINC1 in HEK293 cells with and without 25 μM CM1tf. [Figure 1C] Figure 1C shows an example heat map showing the percentage of HDR in SERPINC1 in K562 cells with and without 50 μM CM1tf. Figures 1A-C show that when donor templates are delivered using AAV vectors, the ubiquitin mutant CM1tf, when delivered with Cas9 RNP, promotes HDR. The heat map shows the percentage of HDR measured by EcoR1 cleavage assay with and without CM1tf. Cas9 RNP (2 μM) was delivered to cells by Lonza nucleofection using 4 μM Alt-R Cas9 electroporation enhancer, with and without CM1tf. An AAV donor containing an EcoR1 cleavage site insert with 500 bp homology arms was added to cells at various multiplicities of infection (MOI) 24 h after RNP delivery. Editing is shown as the average percent HDR ± standard deviation for three biological replicates. [Figure 2A] FIG. 2A shows an example heatmap showing the percentage of HDR in HPRT1 in HEK293 cells with no enhancer, CM1tf, Alt-R HDR enhancer V2 (“V2”), or CM1tf+V2. [Figure 2B]Figure 2B shows an example heat map showing the rate of HDR in SERPINC1 in HEK293 cells using no enhancer, CM1tf, V2, or CM1tf+V2. In Figure 2A-B, HDR is further enhanced when the ubiquitin mutant CM1tf is used in combination with the Alt-R HDR enhancer V2. The heat map shows the rate of HDR measured by EcoR1 cleavage assay using CM1tf, the Alt-R HDR enhancer V2 (V2), or both. Cas9 RNP was 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, with or without CM1tf. An AAV donor containing an EcoR1 cleavage site insert with 500 bp homology arms was added to cells at a range of MOIs 24 h after RNP delivery. The V2 enhancer was added to the medium at a final concentration of 1 μM for 24 h after nucleofection. Editing is displayed as the mean percent HDR ± standard deviation from three biological replicates. [Figure 3] Figure 3 summarizes an example heat map showing that the IDT ubiquitin variant CM1tf promotes HDR when used with AAV donors with different homology arm lengths. The heat map shows the percentage of HDR at STAT3 as measured by EcoR1 cleavage assay with or without CM1tf. Cas9 RNP was co-delivered to cells by Lonza nucleofection using 2 μM Cas9 RNP, 4 μM Alt-R Cas9 electroporation enhancer, and 0 or 25 μM CM1tf, with or without CM1tf. AAV donors with 500 base pairs (bp), 300 bp, or 100 bp homology arms were added to cells at a range of MOIs 24 h after RNP delivery. Editing is displayed as the average percent HDR ± standard deviation obtained from three replicates (single nucleofection, separate AAV delivery, and downstream processing). [Figure 4]Figure 4 demonstrates that CM1tf outperforms i53 in its ability to promote HDR using AAV donors. The graph shows the percentage of HDR measured by EcoR1 cleavage assay using CM1tf. Cas9 RNP was co-delivered into cells by Lonza nucleofection using 2 μM Cas9 RNP, 4 μM Alt-R Cas9 electroporation enhancer, and CM1tf at concentrations ranging from 200 μM to 6.25 μM. An AAV donor containing an EcoR1 cleavage site insert with 500 bp homology arms was added to cells at an MOI of 20,000 24 h after RNP delivery. Editing is displayed as the average percent HDR, and the bars indicate the standard deviation from three biological replicates. DETAILED DESCRIPTION OF THE INVENTION

[0013] The present invention identifies the extent to which CM1 can improve HDR when using adeno-associated virus (AAV) for template delivery into cells. Consideration of the type of donor to use is particularly important when working with primary cells, as different donors, including plasmid DNA, linear dsDNA, and ssODN, cause different amounts of cytotoxicity, which can significantly affect overall cell yield [15-17]. The use of AAV is often the preferred method for delivering DNA templates into cells due to its ability to introduce long 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 achieve higher editing levels, but at the expense of increased toxicity, which results in lower cell yields. AAV production is also time-consuming and expensive. Therefore, products that can reduce the amount of AAV required to achieve high levels of editing could improve cell yields and HDR rates while reducing production costs.

[0014] Purpose In a first aspect, there is provided an isolated nucleic acid sequence encoding a tag-free CM1 polypeptide. In a first aspect, the isolated nucleic acid sequence encodes a CM1tf polypeptide.

[0015] In a second aspect, an isolated protein sequence of a tag-free CM1 polypeptide is provided. In a first aspect, the tag-free CM1 polypeptide comprises a CM1tf polypeptide.

