Using inhibitors to increase the efficiency of CRISPR / Cas insertion

Abstract

The present disclosure provides a method for inserting a polynucleotide of interest into the genome of a eukaryotic cell, the method comprising improving the efficiency of CRISPR / Cas-mediated polynucleotide insertion by the addition of an inhibitor of the microhomology-mediated end joining (MMEJ) pathway to the eukaryotic cell. The present disclosure further provides compositions for inserting a polynucleotide of interest into the genome of a eukaryotic cell, and kits for inserting a gene of interest into the genome of a eukaryotic cell.

Description

[Technical field]

[0001] The present disclosure provides a method for inserting a polynucleotide of interest into the genome of a eukaryotic cell, the method comprising improving the efficiency of CRISPR / Cas-mediated polynucleotide insertion by the addition of an inhibitor of the microhomology-mediated end joining (MMEJ) pathway to the eukaryotic cell. The present disclosure further provides compositions for inserting a polynucleotide of interest into the genome of a eukaryotic cell, and kits for inserting a gene of interest into the genome of a eukaryotic cell. [Background technology]

[0002] It has been a long-standing goal to develop cost-effective and reliable methods for making precisely targeted changes to the genome of living cells. Genome editing has the potential to provide a means to eliminate genes responsible for specific disorders (i.e., gene "knockout"), or to manipulate or insert genes to correct genetic defects or enhance biological processes by gene "knock-in". Genome editing can be applied to the treatment of many disorders, including the treatment of genetic disorders, hematological disorders, and cancer, as well as in immunotherapy methods.

[0003] Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) systems are prokaryotic immune systems first discovered by Ishino in Escherichia coli (E. coli) (Non-Patent Document 1). Prokaryotic immune systems provide immunity against viruses and plasmids by sequence-specifically targeting the nucleic acids of viruses and plasmids. See also (Non-Patent Document 2).

[0004] Since its initial discovery, multiple groups have conducted extensive research into the potential applications of the CRISPR system in genetic engineering, including gene editing (Non-Patent Document 3, Non-Patent Document 4, and Non-Patent Document 5). The CRISPR-Cas9 gene editing system has been successfully used in a wide range of organisms and cell lines. In addition to genome editing, the CRISPR system has multiple other applications, including, inter alia, regulating gene expression, constructing genetic circuits, and functional genomics (reviewed in Non-Patent Document 6).

[0005] Cas9 endonuclease generates a double-stranded DNA break upstream of the protospacer adjacent motif (PAM) of the target sequence. The target sequence can then be removed or a sequence of interest can be inserted into the target sequence using the cell's endogenous repair pathways. Endogenous DNA repair pathways include the non-homologous end joining (NHEJ) pathway, the microhomology-mediated end joining (MMEJ) pathway, and the homology-directed repair (HDR) pathway. NHEJ, MMEJ, and HDR pathways repair double-stranded DNA breaks, but repair of such double-stranded DNA breaks can result in insertions or deletions at the double-stranded break site. In NHEJ, a homologous template is not required to repair the break in DNA. NHEJ repair can be error-prone, but errors are reduced when the DNA break contains a compatible overhang. NHEJ and MMEJ are DNA repair pathways that are mechanistically distinct due to the different subsets of DNA repair enzymes involved in each of them. Unlike NHEJ, which can be sometimes accurate and sometimes error-prone, MMEJ is always error-prone and results in both deletions and insertions at the site being repaired. MMEJ-associated deletions result from microhomology (2-10 base pairs) on both sides of the double-stranded break. In contrast, HDR requires a homologous template to direct the repair, but HDR repair is typically high fidelity and low error-prone. Thus, HDR-driven repair of double-stranded DNA breaks is preferred over NHEJ or MMEJ-mediated repair. However, in many cell types, HDR is limited by the activity of NHEJ at all stages of the cell cycle, and HDR is primarily utilized in the S phase of cell proliferation (Non-Patent Document 7).

Prior Art Documents

Non-Patent Documents

[0006]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

Non-Patent Document 4

Non-Patent Document 5

Non-Patent Document 6

Non-Patent Document 7

Summary of the Invention

Means for Solving the Problems

[0007] In some embodiments, the present disclosure relates to a method for increasing the efficiency of CRISPR / Cas-mediated gene insertion. In some embodiments, the method includes inserting a polynucleotide of interest into the genome of a eukaryotic cell, the method includes the steps of (a) adding an inhibitor of the MMEJ pathway to a composition comprising the eukaryotic cell, (b) adding a Cas effector protein to the composition, and (c) adding a polynucleotide of interest to the composition, wherein the polynucleotide of interest is inserted into the genome of the eukaryotic cell by homology-directed repair (HDR) or single-stranded template repair (SSTR).

[0008] In some embodiments, step (a) of the method further comprises adding an inhibitor of the non-homologous end joining (NHEJ) pathway.

[0009] In some embodiments, the method further comprises (d) adding to the composition a polynucleotide comprising an RNA guide sequence, a Cas binding region, a DNA template sequence, or a combination thereof.

[0010] In some embodiments, the Cas effector protein and the polynucleotide of (d) are added in the form of a ribonucleoprotein (RNP).

[0011] In some embodiments, the Cas effector protein is added in (b) by adding a Cas polynucleotide encoding the Cas effector protein.

[0012] In some embodiments, the polynucleotide of interest, the polynucleotide of step (d), and the Cas polynucleotide are encoded on a single vector. In some embodiments, the polynucleotide of interest is added as DNA. In some embodiments, the polynucleotide of step (d) is added as DNA. In some embodiments, the polynucleotide of step (d) is added as RNA. In some embodiments, the Cas effector polynucleotide is added as DNA. In some embodiments, the Cas polynucleotide is added as RNA. In some embodiments, the Cas polynucleotide is added as mRNA.

[0013] In some embodiments, the vector is a viral vector, hi some embodiments, the viral vector is a retrovirus, lentivirus, adenovirus, or adeno-associated virus (AAV).

[0014] In some embodiments, the Cas effector protein, the polynucleotide of interest, and the polynucleotide of (d) are added to the eukaryotic cell by microinjection, electroporation, or by lipid nanoparticles, liposomes, exosomes, gold nanoparticles, or DNA nanoclears.

[0015] In some embodiments, the vector is added to the composition comprising the eukaryotic cell by transfecting the eukaryotic cell.

[0016] In some embodiments, the Cas effector protein is a Cas9 nuclease, a Cas12a nuclease, or a Cas12f nuclease. In some embodiments, the Cas effector protein is a Cas9 nuclease. In some embodiments, the Cas9 nuclease is a Cas9 nuclease fused to a reverse transcriptase, a Cas9 nuclease fused to a DNA polymerase, a Cas9 nuclease fused to DN1S, a Cas9 nickase, a Cas9 fused to a geminin degron domain, or a Cas9 nuclease fused to CTIP.

[0017] In some embodiments, the polynucleotide of interest is delivered by a vector. In some embodiments, the vector is a viral vector. In some embodiments, the viral vector is a retrovirus, a lentivirus, an adenovirus, or an adeno-associated virus.

[0018] In some embodiments, the polynucleotide of interest comprises a gene of interest. In some embodiments, the polynucleotide of interest is 1-50 base pairs in length. In some embodiments, the polynucleotide of interest is 1-10 base pairs in length. In some embodiments, the polynucleotide of interest is 50-5000 base pairs in length.

[0019] In some embodiments, the polynucleotide of interest is single stranded. In some embodiments, the polynucleotide of interest is double stranded. In some embodiments, the polynucleotide of interest is a hybrid polynucleotide comprising a single stranded region and a double stranded region. In some embodiments, the hybrid polynucleotide comprises double stranded sequences at the 5' and 3' ends, and an internal single stranded sequence. In some embodiments, the polynucleotide of interest is double stranded with blunt ends. In some embodiments, the polynucleotide of interest is double stranded with a 3' overhang. In some embodiments, the polynucleotide of interest is double stranded with a 5' overhang. In some embodiments, the polynucleotide of interest is a circular polynucleotide.

[0020] In some embodiments, the polynucleotide of interest comprises a chemical modification that enhances the activity, distribution, or uptake of the polynucleotide.

[0021] In some embodiments, the inhibitor of the MMEJ pathway is an inhibitor of POLQ / DNA polymerase θ. In some embodiments, the inhibitor of POLQ is PolQ1, PolQ2, PolQ3, PolQ4, PolQ5, PolQ6 PolQ7, or a combination thereof. In some embodiments, the inhibitor of POLQ is a peptide.

[0022] In some embodiments, the inhibitor of the MMEJ pathway in a composition comprising a eukaryotic cell is from about 0.01 μM to about 1 mM, from about 0.1 μM to about 1 mM, from about 0.1 μM to about 0.5 mM, from about 0.1 μM to about 100 μM, or from about 1 μM to about 50 μM.

[0023] In some embodiments, the inhibitor of the NHEJ pathway is an inhibitor of DNA-dependent protein kinase (DNA-PK). In some embodiments, the inhibitor of DNA-PK is M3814, M9831 / VX984, Nu7441, KU0060648, AZD7648, or a combination thereof. In some embodiments, the inhibitor of DNA-PK is AZD7648. In some embodiments, the inhibitor of DNA-PK is a peptide.

[0024] In some embodiments, the inhibitor of the NHEJ pathway in the composition comprising a eukaryotic cell is from about 0.01 μM to about 1 mM, from about 0.1 μM to about 1 mM, from about 0.1 μM to about 0.5 mM, from about 0.1 μM to about 100 μM, or from about 1 μM to about 50 μM.

[0025] In some embodiments, the inhibitor of the MMEJ pathway is added to the composition comprising the eukaryotic cells 0 minutes to about 48 hours, 0 minutes to about 24 hours, 0 minutes to about 12 hours, 0 minutes to about 6 hours, or 0 minutes to about 1 hour before the Cas effector protein is added to the composition. In some embodiments, the inhibitor of the MMEJ pathway is added to the composition comprising the eukaryotic cells 0 minutes to about 1 hour after the Cas effector protein is added to the composition comprising the eukaryotic cells.

[0026] In some embodiments, the inhibitor of the NHEJ pathway is added to the composition comprising the eukaryotic cells 0 minutes to about 48 hours, 0 minutes to about 24 hours, 0 minutes to about 12 hours, 0 minutes to about 6 hours, or 0 minutes to about 1 hour before the Cas effector protein is added to the composition. In some embodiments, the inhibitor of the NHEJ pathway is added to the composition comprising the eukaryotic cells 0 minutes to about 1 hour after the Cas effector protein is added to the composition comprising the eukaryotic cells.

[0027] In some embodiments, the inhibitor of the MMEJ pathway and the inhibitor of the NHEJ pathway are added to the composition comprising the eukaryotic cells at the same time. In some embodiments, the inhibitor of the MMEJ pathway and the inhibitor of the NHEJ pathway are added to the composition comprising the eukaryotic cells at different times.

[0028] In some embodiments, the inhibitor of the MMEJ pathway, the inhibitor of the NHEJ pathway, and the Cas effector protein are added simultaneously to a composition comprising eukaryotic cells.

[0029] In some embodiments, the inhibitor of the MMEJ pathway is present in the composition comprising the eukaryotic cell for about 1 to about 300 hours, about 10 to about 100 hours, or about 20 to about 80 hours.

[0030] In some embodiments, the inhibitor of the MMEJ pathway is added to the composition comprising the eukaryotic cell at least once, at least twice, or at least three times.

[0031] In some embodiments, the inhibitor of the NHEJ pathway is present in the composition comprising the eukaryotic cell for about 1 to about 300 hours, about 10 to about 100 hours, or about 20 to about 80 hours.

[0032] In some embodiments, the inhibitor of the NHEJ pathway is added to the composition comprising the eukaryotic cell at least once, at least twice, or at least three times.

[0033] In some embodiments, the composition comprising the eukaryotic cells is a cell culture. In some embodiments, the cell culture is an in vitro cell culture or an ex vivo cell culture. In some embodiments, the eukaryotic cells are in vivo.

[0034] In some embodiments, the cell culture comprises a cell extract.

[0035] In some embodiments, the eukaryotic cell is a lymphocyte. In some embodiments, the lymphocyte comprises a chimeric antigen receptor (CAR) or a T cell receptor (TCR).

[0036] In some embodiments, the eukaryotic cell is a pluripotent stem cell. In some embodiments, the pluripotent stem cell is an induced pluripotent stem cell (iPSC).

[0037] In some embodiments, the cell culture is a mammalian cell culture.