[0016] In a third aspect, a method for improving homology-directed repair (HDR) in a recipient cell is provided. The method comprises introducing a nucleic acid donor template and an isolated tag-free CM1 polypeptide into the recipient cell. In a first aspect, the nucleic acid donor template comprises an adenovirus-associated vector. In a second aspect, the isolated tag-free CM1 polypeptide comprises a CM1tf polypeptide. [Example]

[0017] Example 1 Tag-free CM1 (CM1tf) improves HDR when AAV is used for repair template delivery. We developed a tag-free version of CM1 (CM1tf) and tested its ability to enhance HDR in cell lines using AAV donors (see Table 1). AAV-DJ, a synthetic AAV serotype most closely related to AAV-2, a type 2 / type 8 / type 9 chimera, was selected for testing due to its high in vitro transduction efficiency across a wide range of cell types

[22] . The dose of CM1tf was kept constant, while a range of MOIs was used for the AAV donor. The AAV donor was constructed with a 6-base-pair insert consisting of an EcoR1 cleavage site (GAATTC) flanked by 500-base-pair homology arms matching the genomic sequence on either side of the target cleavage site. Briefly, Cas9 V3 protein (IDT) and Alt-R sgRNA (IDT) were mixed at a 1:1.2 ratio and incubated for 10 minutes. Alt-R Cas9 electroporation enhancer (EE) (IDT), 1X PBS (Gibco), and CM1tf diluted in 1X PBS were then added. HEK293 or K562 cells were washed and resuspended in Lonza SF Nucleofection Buffer and added to the RNP+DNA mixture to achieve final concentrations of 2 μM Cas9 RNP, 4 μM EE, and 25 μM (HEK293 cells) or 50 μM (K562 cells). Cells and RNP+DNA were electroporated using the Lonza Nucleofector System, program DS-150 (HEK293 cells) or FF-120 (K562 cells). Cells were then seeded at 20,000 cells / well in 96-well plates, and AAV was added to wells at an MOI ranging from 0 (no virus) to 160,000. The MOI was calculated as viral genomes (vg) per cell using qPCR. Cells were harvested, plated in serum-free medium, and AAV was added. Serum-containing medium was then added 4 hours later. Genomic DNA was isolated 48 hours after RNP delivery using QuickExtract (Lucigen). The results are shown in Figure 1.The use of CM1tf dramatically enhanced HDR rates, requiring approximately fourfold fewer AAVs to achieve the same level of editing in HEK293 cells as without the CM1tf HDR enhancer.

[0018] Example 2 CM1tf can be used in combination with Alt-R HDR enhancer V2 to further promote HDR when using AAV donors. To optimize HDR, it is often desirable to use a combination of HDR enhancers that act by different mechanisms to further promote HDR rates beyond those achievable by any single enhancer alone

[23] . Because CM1tf acts by facilitating end resection and thus promoting HDR, it may be possible to further promote HDR by combining it with the IDT Alt-R HDR enhancer V2, an inhibitor of NHEJ. To test this, Cas9 RNP with or without CM1tf was delivered to HEK293 cells as described in Example 1. The cells were then plated in media with or without 1 μM V2 enhancer. AAV was then added to each well at a range of MOIs, as previously described. The results of this study are shown in Figure 2.

[0019] Although the use of the CM1tf or V2 enhancer resulted in roughly equivalent improvements in HDR rates, their combination was able to further improve HDR beyond what either enhancer could achieve individually.

[0020] Example 3 CM1tf promotes HDR when used with donor DNA templates packaged into AAVs with various homology arm lengths. To test the compatibility of CM1tf with AAV donors with different homology arm lengths and to identify the optimal HA length for short inserts, we tested the effect of CM1tf in combination with AAV donors containing 100, 300, or 500 bp homology arms. RNP and CM1tf were delivered to HEk293 cells as described in Example 1. The results are shown in Figure 3. The 300 bp homology arm donor resulted in the highest HDR rate without an enhancer, and this trend was maintained by the addition of CM1tf. The use of CM1tf promoted HDR rates to similar levels regardless of homology arm length.

[0021] Example 4 CM1tf outperforms i53 in its ability to promote HDR in AAV donors. Achieving the best possible HDR rates using an AAV donor may require a different optimal dose of CM1tf compared to using an ssDNA donor. Furthermore, the nature of the donor may affect the benefits of using CM1tf compared to i53. To test this, we co-delivered Cas9 RNP with either CM1tf or i53 into HEK293 cells at a fixed dose, as described in Example 1, and then added AAV to the cells at a set MOI of 20,000 for 24 hours. The results are shown in Figure 4. Previous testing of CM1tf using an ssDNA donor showed that CM1tf exhibited similar HDR efficacy across a dose range of 6.25–50 μM, while the optimal concentration of i53 was approximately 100–150 μM, with reduced efficacy at 200 μM. Here, we show that while CM1tf continues to outperform i53 with AAV donors, using higher doses of CM1tf up to 200 μM provided a slightly greater effect on HDR with increasing dose. This was even more pronounced with i53, where a significant improvement in performance was observed from 100 μM to 200 μM, a trend not observed with ssDNA donors. These results indicate that the nature of the donor used is an important consideration when dosing CM1tf. However, the HDR benefit of CM1tf was much less affected by dose compared to i53, and overall higher editing levels were achieved with CM1tf compared to i53.