[0038] In some embodiments, the present disclosure relates to a method for increasing the efficiency of CRISPR / Cas-mediated gene insertion, comprising inserting a polynucleotide of interest into the genome of a eukaryotic cell comprising a Cas polynucleotide integrated into the genome. In some embodiments, the present disclosure provides a method for inserting a polynucleotide of interest into the genome of a eukaryotic cell, comprising (a) adding an inhibitor of the microhomology-mediated end joining (MMEJ) pathway to a composition comprising a eukaryotic cell, and (b) adding a polynucleotide of interest to the composition, wherein the genome comprises a Cas polynucleotide integrated into the genome, and the polynucleotide of interest is inserted into the genome by homology-directed repair (HDR) or single-stranded template repair (SSTR). In some embodiments, the Cas polynucleotide integrated into the genome is inducible.

[0039] In some embodiments, the method further comprises adding an inhibitor of the non-homologous end joining (NHEJ) pathway to the composition.

[0040] In some embodiments, the method further comprises (c) adding to the composition a polynucleotide comprising an RNA guide sequence, a Cas binding region, a DNA template sequence, or a combination thereof.

[0041] In some embodiments, (i) the polynucleotide of interest and (ii) the polynucleotide of (c) are encoded on a vector. In some embodiments, the polynucleotide of interest is added as DNA. In some embodiments, the polynucleotide of (c) is added as DNA. In some embodiments, the polynucleotide of (c) is added as RNA.

[0042] In some embodiments, the vector is a viral vector. In some embodiments, the viral vector is a retrovirus, lentivirus, adenovirus, or adeno-associated virus (AAV). In some embodiments, the vector is added to the composition comprising the eukaryotic cell by transfecting the eukaryotic cell.

[0043] In some embodiments, the Cas effector protein is a Cas9 nuclease, a Cas12a nuclease, or a Cas12f nuclease. In some embodiments, the Cas effector protein is a Cas9 nuclease. In some embodiments, the Cas9 nuclease is a Cas9 nuclease fused to a reverse transcriptase, a Cas9 nuclease fused to a DNA polymerase, a Cas9 nuclease fused to DN1S, a Cas9 nickase, a Cas9 fused to a geminin degron domain, or a Cas9 nuclease fused to CTIP.

[0044] In some embodiments, the polynucleotide of interest is added by a vector. In some embodiments, the vector is a viral vector. In some embodiments, the viral vector is a retrovirus, a lentivirus, an adenovirus, or an adeno-associated virus (AAV).

[0045] In some embodiments, the polynucleotide of interest comprises a gene of interest. In some aspects, the polynucleotide of interest is 1-50 base pairs in length, 1-10 base pairs in length, or 50-5000 base pairs in length.

[0046] In some embodiments, the polynucleotide of interest is single stranded. In some embodiments, the polynucleotide of interest is double stranded. In some embodiments, the polynucleotide of interest is a hybrid polynucleotide comprising a single stranded region and a double stranded region. In some embodiments, the hybrid polynucleotide comprises double stranded sequences at the 5' and 3' ends, and an internal single stranded sequence. In some embodiments, the polynucleotide of interest is double stranded with blunt ends. In some embodiments, the polynucleotide of interest is double stranded with a 3' overhang. In some embodiments, the polynucleotide of interest is double stranded with a 5' overhang. In some embodiments, the polynucleotide of interest is a circular polynucleotide.

[0047] In some embodiments, the polynucleotide comprises a chemical modification that enhances activity, distribution, or uptake of the polynucleotide.

[0048] In some embodiments, the inhibitor of the MMEJ pathway is an inhibitor of POLQ / DNA polymerase θ. In some embodiments, the inhibitor of POLQ is PolQ1, PolQ2, PolQ3, PolQ4, PolQ5, PolQ6 PolQ7, or a combination thereof. In some embodiments, the inhibitor of POLQ is a peptide.

[0049] In some embodiments, the inhibitor of the MMEJ pathway in a composition comprising a eukaryotic cell is from about 0.01 μM to about 1 mM, from about 0.1 μM to about 1 mM, from about 0.1 μM to about 0.5 mM, from about 0.1 μM to about 100 μM, or from about 1 μM to about 50 μM.

[0050] In some embodiments, the inhibitor of the NHEJ pathway is an inhibitor of DNA-dependent protein kinase (DNA-PK). In some embodiments, the inhibitor of DNA-PK is M3814, M9831 / VX984, Nu7441, KU0060648, AZD7648, or a combination thereof. In some embodiments, the inhibitor of DNA-PK is AZD7648. In some embodiments, the inhibitor of DNA-PK is a peptide.

[0051] In some embodiments, the inhibitor of the NHEJ pathway in the composition comprising a eukaryotic cell is from about 0.01 μM to about 1 mM, from about 0.1 μM to about 1 mM, from about 0.1 μM to about 0.5 mM, from about 0.1 μM to about 100 μM, or from about 1 μM to about 50 μM.

[0052] In some embodiments, an inhibitor of the MMEJ pathway is added to a composition comprising a eukaryotic cell comprising a genomically integrated Cas polynucleotide from 0 minutes to about 48 hours, 0 minutes to about 24 hours, 0 minutes to about 12 hours, 0 minutes to about 6 hours, or 0 minutes to about 1 hour prior to induction of the genomically integrated Cas polynucleotide.

[0053] In some embodiments, an inhibitor of the NHEJ pathway is added to a composition comprising a eukaryotic cell comprising a genomically integrated Cas polynucleotide from 0 minutes to about 48 hours, 0 minutes to about 24 hours, 0 minutes to about 12 hours, 0 minutes to about 6 hours, or 0 minutes to about 1 hour prior to induction of the genomically integrated Cas polynucleotide.

[0054] In some embodiments, the inhibitor of the MMEJ pathway and the inhibitor of the NHEJ pathway are added simultaneously to a composition comprising a eukaryotic cell comprising a Cas polynucleotide integrated into its genome. In some embodiments, the inhibitor of the MMEJ pathway and the inhibitor of the NHEJ pathway are added at different times to a composition comprising a eukaryotic cell comprising a Cas polynucleotide integrated into its genome.

[0055] In some embodiments, an inhibitor of the MMEJ pathway is added to a composition comprising a eukaryotic cell comprising a genomically integrated Cas polynucleotide simultaneously with induction of the genomically integrated Cas polynucleotide.

[0056] In some embodiments, an inhibitor of the NHEJ pathway is added to a composition comprising a eukaryotic cell comprising a genomically integrated Cas polynucleotide simultaneously with induction of the genomically integrated Cas polynucleotide.

[0057] In some embodiments, an inhibitor of the MMEJ pathway and an inhibitor of the NHEJ pathway are added to a composition comprising a eukaryotic cell comprising a genomically integrated Cas polynucleotide simultaneously with induction of the genomically integrated Cas polynucleotide.

[0058] In some embodiments, the inhibitor of the MMEJ pathway is present in the composition comprising a eukaryotic cell comprising a Cas polynucleotide integrated into its genome for about 1 to about 300 hours, about 10 to about 100 hours, or about 20 to about 80 hours.

[0059] In some embodiments, an inhibitor of the MMEJ pathway is added at least once, at least twice, or at least three times to a composition comprising a eukaryotic cell comprising a Cas polynucleotide integrated into its genome.

[0060] In some embodiments, the inhibitor of the NHEJ pathway is present in the composition comprising a eukaryotic cell comprising a Cas polynucleotide integrated into its genome for about 1 to about 300 hours, about 10 to about 100 hours, or about 20 to about 80 hours.

[0061] In some embodiments, an inhibitor of the NHEJ pathway is added at least once, at least twice, or at least three times to a composition comprising a eukaryotic cell comprising a Cas polynucleotide integrated into its genome.

[0062] In some embodiments, the composition comprising a eukaryotic cell comprising a Cas polynucleotide integrated into its genome is a cell culture, hi some embodiments, the cell culture is an in vitro cell culture or an ex vivo cell culture.

[0063] In some embodiments, the eukaryotic cell comprising the Cas polynucleotide integrated into its genome is in vivo.

[0064] In some embodiments, the cell culture comprises a cell extract, hi some embodiments, the cell culture is a mammalian cell culture.

[0065] In some embodiments, the eukaryotic cell comprising a Cas polynucleotide integrated into its genome is a lymphocyte. In some embodiments, the lymphocyte comprises a chimeric antigen receptor (CAR) or a T cell receptor (TCR).

[0066] In some embodiments, the eukaryotic cell comprising a Cas polynucleotide integrated into its genome is a pluripotent stem cell. In some embodiments, the pluripotent stem cell is an induced pluripotent stem cell (iPSC).

[0067] In some embodiments, the disclosure provides a method of inserting a polynucleotide of interest into the genome of a eukaryotic cell, the method comprising: (a) adding an inhibitor of the microhomology-mediated end joining (MMEJ) pathway to a composition comprising the eukaryotic cell; and (b) adding to the composition comprising the eukaryotic cell: (i) a Cas effector protein, (ii) a polynucleotide of interest, and (iii) a polynucleotide comprising an RNA guide sequence, a Cas binding region, a DNA template sequence, or a combination thereof, wherein the polynucleotide of interest is inserted into the genome by homology directed repair (HDR) or single-stranded template repair (SSTR).

[0068] In some embodiments, the method further comprises adding an inhibitor of the non-homologous end joining (NHEJ) pathway to the composition comprising the eukaryotic cell.

[0069] In some embodiments, the Cas effector protein and the polynucleotide comprising an RNA guide sequence, a Cas binding region, a DNA template sequence, or a combination thereof are added in the form of a ribonucleoprotein (RNP).

[0070] In some embodiments, the Cas effector protein is encoded by a Cas polynucleotide. In some embodiments, the Cas effector protein and the polynucleotide of interest are encoded on a vector. In some embodiments, the Cas effector protein and the polynucleotide of (iii) are encoded on a vector. In some embodiments, the Cas effector protein, the polynucleotide of interest, and the polynucleotide of (iii) are encoded on a vector. In some embodiments, the polynucleotide is present on a vector.

[0071] In some embodiments, the present disclosure relates to a method of increasing the gene insertion efficiency of homology directed repair (HDR) and single stranded template repair (SSTR) in eukaryotic cells, comprising adding an inhibitor of the microhomology-mediated end joining (MMEJ) pathway when performing CRISPR / Cas-mediated gene insertion in eukaryotic cells.

[0072] In some embodiments, the method further comprises adding an inhibitor of the non-homologous end joining (NHEJ) pathway.

[0073] In some embodiments, the CRISPR / Cas-mediated gene insertion is CRISPR / Cas9-mediated gene insertion.

[0074] In some embodiments, the disclosure relates to a method of reducing microhomology-mediated end-joining (MMEJ) pathway recombination during CRISPR / Cas-mediated gene insertion in a cell, the method comprising adding an inhibitor of the MMEJ pathway to the cell when performing Cas-mediated gene insertion.

[0075] In some embodiments, the method further comprises reducing non-homologous end joining (NHEJ) recombination during CRISPR / Cas-mediated gene insertion in the cell, comprising adding an inhibitor of the NHEJ pathway to the cell.

[0076] In some embodiments, the CRISPR / Cas-mediated gene insertion is CRISPR / Cas9-mediated gene insertion.

[0077] In some embodiments, the disclosure relates to a composition comprising a Cas effector protein, or a vector encoding a Cas effector protein, and an inhibitor of the microhomology-mediated end joining (MMEJ) pathway. In some embodiments, the method further comprises an inhibitor of the non-homologous end joining (NHEJ) pathway.

[0078] In some embodiments, the composition further comprises a polynucleotide comprising at least one RNA guide sequence, a Cas binding region, a DNA template sequence, or a combination thereof.

[0079] In some embodiments, the Cas effector protein is a Cas9 nuclease, a Cas12a nuclease, or a Cas12f nuclease. In some embodiments, the Cas effector protein is a Cas9 nuclease. In some aspects, the Cas9 nuclease is a Cas9 nuclease fused to a reverse transcriptase, a Cas9 nuclease fused to a DNA polymerase, a Cas9 fused to a DN1S, a Cas9 nickase, a Cas9 fused to a geminin degron domain, or a Cas9 nuclease fused to a CTIP.

[0080] In some embodiments, the vector encoding the Cas effector protein is a viral vector.

[0081] In some embodiments, the polynucleotide comprising at least one RNA guide sequence, Cas binding region, DNA template sequence, or combinations thereof is encoded on a vector, hi some embodiments, the vector is a viral vector.

[0082] In some embodiments, the polynucleotide comprising the Cas effector protein and at least one RNA guide sequence, Cas binding region, DNA template sequence, or combinations thereof is in the form of a ribonucleoprotein (RNP).

[0083] In some embodiments, the composition further comprises a pharma- ceutically acceptable carrier, diluent, or excipient.

[0084] In some embodiments, the disclosure relates to a kit comprising a Cas effector protein, or a vector encoding a Cas effector protein, and an inhibitor of the microhomology-mediated end joining (MMEJ) pathway.

[0085] In some embodiments, the kit further comprises an inhibitor of the non-homologous end joining (NHEJ) pathway.

[0086] In some embodiments, the kit further comprises a polynucleotide comprising at least one RNA guide sequence, a Cas binding region, a DNA template sequence, or a combination thereof.

[0087] In some embodiments, the Cas effector protein is a Cas9 nuclease, a Cas12a nuclease, or a Cas12f nuclease. In some embodiments, the Cas effector protein is a Cas9 nuclease. In some embodiments, the Cas9 nuclease is a Cas9 nuclease fused to a reverse transcriptase, a Cas9 nuclease fused to a DNA polymerase, a Cas9 fused to a DN1S, a Cas9 nickase, a Cas9 fused to a geminin degron domain, or a Cas9 nuclease fused to a CTIP.

[0088] In some embodiments, the polynucleotide comprising at least one RNA guide sequence, Cas binding region, DNA template sequence, or combinations thereof is encoded on a vector, hi some embodiments, the vector is a viral vector.

[0089] In some embodiments, the polynucleotide comprising the Cas effector protein and at least one RNA guide sequence, Cas binding region, DNA template sequence, or combinations thereof is in the form of a ribonucleoprotein (RNP). [Brief description of the drawings]

[0090] [Figure 1] Figure 1 is a schematic diagram showing the manipulation of DNA repair by small molecule inhibitors. In this schematic, components of the CRISPR / Cas genome editing system provide double-strand breaks (DSBs) at specific sequences. DSBs can be repaired by the imprecise and error-prone microhomology-mediated end joining (MMEJ) or non-homologous end joining (NHEJ) pathways, or by the more precise homology-directed repair (HDR) pathway. [Diagram 2] 2A-2B show an exemplary method described in the embodiments herein. FIG. 2A shows an example where cells are pretreated with pharmacological inhibitors of POL Q / DNA polymerase theta (PolQi) and / or DNA-dependent protein kinase (DNA-PKi) for 3 hours. The CRISPR / Cas gene editing system is then added to the cells. After 60 hours, genomic DNA is isolated from the cells and deep target sequencing is performed. The sequencing results are then analyzed by Rational InDel Meta-Analysis (RIMA) to determine the frequency of MMEJ and NHEJ repair. FIG. 2B shows a graphical representation of the RIMA results, where the deletions associated with microhomology are visualized by the bars shown in the figure. [Diagram 3] FIG. 3 shows the chemical structures of representative POL Q / DNA polymerase θ inhibitors. [Figure 4]Figure 4 shows that inhibition of the MMEJ and NHEJ pathways results in increased HDR repair of DSBs. HEK293T cells were treated with the DNA-PK inhibitor AZD7648 (1 μM) alone and in combination with the indicated Pol Q inhibitors, followed by CRISPR / Cas9-mediated gene targeting. As assessed by the percentage of correct DNA repair, the addition of DNA-PK and Pol Q inhibitors reduced DNA repair by MMEJ and NHEJ, while increasing HDR-mediated DNA repair. [Diagram 5] FIG. 5 shows the effect of MMEJ and NHEJ pathway inhibition on CRISPR / Cas editing efficiency, as described in Example 1. [Figure 6] FIG. 6 shows the effect of MMEJ and NHEJ pathway inhibition on CRISPR / Cas-mediated gene knock-in efficiency as measured by mutational leads, as described in Example 2. [Figure 7] FIG. 7 shows the effect of MMEJ and NHEJ pathway inhibition on CRISPR / Cas-mediated gene knock-in efficiency as measured by mapping reads, as described in Example 2. [Figure 8] FIG. 8 shows the effect of Pol Q inhibition on MMEJ in mutant leads, as described in Example 3. [Figure 9] 9 shows the effect of Pol Q inhibition on MMEJ in mapping reads, as described in Example 3. HEK293T cells were treated with the DNA-PK inhibitor AZD7648 (1 μM) alone and in combination with the indicated Pol Q inhibitors, followed by CRISPR / Cas9-mediated gene knock-in. Addition of Pol Q inhibitors resulted in a dose-dependent decrease in MMEJ in mapping reads. [Figure 10] FIG. 10 shows the effect of MMEJ and NHEJ pathway inhibition on cell confluence, as described in Example 4. [Figure 11] FIG. 11 shows the effect of MMEJ and NHEJ pathway inhibition on transfection efficiency, as described in Example 4. [Figure 12]Figure 12 shows that inhibition of MMEJ and NHEJ pathways leads to increased HDR repair of DSBs in induced pluripotent stem cells (iPSCs). Cas9-induced iPSCs were treated with DNA-PK inhibitor AZD7648 (1 μM) and / or the indicated Pol Q inhibitors, followed by induction of Cas9-mediated gene targeting. As assessed by the percentage of correct DNA repair at three separate target sites, the addition of DNA-PK inhibitors and Pol Q inhibitors reduced DNA repair by MMEJ and NHEJ, while increasing HDR-mediated DNA repair. [Figure 13] Figure 13 shows the effect of Pol Q inhibition on single strand template repair (SSTR)-mediated knock-in efficiency in Cas9-induced iPSCs. Cas9-induced iPSCs were treated with 1 μM DNA-PK inhibitor ZAD7648 and / or the indicated Pol Q inhibitors, followed by induction of Cas9-mediated gene knock-in at three separate target sites. Addition of DNA-PK inhibitors and / or Pol Q inhibitors increased SSTR-mediated knock-in at all three target sites. [Figure 14] 14A-14C show the effect of inhibiting MMEJ and NHEJ pathways on gene editing in primary human T cells. Green fluorescent protein (GFP) was inserted by knock-in into primary human T cells transfected with Cas9 in the form of a ribonucleoprotein (RNP) targeting TRAC. Cells were treated with 1 μM of the DNA-PK inhibitor AZD7648 alone and in combination with the indicated Pol Q inhibitors. (A) The effect of NHEJ and / or MMEJ pathway inhibition on cell viability. (B) The effect of NHEJ and / or MMEJ pathway inhibition on cell number. (C) The effect of NHEJ and / or MMEJ pathway inhibition on GFP knock-in efficiency. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0091] The present disclosure relates to methods, compositions, and kits for improving CRISPR / Cas-mediated gene insertion (i.e., gene "knock-in") in eukaryotic cells. In general, CRISPR systems, e.g., CRISPR / Cas systems, contain elements that promote the formation of a CRISPR complex, such as a guide polynucleotide and a Cas protein, at the site of a target polynucleotide, e.g., a target DNA sequence. In naturally occurring CRISPR systems (e.g., bacterial immune CRISPR / Cas9 systems), foreign DNA is integrated into a CRISPR array, which then generates a CRISPR-RNA (crRNA). The crRNA contains an RNA guide sequence region that is complementary to the foreign DNA site and hybridizes with a trans-activating CRISPR-RNA (tracrRNA), which is also encoded by the CRISPR system. The tracrRNA can form a secondary structure, such as a stem-loop, and bind to a Cas9 protein. The crRNA / tracrRNA hybrid associates with Cas9, and the crRNA / tracrRNA / Cas9 complex recognizes and cleaves foreign DNA containing the protospacer sequence, thereby conferring immunity against invading viruses or plasmids. The CRISPR / Cas system is further described, for example, in Jinek et al., Science 337(6096):816-821(2012); Cong et al., Science 339(6121):819-823(2013); Mali et al., Science 339(6121):823-826(2013); and Sander et al., Nat Biotechnol 32:347-355(2014).

[0092] CRISPR / Cas systems are engineered to introduce insertions into target polynucleotides, also known as targeted insertions. Typically, a guide polynucleotide is designed such that a Cas protein creates a double-strand break in the target polynucleotide, and a separate donor template containing a sequence of interest is inserted into the cleaved target polynucleotide by the cell's DNA repair mechanisms, such as non-homologous end joining (NHEJ) or homology-directed repair (HDR). The efficiency of insertion depends on several factors, including the transfection ratio of the donor template, Cas protein, and guide polynucleotide; the sequence and size of the donor template; and the type of DNA repair mechanism induced. For example, HDR provides high fidelity DNA repair but low insertion frequency, while NHEJ provides high insertion frequency but may introduce mutations into the target DNA.

[0093] In some embodiments, the present disclosure provides compositions, polynucleotides, and / or fusion proteins for improved targeted insertion methods. In some embodiments, the compositions, polynucleotides, and / or fusion proteins of the present disclosure provide highly accurate insertion of a sequence of interest. In some embodiments, the compositions, polynucleotides, and fusion proteins of the present disclosure provide highly efficient insertion of a sequence of interest.

[0094] Scientific and technical terms used in this disclosure shall have the meanings commonly understood by those skilled in the art, unless otherwise defined herein. Furthermore, unless the context requires otherwise, singular terms shall include the plural and plural terms shall include the singular. As used herein, "a" or "an" may mean one or more. As used herein, and when used in conjunction with the word "comprising", the word "a" or "an" may mean one or more. As used herein, "another" or "further" may mean at least a second or more.

[0095] Throughout this application, the term "about" is used to indicate that a value includes the inherent variation of error for the method / device being used to determine the value or the variation that exists between test subjects. Typically, the term "about" is meant to encompass a variability of approximately 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20%, or less than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20%, depending on the context.

[0096] Use of the term "or" in the claims is used to mean "and / or" unless expressly stated to refer to alternatives only or the alternatives are not mutually exclusive, however, the present disclosure supports a definition that refers to alternatives only and "and / or."

[0097] As used herein, the terms "comprising" (and any variant or form of comprising, e.g., "comprise" and "comprises"), "having" (and any variant or form of having, e.g., "have" and "has"), "including" (and any variant or form of including, e.g., "includes" and "include"), or "containing" (and any variant or form of containing, e.g., "contains" and "contain") are inclusive or open ended and do not exclude additional unrecited elements or method steps. It is contemplated that any of the embodiments described herein can be implemented with respect to any of the proteins, compositions, polynucleotides, vectors, cells, methods, and / or kits of the present disclosure. Additionally, the compositions, polynucleotides, vectors, cells, and / or kits of the present disclosure can be used to obtain the methods and proteins of the present disclosure.

[0098] Use of the term "for example" and its corresponding abbreviation "eg" (whether italicized or not) means that the particular term recited is a representative example and embodiment of the present disclosure that is not limited to the specific example referenced or recited, unless expressly stated otherwise.

[0099] As used herein, "between" refers to a range that includes both ends of the range, for example, a number between x and y explicitly includes numbers x and y, and all numbers between x and y.

[0100] "Nucleic acid", "nucleic acid molecule", "nucleotide", "nucleotide sequence", "oligonucleotide" or "polynucleotide" refers to a polymeric compound comprising covalently linked nucleotides. The term "nucleic acid" includes ribonucleic acid (RNA) and deoxyribonucleic acid (DNA), both of which may be single-stranded or double-stranded. Polynucleotides may contain naturally occurring nucleobases (e.g., guanine, adenine, cytosine, thymine, and uracil), modified nucleobases (e.g., hypoxanthine, xanthine, 7-methylguanine, dihydrouracil, 5-methylcytosine, 5-hydroxymethylcytosine), and / or artificial nucleobases (e.g., isoguanine or isocytosine). Nucleic acids are transcribed from the 5' end to the 3' end. In some embodiments, the present disclosure provides polynucleotides comprising RNA and DNA nucleotides. Methods for generating polynucleotides comprising both RNA and DNA nucleotides are known in the art and include, for example, ligation or oligonucleotide synthesis methods. In some embodiments, the disclosure provides a polynucleotide that can form a complex with a Cas nuclease or a Cas nickase as described herein. In some embodiments, the disclosure provides a polynucleotide that encodes any one of the proteins disclosed herein, such as a Cas nuclease or a Cas nickase.

[0101] "Gene" refers to a collection of nucleotides that encodes a polypeptide, and includes cDNA and genomic DNA nucleic acid molecules. In some embodiments, "gene" also refers to non-coding nucleic acid fragments that can function as regulatory sequences preceding (i.e., 5') and following (i.e., 3') the coding sequence.