[0022] Example 5 Sequence.

[0023] [Table 1]

[0024] [Table 2]

[0025] [Table 3]

[0026] [Table 4]

[0027] [Table 5]

[0028] [Table 6]

[0029] [Table 7]

[0030] definition To aid in the understanding of this invention, several terms are defined below.

[0031] Use of the terms "a," "an," and "the" and similar referents in the context of describing the present invention (particularly in the context of the appended claims) is to be construed as including both the singular and the plural unless otherwise indicated herein or clearly contradicted by context. The terms "comprising," "having," "including," and "containing" are to be construed as open-ended terms (i.e., meaning "including, but not limited to") unless otherwise indicated. The recitation of numerical ranges herein is intended merely to serve as a shorthand method for individually referencing each value falling within the range, unless otherwise indicated herein, and each value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or clearly contradicted by context. Any examples or exemplary language used herein (e.g., "such as") is intended merely to better illustrate the invention and does not pose a limitation on the scope of the invention, unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0032] The term "CRISPR" refers to the Clustered Regularly Interspaced Short Palindromic Repeat bacterial adaptive immune system.

[0033] The terms "Cas" and "Cas endonuclease" generally refer to a CRISPR-associated endonuclease.

[0034] The term "Cas protein" generally refers to the wild-type protein (including variants thereof) of a CRISPR-associated endonuclease (including the interchangeable terms Cas and Cas endonuclease).

[0035] The term "Cas nucleic acid" generally refers to a CRISPR-associated endonuclease nucleic acid, including a guide RNA, sgRNA, crRNA, or tracrRNA.

[0036] The terms "Cas9" and "CRISPR / Cas9" refer to the CRISPR-associated bacterial adaptive immune system of Steptococcus pyogenes. Examples of this system are disclosed in U.S. Patent Application Nos. 15 / 729,491 and 15 / 964,041, filed October 10, 2017 and April 26, 2018, respectively (Attorney Docket Nos. IDT01-009-US and IDT01-009-US-CIP, respectively), the contents of which are incorporated herein by reference.

[0037] The term "mutant" refers to a protein (e.g., ubiquitin) that is modified and includes at least one amino acid substitution, addition of an amino acid (e.g., an affinity tag or nuclear localization signal), or combination thereof, relative to a reference, typically wild-type, protein amino acid sequence.

[0038] The term "polypeptide" refers to any linear or branched peptide containing two or more amino acids. Polypeptides include proteins or fragments thereof or fusions thereof, provided that such proteins, fragments, or fusions retain useful biochemical or biological activity.