[0102] A nucleic acid molecule is "hybridizable" or "hybridized" to another nucleic acid molecule, e.g., cDNA, genomic DNA or RNA, if the single-stranded form of the nucleic acid molecule can anneal to the other nucleic acid molecule under suitable conditions of temperature and solution ionic strength. Hybridization and washing conditions are known and are exemplified in Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor (1989), particularly Chapter 11 and Table 11.1 therein. The conditions of temperature and ionic strength determine the stringency of hybridization. The stringency of hybridization conditions can be selected to provide the selective formation or maintenance of the desired hybridization product of two complementary polynucleotides in the presence of other potentially cross-reactive or interfering polynucleotides. Stringent conditions are sequence-dependent, and typically, longer complementary sequences hybridize specifically at higher temperatures than shorter complementary sequences. Typically, stringent hybridization conditions are those that, for a particular polynucleotide, have a thermal melting point (T m) (i.e., the temperature at which 50% of the sequence hybridizes to a substantially complementary sequence). Typically, a nucleotide sequence containing a higher percentage of G and C bases will hybridize under more stringent conditions than a nucleotide sequence containing a lower percentage of G and C bases. Generally, stringency can be increased by increasing the temperature, increasing the pH, decreasing the ionic strength, and / or increasing the concentration of chemical nucleic acid denaturants (e.g., formamide, dimethylformamide, dimethylsulfoxide, ethylene glycol, propylene glycol, and ethylene carbonate). Stringent hybridization conditions typically include salt concentrations or ionic strengths of less than about 1 M, 500 mM, 200 mM, 100 mM, or 50 mM; hybridization temperatures of greater than about 20° C., 30° C., 40° C., 60° C., or 80° C.; and chemical denaturing agent concentrations of greater than about 10%, 20%, 30%, 40%, or 50%. Many factors can affect the stringency of hybridization, and therefore combinations of parameters may be more important than the absolute value of any parameter alone.

[0103] The term "complementary" is used to describe the relationship between nucleotide bases that can hybridize to each other. For example, with respect to DNA, adenosine is complementary to thymine, and cytosine is complementary to guanine. When two nucleic acids are "complementary," it means that the first nucleic acid, or one or more regions thereof, can hydrogen bond with the second nucleic acid, or one or more regions thereof. Complementary nucleic acids need not have complementarity at every nucleotide, but may contain one or more nucleotide mismatches, i.e., points where no hydrogen bonding occurs. For example, complementary oligonucleotides may have hydrogen bonds at at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the nucleotides. In contrast, "fully complementary" or "100% complementary" with respect to an oligonucleotide means that each nucleotide is hydrogen bonded without any nucleotide mismatches.

[0104] The term "homologous recombination" refers to the insertion of an exogenous polynucleotide (e.g., DNA) into another nucleic acid (e.g., DNA) molecule, such as the insertion of a vector, a polynucleotide fragment, or a gene in a chromosome. In some instances, the exogenous polynucleotide targets a specific chromosomal site for homologous recombination. In specific homologous recombination, the exogenous polynucleotide typically contains a sufficiently long region that is homologous to a sequence of the chromosome so that the exogenous polynucleotide can complementarily bind and integrate into the chromosome. Longer complementary regions and greater degrees of sequence similarity can increase the efficiency of homologous recombination. In some embodiments, the polynucleotides or compositions described herein promote homologous recombination by generating breaks, e.g., double-strand breaks, in the nucleic acid sequence.

[0105] The term "homologous recombination repair" or "HDR" refers to a mechanism that uses a template nucleic acid sequence to repair double-stranded breaks in DNA. The most common form of HDR is homologous recombination. In HDR, double-stranded breaks are repaired by a process that involves excision of the 5'-terminal DNA strand of the break to generate a 3' overhang, which serves as both a substrate for proteins required for strand invasion and a primer for DNA repair synthesis. The invasive strand then displaces one strand of a double-stranded DNA template sequence that contains a homologous sequence and pairs with the other strand, forming a hybrid DNA known as a displacement loop. These recombination intermediates are then separated to complete the DNA repair process.

[0106] The term "single-stranded template repair" or "SSTR" refers to another mechanism that uses a template nucleic acid sequence to repair double-stranded breaks in DNA. In contrast to HDR, SSTR utilizes a single-stranded template nucleic acid sequence for double-stranded DNA break repair.

[0107] The term "non-homologous end joining pathway" or "NHEJ pathway" refers to another mechanism that repairs double-stranded breaks in DNA. In NHEJ, Ku80 / 70 heterodimers recognize and bind to blunt ends formed by double-stranded breaks, and the resulting complex activates the activity of DNA-PK. Activation of DNA-PK recruits Artemis nuclease, DNA polymerase, and DNA ligase to ultimately repair the double-stranded break. NHEJ differs from HDR and homologous recombination in that it does not require a homologous template sequence for repair.

[0108] The term "microhomology-mediated end joining pathway" or "MMEJ pathway" refers to another mechanism for repairing double-stranded breaks in DNA. MMEJ is similar to NHEJ in that no homologous template sequence is utilized for double-stranded break repair. However, MMEJ is distinguished from other repair mechanisms by utilizing microhomology sequences to align the broken DNA strands. MMEJ does not rely on Ku proteins or DNA-PK, but it has been shown that DNA polymerase θ (Pol Q) is required for MMEJ. MMEJ is also known as "alternative end joining" or "alternative non-homologous end joining" or "Alt-NHEJ".

[0109] As used herein, the term "operably linked" means that a polynucleotide of interest, e.g., a polynucleotide encoding a nuclease, is linked to a regulatory element in such a manner that the polynucleotide can be expressed. The regulatory element may be a cis-regulatory element or a trans-regulatory element. Regulatory elements include, for example, promoters, enhancers, terminators, 5'UTRs and 3'UTRs, insulators, silencers, operators, and the like. In some embodiments, the regulatory element is a promoter. In some embodiments, the polynucleotide expressing a protein of interest is operably linked to a promoter on an expression vector.

[0110] As used herein, a "promoter", "promoter sequence" or "promoter region" refers to a DNA regulatory region or polynucleotide that can bind RNA polymerase and is responsible for initiating transcription of a downstream coding or non-coding sequence. In some embodiments, the promoter sequence includes a transcription initiation site and extends upstream to include a minimum number of bases or elements used to initiate transcription at a level detectable above background. In some embodiments, the promoter sequence includes a transcription initiation site and a protein binding domain responsible for binding RNA polymerase. Eukaryotic promoters typically contain "TATA" and "CAT" boxes. A variety of promoters, including inducible promoters, may be used to drive expression of the various vectors of the present disclosure.

[0111] A "vector" is any means of cloning and / or transferring a nucleic acid into a host cell. A vector can be a replicon to which another DNA segment can be attached resulting in replication of the attached segment. A "replicon" is any genetic element (e.g., plasmid, phage, cosmid, chromosome, virus) that functions as an autonomous unit of DNA replication in vivo, i.e., that can replicate under its own control. In some embodiments, the vector is an episomal vector that is removed / erased from the cell population after many cell generations, e.g., by asymmetric partitioning. The term "vector" includes both viral and non-viral means for introducing nucleic acids into cells in vitro, ex vivo, or in vivo. Numerous vectors known in the art can be used to manipulate nucleic acids, to incorporate response elements and promoters into genes, etc. A vector can include one or more regulatory regions and / or selection markers useful for selection, measurement, and monitoring of the nucleic acid introduction results (tissue introduction, duration of expression, etc.).

[0112] Possible vectors include, for example, plasmids or modified viruses, including, for example, bacteriophages, such as lambda derivatives, or plasmids, such as PBR322 or pUC plasmid derivatives, or Bluescript vectors. For example, insertion of a DNA fragment corresponding to the response element and promoter into a suitable vector can be accomplished by ligating the appropriate DNA fragment into a selected vector with complementary cohesive ends. Alternatively, the ends of the DNA molecules may be enzymatically modified, or any site may be generated by ligating a polynucleotide (linker) onto the DNA termini. Such vectors may be modified to contain a selectable marker gene that provides for selection of cells that have incorporated the marker into the cell genome. Such markers allow for the identification and / or selection of host cells that have incorporated the marker and are expressing the protein encoded by the marker.

[0113] Viral vectors, particularly retroviral vectors, have been used in a wide variety of gene delivery applications in cells as well as in live animal subjects. Viral vectors that can be used include, but are not limited to, retroviral vectors, lentiviral vectors, adenoviral vectors, adeno-associated viral vectors, pox vectors, baculoviral vectors, vaccinia vectors, herpes simplex vectors, Epstein-Barr vectors, adenoviral vectors, geminiviral vectors, and caulimoviral vectors. In some embodiments, viral vectors are utilized to provide the polynucleotides described herein. In some embodiments, viral vectors are utilized to provide the polynucleotides encoding the proteins described herein.

[0114] The vector can be introduced into the desired host cell by known methods, including but not limited to transfection, transduction, cell fusion, and lipofection. The vector can include various regulatory elements, including a promoter. In some embodiments, the design of the vector can be based on the construct designed by Mali et al., Nat Methods 10:957-63 (2013).

[0115] Methods known in the art may be used to propagate the polynucleotides and / or vectors provided herein. Once a suitable host system and growth conditions are established, recombinant expression vectors can be propagated and prepared in large quantities. As described herein, expression vectors that can be used include, but are not limited to, the following vectors or their derivatives: human or animal viruses, such as vaccinia virus or adenovirus, insect viruses, such as baculovirus, yeast vectors, bacteriophage vectors (e.g., lambda), and plasmid and cosmid DNA vectors.

[0116] The term "plasmid" refers to an extrachromosomal element that often carries genes that are not part of the central metabolism of the cell, and is usually in the form of a circular double-stranded DNA molecule. Such elements can be autonomously replicating sequences, genome-integrating sequences, phage or nucleotide sequences, linear, circular or supercoiled, single-stranded or double-stranded DNA or RNA from any source, in which multiple polynucleotides are combined or recombined into a unique construct that allows the introduction of promoter fragments and DNA sequences for producing selected genes with appropriate 3' non-translated sequences into cells. In some embodiments, plasmids are utilized to provide the polynucleotides described herein. In some embodiments, viral vectors are utilized to provide polynucleotides encoding the proteins described herein.

[0117] As used herein, the term "transfection" refers to the introduction of an exogenous nucleic acid molecule, including a vector, into a cell. For example, methods of transfection of the components of the CRISPR / Cas compositions described herein are known to those of skill in the art. A "transfected" cell includes an exogenous nucleic acid molecule inside the cell, and a "transformed" cell is a cell in which the exogenous nucleic acid molecule induces a phenotypic change in the cell. The transfected nucleic acid molecule can be integrated into the genomic DNA of the host cell and / or can be maintained by the cell extrachromosomally for a temporary or long term period. A host cell or organism that expresses an exogenous nucleic acid molecule or fragment is referred to herein as a "recombinant," "transformed," or "transgenic" organism. In some embodiments, the disclosure provides a host cell that includes any of the vectors described herein, such as a vector including a Cas polynucleotide, a vector including a polynucleotide of interest, or a vector including a polynucleotide including an RNA guide sequence, a CAS binding region, a DNA template sequence, or a combination thereof.

[0118] The term "host cell" can refer to a cell into which a recombinant expression vector has been introduced, or to the progeny of such a cell. Such progeny may not be identical to the parent cell because, for example, modifications due to mutations or environmental influences may occur in subsequent generations, but are still included within the scope of the term "host cell."

[0119] The terms "peptide," "polypeptide," and "protein" are used interchangeably herein to refer to polymeric forms of amino acids of any length, and include coded and non-coded amino acids, unnatural amino acids, amino acids that have been chemically or biochemically modified or derivatized, peptides and polypeptides with modified peptide backbones, and circular / cyclic peptides and polypeptides.

[0120] The beginning of a protein or polypeptide is known as the "N-terminus" (also referred to as the amino terminus, NH2-terminus, N-terminal end, or amine terminus) and refers to the free amine (-NH2) group of the first amino acid residue of the protein or polypeptide. The end of a protein or polypeptide is known as the "C-terminus" (also referred to as the carboxy terminus, carboxyl terminus, C-terminal end, or COOH-terminus) and refers to the free carboxyl group (-COOH) of the last amino acid residue of the protein or polypeptide.

[0121] "Amino acid" as used herein refers to a compound containing both a carboxyl (-COOH) and an amino (-NH2) group. "Amino acid" refers to both natural and unnatural (i.e., synthetic) amino acids. Natural amino acids with three-letter and one-letter abbreviations include alanine (Ala; A); arginine (Arg, R); asparagine (Asn; N); aspartic acid (Asp; D); cysteine ​​(Cys; C); glutamine (Gln; Q); glutamic acid (Glu; E); glycine (Gly; G); histidine (His; H); isoleucine (Ile; I); leucine (Leu; L); lysine (Lys; K); methionine (Met; M); phenylalanine (Phe; F); proline (Pro; P); serine (Ser; S); threonine (Thr; T); tryptophan (Trp; W); tyrosine (Tyr; Y); and valine (Val; V). Unnatural or synthetic amino acids include side chains that are different from the natural amino acids provided above, and may include, for example, fluorophores, post-translational modifications, metal ion chelators, photocaged and photocrosslinking moieties, unique reactive functional groups, and NMR, IR, and X-ray crystallography probes. Exemplary unnatural or synthetic amino acids are provided, for example, in Mitra et al., Mater Methods 3:204 (2013) and Wals et al., Front Chem 2:15 (2014). Unnatural amino acids may also include naturally occurring compounds that are not normally incorporated into proteins or polypeptides, such as, for example, citrulline (Cit), selenocysteine ​​(Sec), and pyrrolysine (Pyl).

[0122] "Amino acid substitution" refers to a polypeptide or protein that contains one or more substitutions at its amino acid residues of a wild-type or natural amino acid with an amino acid different from the wild-type or natural amino acid. The substituted amino acid may be a synthetic or naturally occurring amino acid. In some embodiments, the substituted amino acid is a natural amino acid selected from the group consisting of A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y, and V. In some embodiments, the substituted amino acid is a non-natural or synthetic amino acid. Substitution variants may be described using an abbreviation system. For example, a substitution mutation in which the fifth amino acid residue has been substituted may be abbreviated as "X5Y," where "X" is the wild-type or natural amino acid that is replaced, "5" is the position of the amino acid residue in the amino acid sequence of the protein or polypeptide, and "Y" is the substituted amino acid or the non-wild-type or non-natural amino acid.

[0123] An "isolated" polypeptide, protein, peptide, or nucleic acid is a molecule that has been removed from its natural environment. It is also understood that an "isolated" polypeptide, protein, peptide, or nucleic acid may be formulated with an excipient, such as a diluent or auxiliary, and still be considered isolated. As used herein, "isolated" does not necessarily refer to any particular level of purity of the polypeptide, protein, peptide, or nucleic acid.

[0124] The term "recombinant" when used in reference to a nucleic acid molecule, peptide, polypeptide, or protein means or results from a new combination of genetic material not known to exist in nature. Recombinant molecules can be produced by any of the techniques available in the field of recombinant technology, including, but not limited to, polymerase chain reaction (PCR), gene splicing (e.g., using restriction endonucleases), and solid phase synthesis of nucleic acid molecules, peptides, or proteins.

[0125] The term "exogenous" refers to a reference molecule or activity that is introduced into a host cell. The molecule can be introduced, for example, by introducing an encoding nucleic acid into the genetic material of the host, for example, by integration into a chromosome of the host, or as non-chromosomal genetic material, for example, a plasmid. A "foreign" protein can be introduced into a host cell by an "exogenous" nucleic acid that encodes the protein. The term "endogenous" refers to a reference molecule or activity that is naturally present in a host cell. An "endogenous" protein is expressed by a nucleic acid contained within the host cell. The term "heterologous" refers to a molecule or activity that originates from a source other than the reference organism / species, while "homologous" refers to a molecule or activity that originates from the host organism / species. Thus, exogenous expression of an encoding nucleic acid can utilize either a heterologous encoding nucleic acid or a homologous encoding nucleic acid, or both.

[0126] The term "domain" when used in reference to a polypeptide or protein refers to a distinct functional and / or structural unit in a protein. A domain may be responsible for a specific function or interaction that contributes to the overall role of the protein. Domains may exist in a variety of biological contexts. Similar domains may be found in proteins with different functions. Alternatively, domains with low sequence identity (i.e., less than about 50%, less than about 40%, less than about 30%, less than about 20%, less than about 10%, less than about 5%, or less than about 1% sequence identity) may have the same function.

[0127] The term "motif" when used in reference to a polypeptide or protein generally refers to conserved amino acid residues, typically less than 20 amino acids in length, that may be important for the function of the protein. Certain sequence motifs can mediate common functions in various proteins, such as binding or targeting of proteins to specific subcellular locations. Examples of motifs include, but are not limited to, nuclear localization signals, microbody targeting motifs, motifs that block or promote secretion, and motifs that promote protein recognition and binding. Motif databases and / or motif search tools are known in the art, and include, for example, PROSITE, PFAM, PRINTS, and MiniMotif Miner.

[0128] "Modified" protein, as used herein, refers to a protein that contains one or more modifications to achieve a desired property in the protein. Exemplary modifications include, but are not limited to, insertions, deletions, substitutions, and / or fusion with another domain or protein. A "fusion protein" (also called a "chimeric protein") is a protein that typically contains at least two domains, encoded by two separate genes, that are linked so as to be transcribed and translated as a single unit, thereby producing a single polypeptide with the functional properties of each of the domains. Modified proteins of the present disclosure include Cas nucleases, Cas nickases, and fusions of Cas proteins with DNA polymerases, DNA ligases, and / or DNA polymerase-binding proteins.

[0129] In some embodiments, the engineered protein is generated from a wild-type protein. As used herein, a "wild-type" protein or nucleic acid is a naturally occurring, unmodified protein or nucleic acid. For example, a wild-type Cas9 protein can be isolated from the organism Streptococcus pyogenes. A wild-type can be contrasted with a "mutant," which includes one or more modifications to the amino acid and / or nucleotide sequence of a protein or nucleic acid. In some embodiments, the engineered protein can have substantially the same activity as the wild-type protein, e.g., greater than about 80%, greater than about 85%, greater than about 90%, greater than about 95%%, or greater than about 99% of the activity of the wild-type protein. In some embodiments, the Cas nuclease of the fusion protein described herein has substantially the same activity as a wild-type Cas nuclease.

[0130] In some embodiments, the engineered protein, e.g., Cas9 protein, may have substantially the same amino acid sequence as the wild-type protein, e.g., greater than about 80%, greater than about 85%, greater than about 90%, greater than about 95%%, or greater than about 99% identity to the wild-type protein. As used herein, the term "sequence similarity" or "% similarity" refers to the degree of identity or correspondence between nucleic acid or amino acid sequences. In the context of polynucleotides, "sequence similarity" may refer to nucleic acid sequences in which one or more nucleotide base changes result in the substitution of one or more amino acids, but do not affect the functional properties of the protein encoded by the polynucleotide. "Sequence similarity" may also refer to modifications of a polynucleotide that do not substantially affect the functional properties of the resulting transcript, e.g., deletion or insertion of one or more nucleotide bases. Thus, it is understood that the present disclosure encompasses more than the specific exemplary sequences. Methods for making nucleotide base substitutions are known, as are methods for determining retention of biological activity of the encoded polypeptide.

[0131] Moreover, one of skill in the art will recognize that analogous polynucleotides encompassed by the present disclosure are defined by their ability to hybridize under stringent conditions to the sequences exemplified herein. Analogous polynucleotides of the present disclosure are about 70%, at least about 70%, about 75%, at least about 75%, about 80%, at least about 80%, about 85%, at least about 85%, about 90%, at least about 90%, about 95%, at least about 95%, about 99%, at least about 99%, or about 100% identical to the polynucleotides disclosed herein.

[0132] In the context of polypeptides, "sequence similarity" refers to two or more polypeptides in which more than about 40% of the amino acids are identical or more than about 60% of the amino acids are functionally identical. "Functionally identical" or "functionally similar" amino acids have chemically similar side chains. For example, amino acids can be grouped by functional similarity as follows: (i) positively charged side chains: Arg, His, Lys; (ii) negatively charged side chains: Asp, Glu; (iii) polar uncharged side chains: Ser, Thr, Asn, Gln; (iv) hydrophobic side chains: Ala, Val, Ile, Leu, Met, Phe, Tyr, Trp; and (v) other: Cys, Gly, Pro.

[0133] In some embodiments, similar polypeptides of the disclosure have amino acids that are about 40%, at least about 40%, about 45%, at least about 45%, about 50%, at least about 50%, about 55%, at least about 55%, about 60%, at least about 60%, about 65%, at least about 65%, about 70%, at least about 70%, about 75%, at least about 75%, about 80%, at least about 80%, about 85%, at least about 85%, about 90%, at least about 90%, about 95%, at least about 95%, about 97%, at least about 97%, about 98%, at least about 98%, about 99%, at least about 99%, or about 100% identical. In some embodiments, similar polypeptides of the disclosure have amino acids that are about 60%, at least about 60%, about 65%, at least about 65%, about 70%, at least about 70%, about 75%, at least about 75%, about 80%, at least about 80%, about 85%, at least about 85%, about 90%, at least about 90%, about 95%, at least about 95%, about 97%, at least about 97%, about 98%, at least about 98%, about 99%, at least about 99%, or about 100% functionally identical.

[0134] Sequence similarity can be determined by sequence alignment using methods known in the art, such as, for example, BLAST, MUSCLE, Clustal (including ClustalW and ClustalX), and T-Coffee (including variants such as M-Coffee, R-Coffee, and Expresso).

[0135] Percent identity of polynucleotides or polypeptides can be determined when polynucleotide or polypeptide sequences are aligned over a specified comparison window. In some embodiments, only certain portions of two or more sequences are aligned to determine sequence identity. In some embodiments, only certain domains of two or more sequences are aligned to determine sequence similarity. A comparison window can be a segment of at least 10 to more than 1000 residues, at least 20 to about 1000 residues, or at least 50 to 500 residues over which sequences can be aligned and compared. Alignment methods for determining sequence identity are well known and can be performed using publicly available databases such as BLAST. For example, in some embodiments, the "percent identity" of two amino acid sequences is determined using the algorithm of Karlin and Altschul, Proc Nat Acad Sci USA 87:2264-2268 (1990) (as modified as in Karlin and Altschul, Proc Nat Acad Sci USA 90:5873-5877 (1993)). Such an algorithm is incorporated into the BLAST programs described in Altschul et al., J Mol Biol, 215:403-410 (1990), such as the BLAST+ or NBLAST and XBLAST programs. BLAST protein searches can be performed, for example, with a program such as the XBLAST program (score=50, wordlength=3) to obtain amino acid sequences homologous to the protein molecules of the present disclosure. When gaps exist between the two sequences, Gapped BLAST can be utilized as described in Altschul et al., Nucleic Acids Res 25(17):3389-3402 (1997). When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used.

[0136] In some embodiments, a polypeptide or polynucleotide has 70%, at least 70%, 75%, at least 75%, 80%, at least 80%, 85%, at least 85%, 90%, at least 90%, 95%, at least 95%, 97%, at least 97%, 98%, at least 98%, 99%, or at least 99%, or 100% sequence identity to a reference polypeptide or polynucleotide (or a fragment of a reference polypeptide or polynucleotide) provided herein. In some embodiments, a polypeptide or polynucleotide has about 70%, at least about 70%, about 75%, at least about 75%, about 80%, at least about 80%, about 85%, at least about 85%, about 90%, at least about 90%, about 95%, at least about 95%, about 97%, at least about 97%, about 98%, at least about 98%, about 99%, at least about 99%, or about 100% sequence identity to a reference polypeptide or polynucleotide (or a fragment of a reference polypeptide or nucleic acid molecule) provided herein.

[0137] As used herein, a "complex" refers to a group of two or more polynucleotides and / or polypeptides that are associated. In the context of forming a complex, the term "associated" or "association" refers to molecules that are not covalently bound to one another but are bound to one another by electrostatic interactions, hydrophobic / hydrophilic interactions, and / or hydrogen bonding interactions. Molecules that include different moieties that are covalently bound to one another are known. In some embodiments, a complex forms when all components of the complex are present together, i.e., a self-assembled complex. In some embodiments, a complex forms by chemical interactions between different components of the complex, such as hydrogen bonding. In some embodiments, a polynucleotide provided herein forms a complex with a protein provided herein due to secondary structure recognition of the polynucleotide by the protein. In some embodiments, the Cas binding region of a polynucleotide provided herein comprises a secondary structure that is recognized by a Cas nuclease, Cas nickase, or fusion protein provided herein.

[0138] Cas proteins As used herein, "Cas effector protein" is also referred to herein as "Cas protein" and includes both Cas nuclease and Cas nickase. Cas effector protein is part of the CRISPR / Cas system described herein. CRISPR / Cas system including Cas effector protein and polynucleotide (also referred to as "guide polynucleotide") can be utilized for site-specific genome modification. In some embodiments, CRISPR / Cas system includes Cas effector protein and guide polynucleotide including Cas binding region (which binds to and / or activates Cas protein) and guide sequence (which hybridizes to target sequence), where Cas effector protein and guide polynucleotide form a complex as described herein. In some embodiments, CRISPR / Cas system includes Cas effector protein, first polynucleotide including guide sequence, and second polynucleotide including Cas binding region, where first and second polynucleotide hybridize to each other and form a complex with Cas protein.