[0039] Fusion proteins typically contain additional amino acid information not native to the protein to which they are covalently attached. Such additional amino acid information may include a tag that allows for purification or identification of the fusion protein. Such additional amino acid information may include a peptide that allows for transport of the fusion protein into cells and / or to a specific location within the cell. Examples of tags for these purposes include the following: AviTag (GLNDIFEAQKIEWHE; SEQ ID NO: 21), a peptide that allows biotinylation by the enzyme BirA, so that the protein can be isolated with streptavidin; Calmodulin-tag (KRRWKKNFIAVSAANRFKKISSSGAL; SEQ ID NO: 22), a peptide that is bound by the protein calmodulin; E-tag (GAPVPYPDPLEPR; SEQ ID NO: 23), a peptide that is recognized by an antibody; FLAG-tag (DYKDDDDK; SEQ ID NO: 24), a peptide that is recognized by an antibody; HA-tag (YPYDVPDYA; SEQ ID NO: 25), a peptide derived from hemagglutinin that is recognized by an antibody; His-tag (e.g., HHHHHH), which is typically 5-10 histidines attached by a nickel or cobalt chelate. ; SEQ ID NO: 26); Myc-tag (EQKLISEEDL; SEQ ID NO: 27), a peptide derived from c-myc that is recognized by antibodies; NE-tag, a novel 18-amino acid synthetic peptide (TKENPRSNQEESYDDNES; SEQ ID NO: 28) that is recognized by a monoclonal IgG1 antibody and is useful in a wide range of applications including Western blotting, ELISA, flow cytometry, immunocytochemistry, immunoprecipitation, and affinity purification of recombinant proteins; S-tag (KETAAAKFERQHMDS; SEQ ID NO: 29), a peptide derived from ribonuclease A; SBP-tag (MDEKTTGWRGGHVVEGLAGELEQLRARLEHHPQGQREP; SEQ ID NO: 30)), a peptide that binds to streptavidin; Soft tag 1 (SLAELLNAGLGGS; SEQ ID NO: 31), intended for mammalian expression;Soft tag 3 (TQDPSRVG; SEQ ID NO: 32), intended for prokaryotic expression; Strep-tag (Strep-tag II: WSHPQFEK; SEQ ID NO: 33), a peptide that binds to streptavidin or a modified streptavidin called streptactin; TC-tag (CCPGCC; SEQ ID NO: 34), a tetracysteine ​​tag recognized by the FlAsH and ReAsH viral helical compounds; V5-tag (GKPIPNPLLGLDST; SEQ ID NO: 35), a peptide recognized by an antibody; VSV-tag (YTDIEMNRLGK; SEQ ID NO: 36), a peptide recognized by an antibody; Xpress-tag (DLYDDDDK; SEQ ID NO: 37); Isopep-tag (TDKDMTITFTNKKDAE; SEQ ID NO: 38), a peptide that covalently binds to the pyrin-C protein; SpyTag (AHIVMVDAYKPTK; SEQ ID NO: 39), a peptide that covalently binds to the SpyCatcher protein; and SnoopCatcher SnoopTag (KLGDIEFIKVNK; SEQ ID NO: 40), a peptide that covalently binds to proteins; BCCP (biotin carboxyl carrier protein), a protein domain that is biotinylated by BirA to enable recognition by streptavidin; glutathione-S-transferase-tag, a protein that binds to immobilized glutathione; green fluorescent protein-tag, a protein that is spontaneously fluorescent and can be bound by antibodies; HaloTag, a mutant bacterial haloalkane dehalogenase that covalently binds reactive haloalkane substrates, allowing binding to a wide variety of substrates; maltose-binding protein-tag, a protein that binds to amylose-agarose; Nus-tag; thioredoxin-tag;and an Fc-tag derived from the immunoglobulin Fc domain, which allows dimerization and solubilization and can be used for purification on Protein A Sepharose. A nuclear localization signal (NLS), such as that obtained from SV40, allows the protein to be transported to the nucleus immediately upon entry into the cell. Considering that the native Cas9 protein is of bacterial origin and therefore does not naturally contain an NLS motif, adding one or more NLS motifs to a recombinant Cas9 protein is expected to exhibit improved genome editing activity when used in eukaryotic cells where the target genomic DNA substrate is present in the nucleus. Those skilled in the art will understand these various fusion tag technologies and how to make and use fusion proteins containing them.

[0040] The term "tag-free," as the term modifies a polypeptide, refers to a polypeptide that lacks additional amino acid information not native to the polypeptide.

[0041] The term "ubiquitin" or "human ubiquitin" refers to the wild-type ubiquitin polypeptide amino acid sequence.

[0042] The terms "i53," "i53 ubiquitin," or "ubiquitin i53" refer to a ubiquitin mutant polypeptide amino acid sequence that lacks the carboxy-terminal diglycine of the wild-type ubiquitin polypeptide and contains several amino acid substitutions (Q2L, I44A, Q49S, Q62L, E64D, T66K, L69P, and V70L) compared to the wild-type ubiquitin polypeptide. [Prior art documents] [Non-patent literature]

[0043] [Non-Patent Document 1] Chapman , JR , Taylor , MR & Boulton , SJ Playing the end game: DNA double-strand break repair pathway choice . Molecular cell 47, 497–510 (2012).

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

[0045] Preferred embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of these preferred embodiments may become apparent to those skilled in the art upon reading the foregoing description.

[0046] The inventors expect that skilled artisans will adapt such variations as appropriate, and intend that the invention may be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or clearly contradicted by context.

Claims

1. An isolated nucleic acid sequence encoding a tag-free CM1 polypeptide.

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

3. Isolated tag-free CM1 polypeptide.

4. The isolated tag-free CM1 polypeptide of claim 3, wherein the tag-free CM1 polypeptide comprises a CM1tf polypeptide.

5. 1. A method for improving homology directed repair (HDR) in a recipient cell, comprising: Introducing the nucleic acid donor template and the isolated tag-free CM1 polypeptide into recipient cells A method comprising:

6. 6. The method of claim 5, wherein the nucleic acid donor template is introduced using an adenovirus-associated vector.

7. The method of claim 5, wherein the isolated tag-free CM1 polypeptide comprises a CM1tf polypeptide.

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