[0139] CRISPR / Cas systems can be classified into types I-VI based on the Cas effector proteins in the system. For example, Cas9 is found in type II systems, while Cas12 is found in type V systems. Each type can be further classified into subtypes. For example, type II can include subtypes II-A, II-B, and II-C, and type V can include subtypes VA and VB. Classification of CRISPR / Cas systems and Cas nucleases is further described, for example, in Makarova et al., Methods Mol Biol 1311:47-75 (2015); Makarova et al., The CRISPR Journal Oct 2018; 325-336; and Koonin et al., Phil Trans R Soc B 374:20180087 (2018). The Cas nucleases described herein can encompass any type or variant unless otherwise specified.

[0140] In some embodiments, the Cas effector protein is a Cas nuclease. Generally, the Cas effector nuclease can cause double-stranded polynucleotide cleavage, e.g., double-stranded DNA cleavage. Generally, the Cas nuclease can include one or more nuclease domains, such as RuvC and HNH, and can cleave double-stranded DNA. In some embodiments, the Cas nuclease includes a RuvC domain and an HNH domain, each of which cleaves one strand of double-stranded DNA. In some embodiments, the Cas nuclease generates blunt ends. In some embodiments, the Cas nuclease RuvC and HNH cleave each DNA strand at the same position, thereby generating blunt ends. In some embodiments, the Cas nuclease generates blunt ends. In some embodiments, the Cas nuclease RuvC and HNH cleave each DNA strand at a different position (i.e., cut with an "offset"), thereby generating sticky ends. As used herein, the terms "cohesive ends," "staggered ends," or "sticky ends" refer to nucleic acid fragments having strands of unequal length. In contrast to "blunt ends," cohesive ends are generated by staggered cuts of double-stranded nucleic acids (e.g., DNA). A sticky or cohesive end has a protruding single strand, or "overhang," of unpaired nucleotides, e.g., a 3' or 5' overhang.

[0141] In some embodiments, the Cas nuclease is a Cas9 nuclease. Exemplary Cas9 nucleases include, but are not limited to, Cas9 from Streptococcus pyogenes, Streptococcus thermophilus, Streptococcus mutans, Listeria innocua, Neisseria meningitidis, Staphylococcus aureus, Klebisella pneumoniae, and many other bacteria. Additional exemplary Cas9 nucleases are described, for example, in U.S. Patent Nos. 8,771,945, 9,023,649, 10,000,772, 10,407,697, and U.S. Patent Application Publication No. 2014 / 0068797. In some embodiments, the Cas9 nuclease is derived from S. pyogenes (SpCas9).

[0142] In some embodiments, the Cas9 nuclease comprises a sequence disclosed in UniProt ID G3ECR1 (SEQ ID NO:1), UniProt ID Q99ZW2 (SEQ ID NO:2), or UniProt ID J7RUA5 (SEQ ID NO:3). In some embodiments, the Cas9 comprises a polypeptide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or about 100% sequence identity to any of SEQ ID NOs:1-3. In some embodiments, the disclosure provides a polynucleotide encoding a polypeptide having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or about 100% sequence identity to any of SEQ ID NOs:1-3. In some embodiments, the Cas9 is encoded by a polynucleotide that is codon-optimized for expression in a host cell.

[0143] In some embodiments, the Cas9 nuclease is a type IIB Cas9 nuclease. Generally, type IIB Cas9 proteins are capable of generating sticky ends as described herein. Exemplary type IIB Cas9 proteins include, but are not limited to, Cas9 proteins from Legionella pneumophila, Francisella novicida, Parasutterella excrementihominis, Sutterella wadsworthensis, Wolinella succinogenes, and many other bacteria. Additional type IIB Cas9 proteins are described, for example, in WO 2019 / 099943.

[0144] In some embodiments, the Cas effector protein is a Cas12 nuclease. In some embodiments, the Cas nuclease is a Cas12a nuclease (previously known as "Cpf1" or "C2c1"). In some embodiments, the Cas nuclease is a Cas12f nuclease. Cas12f nuclease is also known in the art as Cas14 (Makarova et al, Nature Rev. Microbiol., 2019, 18:67-83). In some embodiments, the Cas nuclease is a Cas14 nuclease. Cas12 nucleases are generally smaller than Cas9 nucleases and are typically capable of generating sticky ends. Exemplary Cas12 proteins include, but are not limited to, Cas12 proteins from Francisella novicida, Acidaminococcus sp., Lachnospiraceae sp., Prevotella sp., and many other bacteria. Additional Cas12 nucleases are described, for example, in U.S. Pat. No. 9,580,701, U.S. Patent Application Publication No. 2016 / 0208243, Zetsche et al., Cell 163(3):759-771 (2015), and Chen et al., Science 360:436-439 (2018).

[0145] In some embodiments, the Cas12 nuclease comprises a sequence disclosed by UniProt ID A0Q7Q2 (SEQ ID NO:4), UniProt ID U2UMQ6 (SEQ ID NO:5), or UniProt ID T0D7A2 (SEQ ID NO:6). In some embodiments, the Cas12 has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or about 100% sequence identity to any of SEQ ID NOs:4-6. In some embodiments, the disclosure provides a polynucleotide encoding a polypeptide having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or about 100% sequence identity to any of SEQ ID NOs:4-6. In some embodiments, Cas12 is encoded by a polynucleotide that is codon-optimized for expression in a host cell.

[0146] In some embodiments, the Cas effector protein is a Cas nickase. A nickase produces a single-stranded cleavage in a double-stranded polynucleotide (e.g., DNA) and is distinct from a nuclease that cleaves both strands of a double-stranded polynucleotide (e.g., DNA). As described herein, wild-type Cas nucleases typically contain two catalytic nuclease domains, RuvC and HNH, each responsible for cleaving one strand of double-stranded DNA. Thus, in some embodiments, the Cas nickase contains amino acid mutations in the catalytic domain compared to a Cas nuclease. Cas nickases are further described in, for example, Cho et al., Genome Res 24:132-141 (2013), Ran et al., Cell 154:1380-1389 (2013), and Mali et al., Nat Biotechnol 31:833-838 (2013).

[0147] In some embodiments, the Cas nickase is a Cas9 nickase. In some embodiments, the Cas nickase is a Cas12a nickase. In some embodiments, the Cas nickase is a type II-B Cas nickase. In some embodiments, the Cas nickase is generated by mutating a Cas nuclease. For example, the SpCas9 nickase contains a D10A mutation or a H840A mutation compared to a wild-type SpCas9 nuclease. It will be understood by those skilled in the art that the alignment method as described herein can be used to determine the corresponding amino acid residues of other Cas nucleases (e.g., Cas12a or type II-B Cas nucleases) to generate a Cas nickase.

[0148] In some embodiments, the Cas nuclease or Cas nickase of the composition is not fused to a heterologous protein domain, hi some embodiments, the Cas nuclease or Cas nickase is not fused to a DNA polymerase, a DNA ligase, or a reverse transcriptase.

[0149] In some embodiments, the recombinant Cas effector proteins of the present disclosure are part of a fusion protein that includes one or more heterologous protein domains (e.g., about or at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more domains in addition to the recombinant Cas effector protein). The Cas fusion protein may include any additional protein sequences, and optionally a linker sequence between any two domains. Examples of protein domains that may be fused to a recombinant Cas9 protein include, but are not limited to, epitope tags, reporter gene sequences, and protein domains having one or more of the following activities: methylase activity, demethylase activity, transcriptional activation activity, transcriptional repression activity, transcriptional terminator activity, histone modification activity, RNA cleavage activity, and nucleic acid binding activity. Non-limiting examples of epitope tags include histidine (His) tags, V5 tags, FLAG tags, influenza hemagglutinin (HA) tags, Myc tags, VSV-G tags, and thioredoxin (Trx) tags. Examples of reporter genes include, but are not limited to, glutathione-5-transferase (GST), horseradish peroxidase (HRP), chloramphenicol acetyltransferase (CAT), beta-galactosidase, beta-glucuronidase, luciferase, green fluorescent protein (GFP), HcRed, DsRed, cyan fluorescent protein (CFP), yellow fluorescent protein (YFP), autofluorescent proteins such as blue fluorescent protein (BFP) and mCherry. In some embodiments, the recombinant Cas effector protein is fused to a protein or a fragment of a protein that binds to a DNA molecule or binds to another cellular molecule, such as, but not limited to, maltose binding protein (MBP), S-tag, Lex A DNA binding domain (DBD), GAL4 DNA binding domain, and herpes simplex virus (HSV) BP16 protein. Additional domains that may form part of a fusion protein that includes a Cas effector protein are described in US Patent Application Publication No. 2011 / 0059502.In some embodiments, tagged recombinant Cas proteins are used to identify the location of target sequences.

[0150] In some embodiments, the Cas effector protein is fused to a heterologous protein or protein domain. In some embodiments, the Cas effector protein is fused to a reverse transcriptase. In some embodiments, the Cas effector protein is a Cas9 nuclease fused to a reverse transcriptase. Examples of such Cas9-reverse transcriptase fusions are described in Anzalone et al., Nature, 576:149-157 (2019).

[0151] In some embodiments, the Cas effector protein is fused to a DNA polymerase. In some embodiments, the Cas effector protein is a Cas9 nuclease fused to a DNA polymerase.

[0152] In some embodiments, the Cas effector protein is fused to dominant negative 53BP1 (TP53BP1, also known as tumor suppressor p53 binding protein 1). In some embodiments, the Cas effector protein is a Cas9 nuclease fused to a dominant negative 53BP1 protein. In some embodiments, the dominant negative 53BP1 protein is DN1S. In some embodiments, the Cas effector protein is a Cas9 nuclease fused to DN1S.

[0153] In some embodiments, the Cas effector protein is fused to a geminin degron domain. In some embodiments, the Cas effector protein is a Cas9 nuclease fused to a geminin degron domain. Examples of such proteins are described in Gutschner et al., Cell Reports, 14:1555-1566 (2016).

[0154] In some embodiments, the Cas effector protein is fused to a CtIP (C-terminal binding protein 1) protein. In some embodiments, the Cas effector protein is a Cas9 nuclease fused to a CtIP protein.

[0155] In some embodiments, the recombinant Cas effector protein may form a component of an inducible system. The inducibility of the system allows for spatiotemporal control of gene editing or gene expression using forms of energy. The forms of energy may include, but are not limited to, electromagnetic radiation, acoustic energy, chemical energy, and thermal energy:. Non-limiting examples of inducible systems include tetracycline-inducible promoters (Tet-On or Tet-Off), small molecule two-hybrid transcription activation systems (FKBP, ABA, etc.) or light-inducible systems (phytochromes, LOV domains, or cryptochromes). In some embodiments, the Cas effector protein is part of a light-inducible transcriptional effector (LITE) to direct changes in transcriptional activity in a sequence-specific manner. The light components may include the Cas effector protein, a light-responsive cytochrome heterodimer (e.g., from Arabidopsis thaliana), and a transcription activation / repression domain. Further examples of inducible DNA binding proteins and methods of their use are provided in International Patent Application Publication Nos. WO 2014 / 018423 and 2014 / 093635; U.S. Pat. Nos. 8,889,418 and 8,895,308; and U.S. Patent Application Publication Nos. 2014 / 0186919, 2014 / 0242700, 2014 / 0273234, and 2014 / 0335620.

[0156] [Table 1]

[0157] [Table 2]

[0158] [Table 3]

[0159] [Table 4]

[0160] [Table 5]

[0161] nucleotide i. Sequence of interest In some embodiments, the polynucleotide of the present disclosure is an exogenous polynucleotide that comprises a sequence of interest (SOI) to be inserted into the genome of a eukaryotic cell. In some embodiments, the sequence of interest comprises a gene of interest.

[0162] In some embodiments, the polynucleotide comprising the exogenous polynucleotide comprising the SOI is an exogenous polynucleotide template that is inserted into the genome of the eukaryotic cell via CRISPR / Cas-mediated homologous recombination. In some embodiments, the SOI comprises at least one mutation of interest that is inserted into the genome of the eukaryotic cell. In some embodiments, the SOI comprises a gene of interest that is inserted into the genome of the eukaryotic cell. In some embodiments, the SOI can be introduced as an exogenous polynucleotide template. In some embodiments, the SOI is a hybrid polynucleotide that comprises single-stranded and double-stranded regions. In some embodiments, the hybrid polynucleotide comprises double-stranded sequences at the 5' and 3' ends and an internal single-stranded sequence (Shy et al, bioRxiv,2021,preprint published 9 / 2 / 2021). In some embodiments, the exogenous polynucleotide comprises a blunt end. In some embodiments, the exogenous polynucleotide template comprises a sticky end. In some embodiments, the exogenous polynucleotide template comprises a sticky end that is complementary to the sticky end of the target sequence.

[0163] The exogenous polynucleotide template can be of any suitable length, for example, about or at least about 10, 15, 20, 25, 50, 75, 100, 150, 200, 250, 500, 1000, 5000, or 10000 or more nucleotides in length. In some embodiments, the exogenous polynucleotide template is complementary to a portion of the polynucleotide that comprises the target sequence. In some embodiments, the exogenous polynucleotide template overlaps with one or more nucleotides (e.g., about or at least about 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, or 100 or more nucleotides) of the target sequence when optimally aligned. In some embodiments, when the exogenous polynucleotide template and a polynucleotide comprising a target sequence are optimally aligned, the nearest nucleotide of the exogenous polynucleotide template is within about 1, 5, 10, 15, 20, 25, 50, 75, 100, 200, 300, 400, 500, 100, 1500, 2000, 2500, 5000, 10,000 or more nucleotides of the target sequence.

[0164] In some embodiments, the exogenous polynucleotide is DNA, such as a DNA plasmid, a bacterial artificial chromosome (BAC), a yeast artificial chromosome (YAC), a viral vector, a linear single-stranded or double-stranded piece of DNA, an oligonucleotide, a PCR fragment, a naked nucleic acid, or a nucleic acid complexed with a delivery vehicle such as a transfectosome. In some embodiments, the exogenous polynucleotide is RNA. In some embodiments, the RNA is messenger RNA (mRNA).

[0165] In some embodiments, the exogenous polynucleotide is inserted into the target sequence using the cell's endogenous DNA repair pathway. In some embodiments, the endogenous DNA repair pathway is HDR. During the repair process, an exogenous polynucleotide template comprising an SOI can be introduced into the target sequence. In some embodiments, an exogenous polynucleotide template comprising an SOI flanked by upstream and downstream sequences is introduced into the cell, the upstream and downstream sequences sharing sequence similarity with either side of the site of integration in the target sequence. In some embodiments, the exogenous polynucleotide comprising an SOI comprises, for example, a mutated gene. In some embodiments, the exogenous polynucleotide comprises a sequence that is endogenous or exogenous to the cell. In some embodiments, the SOI comprises a polynucleotide that codes for a protein or a non-coding sequence, such as, for example, a microRNA. In some embodiments, the SOI is operably linked to a regulatory element. In some embodiments, the SOI is a regulatory element. In some embodiments, the SOI comprises a resistance cassette, for example, a gene that confers resistance to an antibiotic. In some embodiments, the SOI comprises a mutation of the wild-type target sequence. In some embodiments, the SOI disrupts or corrects the target sequence by creating a frameshift mutation or a nucleotide substitution. In some embodiments, the SOI comprises a marker. The introduction of a marker into the target sequence can facilitate the screening of targeted integration. In some embodiments, the marker is a restriction site, a fluorescent protein, or a selection marker. In some embodiments, the SOI is introduced as a vector comprising the SOI.

[0166] The upstream and downstream sequences in the exogenous polynucleotide template are selected to promote homologous recombination between the target sequence and the exogenous polynucleotide. The upstream sequence is a nucleic acid sequence that shares sequence similarity with the sequence upstream of the target site for integration (i.e., the target sequence). Similarly, the downstream sequence is a nucleic acid sequence that shares sequence similarity with the sequence downstream of the target site for integration. Thus, in some embodiments, the exogenous polynucleotide template that comprises a SOI inserts into the target sequence by homologous recombination at the upstream and downstream sequences. In some embodiments, the upstream and downstream sequences in the exogenous polynucleotide template have at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the upstream and downstream sequences of the targeted genome sequence, respectively. In some embodiments, the upstream or downstream sequence has at least about 20, 50, 100, 150, 200, 250, 300, 350, 400, or 500 base pairs, and up to about 600, 750, 1000, 1250, 1500, 1750, or 2000 base pairs. In some embodiments, the upstream or downstream sequence has about 20-2000 base pairs, or about 50-1750 base pairs, or about 100-1500 base pairs, or about 200-1250 base pairs, or about 300-1000 base pairs, or about 400 to about 750 base pairs, or about 500-600 base pairs. In some embodiments, the upstream or downstream sequence has about 50, about 100, about 250, about 500, about 100, about 1250, about 1500, about 1750, about 2000, about 2250, or about 2500 base pairs.

[0167] In some embodiments, the SOI is a gene of interest. As used herein, the term "gene of interest" refers to a gene that encodes a biomolecule of interest (e.g., a protein or an RNA molecule). In some embodiments, the gene of interest encodes a protein of interest. In some embodiments, the protein of interest comprises an intracellular protein, a membrane protein, an extracellular protein, or a combination thereof. In some embodiments, the protein of interest comprises a nuclear protein, a transcription factor, a nuclear membrane transporter, an organelle-associated protein, a membrane receptor, a catalytic protein, an enzyme, a therapeutic protein, a membrane protein, a membrane trafficking protein, a signaling protein, an immunological protein, or a combination thereof. In some embodiments, the immunological protein comprises an antibody, e.g., IgG, IgA, IgM, IgD, IgE, or a combination thereof. In some embodiments, the immunological protein is a T cell receptor (TCR). In some embodiments, the immunological protein is a chimeric antigen receptor (CAR). In some embodiments, the SOI encodes a copy of a native gene of the host cell. In some embodiments, the SOI encodes a copy of a native gene that is missing in the host cell. In some embodiments, the host cell comprises a mutation in a gene and the SOI encodes a wild-type copy of the gene. In some embodiments, the host cell comprises a wild-type gene and the SOI encodes a copy of the gene that comprises a mutation of interest. In some embodiments, the SOI encodes a heterologous gene that does not naturally occur in the host cell.

[0168] In some embodiments, the gene of interest encodes an RNA of interest. In some embodiments, the RNA of interest comprises a therapeutic RNA. In some embodiments, the RNA of interest comprises messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), small nuclear RNA (snRNA), antisense RNA, microRNA (miRNA), small interfering RNA (siRNA), cell-free RNA (cfRNA), or a combination thereof. In some embodiments, the sequence of interest comprises a regulatory element of interest. In some embodiments, the SOI is inserted into a target polynucleotide of a host cell such that the regulatory element on the sequence of interest can regulate a native gene of the host cell. Regulatory elements are described herein and include, for example, promoters, enhancers, silencers, operators, response elements, 5'UTRs, 3'UTRs, insulators, and the like.

[0169] In some embodiments, the polynucleotide comprising an SOI is from about 1 nucleotide to about 5000 nucleotides in length. In some embodiments, the polynucleotide comprising an SOI is from about 5 nucleotides to about 5000 nucleotides in length. In some embodiments, the polynucleotide comprising an SOI is from about 6 nucleotides to about 1000 nucleotides in length. In some embodiments, the polynucleotide comprising an SOI is from about 7 nucleotides to about 750 nucleotides in length. In some embodiments, the polynucleotide comprising an SOI is from about 8 nucleotides to about 500 nucleotides in length. In some embodiments, the polynucleotide comprising an SOI is from about 9 nucleotides to about 250 nucleotides in length. In some embodiments, the polynucleotide comprising an SOI is from about 10 nucleotides to about 100 nucleotides in length. In some embodiments, the polynucleotide comprising an SOI is from about 15 nucleotides to about 90 nucleotides in length. In some embodiments, the polynucleotide comprising an SOI is from about 20 nucleotides to about 80 nucleotides in length. In some embodiments, the polynucleotide comprising an SOI is from about 25 nucleotides to about 70 nucleotides in length. In some embodiments, the polynucleotide comprising an SOI is from about 30 nucleotides to about 50 nucleotides in length. In some embodiments, the SOI-containing polynucleotide is from about 1 nucleotide to about 10 nucleotides in length. In some embodiments, the SOI-containing polynucleotide is from about 1 nucleotide to about 20 nucleotides in length. In some embodiments, the SOI-containing polynucleotide is from about 1 nucleotide to about 30 nucleotides in length. In some embodiments, the SOI-containing polynucleotide is from about 10 nucleotides to about 40 nucleotides in length. In some embodiments, the SOI-containing polynucleotide is from about 1 nucleotide to about 50 nucleotides in length.In some embodiments, a polynucleotide comprising an SOI is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides in length. In some embodiments, a polynucleotide comprising an SOI is greater than about 10 nucleotides in length, greater than about 15 nucleotides in length, greater than about 20 nucleotides in length, greater than about 25 nucleotides in length, greater than about 30 nucleotides in length, greater than about 35 nucleotides in length, greater than about 40 nucleotides in length, greater than about 45 nucleotides in length, or greater than about 50 nucleotides in length.

[0170] In some embodiments, the SOI is about 3 to about 5000 nucleotides in length. In some embodiments, the SOI is about 4 to about 1000 nucleotides in length. In some embodiments, the SOI is about 5 to about 900 nucleotides in length. In some embodiments, the SOI is about 6 to about 800 nucleotides in length. In some embodiments, the SOI is about 7 to about 700 nucleotides in length. In some embodiments, the SOI is about 8 to about 600 nucleotides in length. In some embodiments, the SOI is about 9 to about 500 nucleotides in length. In some embodiments, the SOI is about 50 to about 5000 nucleotides in length. In some embodiments, the SOI is about 60 to about 1000 nucleotides in length. In some embodiments, the SOI is about 70 to about 900 nucleotides in length. In some embodiments, the SOI is about 8 to about 800 nucleotides in length. In some embodiments, the SOI is about 90 to about 700 nucleotides in length. In some embodiments, the SOI is about 100 to about 500 nucleotides in length. In some embodiments, the SOI is about 100 to about 250 nucleotides in length. In some embodiments, the SOI is about 10 to about 90 nucleotides in length. In some embodiments, the SOI is about 11 to about 80 nucleotides in length. In some embodiments, the SOI is about 12 to about 70 nucleotides in length. In some embodiments, the SOI is about 15 to about 60 nucleotides in length. In some embodiments, the SOI is about 10 to about 50 nucleotides in length. In some embodiments, the SOI is about 1 to about 10 nucleotides in length. In some embodiments, the SOI is about 1 to about 25 nucleotides in length. In some embodiments, the SOI is about 1 to about 50 nucleotides in length. In some embodiments, the SOI is about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 nucleotides in length.In some embodiments, the SOI is greater than about 10 nucleotides in length, greater than about 15 nucleotides in length, greater than about 20 nucleotides in length, greater than about 25 nucleotides in length, greater than about 30 nucleotides in length, greater than about 35 nucleotides in length, greater than about 40 nucleotides in length, greater than about 45 nucleotides in length, or greater than about 50 nucleotides in length.

[0171] ii. Cas and Cas-related polynucleotides In some embodiments, the disclosure encompasses nucleotide or polynucleotide sequences that encode the Cas effector proteins of the disclosure, i.e., Cas polynucleotides.

[0172] In some embodiments, the polynucleotide of the disclosure can be complexed with a Cas effector protein. In some embodiments, the polynucleotide that can be complexed with a Cas effector protein comprises a guide sequence. In some embodiments, the polynucleotide that can be complexed with a Cas effector protein comprises a Cas binding region. In some embodiments, the polynucleotide that can be complexed with a Cas effector protein comprises a DNA template sequence. In some embodiments, the polynucleotide that can be complexed with a Cas effector protein comprises a guide sequence, a Cas binding region, and a DNA template sequence, or any combination thereof. In some embodiments, the polynucleotide comprises, in 5' to 3' order, a guide sequence, a Cas binding region, and a DNA template sequence.

[0173] In some embodiments, the guide sequence can hybridize to a target polynucleotide, e.g., a target polynucleotide in a host cell genome. In some embodiments, the guide sequence is complementary to the target polynucleotide. In some embodiments, the target polynucleotide is a target DNA intended to be cleaved by a Cas nuclease or Cas nickase. In some embodiments, the guide sequence comprises RNA, i.e., is an RNA guide sequence. In some embodiments, the guide sequence comprises a combination of RNA and DNA. Hybrid RNA-DNA guide sequences are further described, for example, in Rueda et al., Nat Comm 8:1610 (2017).

[0174] In some embodiments, the guide sequence is about 10 to about 40 nucleotides in length. In some embodiments, the guide sequence is about 12 to about 30 nucleotides in length. In some embodiments, the guide sequence is about 15 to about 20 nucleotides in length. In some embodiments, the guide sequence is about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, or about 40 nucleotides in length. In some embodiments, the guide sequence is of sufficient length to hybridize to the target polynucleotide.

[0175] In some embodiments, the Cas binding region can bind to a Cas effector protein (e.g., a Cas nuclease or a Cas nickase), thereby forming a complex with the Cas protein. In some embodiments, the Cas binding region comprises RNA. In some embodiments, the Cas binding region comprises a combination of RNA and DNA. Hybrid RNA-DNA sequences that bind to and / or activate Cas proteins are further described, for example, in Rueda et al., Nat Comm 8:1610 (2017).

[0176] In some embodiments, multiple guide RNAs described in the methods, kits, and compositions described herein can be used in the same method, kit, or composition, for example, in some embodiments, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more different guide RNAs can be used simultaneously.

[0177] In some embodiments, the Cas binding region comprises a tracrRNA that binds to and activates a Cas protein. In some embodiments, the Cas binding region can hybridize with a tracrRNA, and the composition further comprises a tracrRNA. In some embodiments, the tracrRNA can bind to a Cas nuclease or Cas nickase. In some embodiments, the tracrRNA can activate a Cas nuclease or Cas nickase. In some embodiments, the activation includes initiating or increasing the cleavage activity of a Cas nuclease or Cas nickase. In some embodiments, the activation includes promoting binding of a Cas nuclease or Cas nickase to a target polynucleotide (e.g., by guiding the Cas nuclease or Cas nickase with a guide sequence). In some embodiments, the activation includes a combination of promoting binding of a Cas nuclease or Cas nickase to a target polynucleotide and initiating or increasing the cleavage activity of a Cas nuclease or Cas nickase. TracrRNA sequences for Cas proteins (e.g., Cas9, Cas12a, or type II-B Cas proteins described herein) are available from public databases including RNAcentral and Rfam, and are further described, for example, in Chylinski et al., RNA Biol 10(5):726-737 (2013) and Gasiunas et al., Nat Comm 11:5512 (2020).

[0178] In some embodiments, the polynucleotide capable of forming a complex with the Cas effector molecule comprises a DNA template sequence at the 3' end of the polynucleotide. In some embodiments, the DNA template sequence comprises a single stranded DNA. In some embodiments, the DNA template sequence comprises a sequence of interest. In some embodiments, the DNA template sequence comprises a primer binding sequence and a sequence of interest. In some embodiments, the DNA template sequence comprises a template for amplification by a DNA polymerase. In some embodiments, the sequence of interest comprises a template for amplification by a DNA polymerase. In some embodiments, the Cas nuclease or Cas nickase of the composition is guided to the target polynucleotide by the guide sequence to cleave the target polynucleotide, and one strand of the cleaved target polynucleotide hybridizes to the primer binding sequence and serves as a primer for the DNA polymerase. In some embodiments, the DNA polymerase can synthesize a DNA strand complementary to the SOI to form a double-stranded sequence including the SOI. In some embodiments, a double-stranded sequence comprising an SOI is inserted into a cleaved target polynucleotide, for example, by ligation or a DNA repair pathway described herein.

[0179] In some embodiments, the DNA template sequence is from about 5 nucleotides to about 5000 nucleotides in length. In some embodiments, the DNA template sequence is from about 6 nucleotides to about 1000 nucleotides in length. In some embodiments, the DNA template sequence is from about 7 nucleotides to about 750 nucleotides in length. In some embodiments, the DNA template sequence is from about 8 nucleotides to about 500 nucleotides in length. In some embodiments, the DNA template sequence is from about 9 nucleotides to about 250 nucleotides in length. In some embodiments, the DNA template sequence is from about 10 nucleotides to about 100 nucleotides in length. In some embodiments, the DNA template sequence is from about 15 nucleotides to about 90 nucleotides in length. In some embodiments, the DNA template sequence is from about 20 nucleotides to about 80 nucleotides in length. In some embodiments, the DNA template sequence is from about 25 nucleotides to about 70 nucleotides in length. In some embodiments, the DNA template sequence is from about 30 nucleotides to about 50 nucleotides in length. In some embodiments, the DNA template sequence is about 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides in length. In some embodiments, the DNA template sequence is greater than about 10 nucleotides in length, greater than about 15 nucleotides in length, greater than about 20 nucleotides in length, greater than about 25 nucleotides in length, greater than about 30 nucleotides in length, greater than about 35 nucleotides in length, greater than about 40 nucleotides in length, greater than about 45 nucleotides in length, or greater than about 50 nucleotides in length.

[0180] In some embodiments, the DNA template sequence comprises a primer binding sequence. In some embodiments, the primer binding sequence is about 3 to about 50 nucleotides in length. In some embodiments, the primer binding sequence is about 4 to about 45 nucleotides in length. In some embodiments, the primer binding sequence is about 5 to about 40 nucleotides in length. In some embodiments, the primer binding sequence is about 6 to about 35 nucleotides in length. In some embodiments, the primer binding sequence is about 7 to about 30 nucleotides in length. In some embodiments, the primer binding sequence is about 8 to about 25 nucleotides in length. In some embodiments, the primer binding sequence is about 10 to about 20 nucleotides in length. In some embodiments, the primer binding sequence is about 4 to about 30 nucleotides in length. In some embodiments, the primer binding sequence is about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 nucleotides in length. In some embodiments, the primer binding sequence is of sufficient length to hybridize with a region of the cleaved target DNA sequence.

[0181] In some embodiments, the polynucleotide comprising the DNA template sequence comprises modified nucleotides, non-B-type DNA structures, DNA polymerase recruiting moieties, DNA ligase recruiting moieties, or combinations thereof.

[0182] In some embodiments, the polynucleotide comprising the DNA template sequence comprises modified nucleotides, hi some embodiments, the modified nucleotides comprise an abasic site, a covalent linker, a xenonucleic acid (XNA), a locked nucleic acid (LNA), a peptide nucleic acid (PNA), a phosphorothioate bond, a DNA lesion, a DNA photoproduct, a modified deoxyribonucleoside, a methylated nucleotide, or a combination thereof.

[0183] In some embodiments, the modified nucleotide reduces or prevents over-extension of the sequence of interest by a DNA polymerase. In some embodiments, reducing or preventing over-extension of the sequence of interest by a DNA polymerase increases the accuracy of insertion of a double-stranded sequence containing the sequence of interest. In some embodiments, the modified nucleotide comprises an abasic site, also known as an apurinated / apyrimidinic site (AP site). In some embodiments, the modified nucleotide comprises a covalent linker. In some embodiments, the covalent linker comprises a triethylene glycol (TEG) linker. In some embodiments, the covalent linker comprises an amino linker. TEG linkers and amino linkers have been shown to inhibit polymerase extension, see, e.g., Strobel et al., bioRxiv doi:10.1101 / 2019.12.26.888743 (23 January 2020).

[0184] In some embodiments, the modified nucleotide reduces or prevents nuclease degradation of the polynucleotides of the present disclosure. In some embodiments, the modified nucleotide comprises a xenonucleic acid (XNA). XNA is a synthetic nucleotide analog with a sugar group different from the deoxyribose of DNA or the ribose of RNA. Exemplary sugar groups of XNA include, but are not limited to, threose, cyclohexene, glycol, or locked ribose. In some embodiments, the XNA comprises 1,5-anhydrohexitol nucleic acid (HNA), cyclohexene nucleic acid (CeNA), threose nucleic acid (TNA), glycol nucleic acid (GNA), locked nucleic acid (LNA), and peptide nucleic acid (PNA). In some embodiments, the modified nucleotide comprises a locked nucleic acid (LNA), also known as a bridged nucleic acid (BNA). LNA is a modified RNA nucleotide in which the ribose moiety is modified by an additional bridge connecting the 2' oxygen and the 4' carbon. In some embodiments, the modified nucleotide comprises a peptide nucleic acid (PNA). Unlike the deoxyribose or ribose backbone of DNA or RNA, the backbone of a PNA polymer comprises N-(2-aminoethyl)-glycine units linked by peptide bonds, with purine and pyrimidine bases linked to the PNA backbone by methylene bridges and carbonyl groups. In some embodiments, the modified nucleotide comprises a phosphorothioate bond. A phosphorothioate bond comprises a sulfur atom in place of one of the oxygens in the phosphate group that connects two nucleotides. In some embodiments, the presence of XNA, such as LNA or PNA, or phosphorothioate bond in a polynucleotide increases the stability of the polynucleotide against nuclease degradation.

[0185] In some embodiments, the presence of modified nucleotides in a polynucleotide (e.g., a polynucleotide of a composition provided herein) can recruit a DNA polymerase to the polynucleotide. In some embodiments, recruiting a DNA polymerase includes, for example, increasing the likelihood that the DNA polymerase will recognize the polynucleotide due to the presence of modified nucleotides in the polynucleotide; promoting the binding of the DNA polymerase to the polynucleotide; and / or activating the DNA polymerase, for example, initiating or increasing the activity of the DNA polymerase. In some embodiments, the recruited DNA polymerase binds to the cleaved strand of the target polynucleotide and extends the sequence of interest on the DNA template sequence, as described herein.

[0186] In some embodiments, the modified nucleotide comprises a DNA lesion. As used herein, "DNA lesion" refers to a region of a DNA polynucleotide that contains base mutations, base deletions, and / or sugar mutations that typically indicate DNA damage. The DNA lesion may result from hydrolysis, oxidation, alkylation, depurination, depyrimidination, and / or deamination of nucleobases. In some embodiments, the DNA lesion is capable of recruiting DNA polymerase. In some embodiments, the DNA lesion comprises 8-oxoguanine, thymine-glycol, N7-(2-hydroxyethyl)guanine (7HEG), 7-(2-oxoethyl)guanine, or a combination thereof. In some embodiments, the DNA lesion comprises 8-oxoguanine, thymine-glycol, or a combination thereof.

[0187] In some embodiments, the modified nucleotide comprises a DNA photoproduct. The DNA photoproduct is an ultraviolet (UV)-induced DNA lesion, as further described, for example, in Yokoyama et al., Int J Mol Sci 15(11):20321-20338 (2014). In some embodiments, the DNA photoproduct can recruit a DNA polymerase. In some embodiments, the DNA photoproduct comprises a pyrimidine dimer, a cyclobutane pyrimidine dimer (CPD), a pyrimidine (6-4) pyrimidone photoproduct (also referred to as "(6-4) photoproduct"), an adenine-thymine heterodimer, a Dewar pyrimidinone, or a combination thereof. In some embodiments, the DNA photoproduct comprises a CPD, a (6-4) photoproduct, or a combination thereof.

[0188] In some embodiments, the modified nucleotide comprises a modified deoxyribonucleoside. In some embodiments, the modified deoxyribonucleoside is capable of recruiting DNA polymerase. In some embodiments, the modified deoxyribonucleoside comprises a base that is not typically present in DNA, i.e., adenine, cytosine, guanine, or thymine. In some embodiments, the modified deoxyribonucleoside comprises deoxyuridine, acrolein-deoxyguanine, malondialdehyde-deoxyguanine, deoxyinosine, deoxyxanthosine, or a combination thereof. In some embodiments, the modified deoxyribonucleoside comprises deoxyuridine.

[0189] In some embodiments, the modified nucleotide comprises one or more methylated nucleotides. In some embodiments, the methylated nucleotide, e.g., methylated cytosine, can recruit DNA polymerase. In some embodiments, the methylated nucleotide comprises 5-hydroxymethylcytosine, 5-methylcytosine, or a combination thereof.

[0190] In some embodiments, the DNA template sequence comprises a non-B DNA structure. As used herein, a "non-B DNA structure" refers to a secondary structural conformation of DNA that is not a standard right-handed B DNA helix. Non-limiting examples of non-B DNA structures include G-quadruplexes, triplex DNA (H-DNA), Z-DNA, cruciforms, slipped DNA strands, A-tract bending, and sticky DNA. Non-B DNA structures are further described, for example, in Guiblet et al., Nucleic Acids Res 49(3):1497-1516 (2021). In some embodiments, the non-B DNA structure can recruit DNA polymerase. In some embodiments, the non-B DNA structure comprises a hairpin, a cruciform, a Z-DNA, an H-DNA (triple-stranded DNA), a G-quadruplex DNA (quadruplex DNA), slipped DNA, sticky DNA, or a combination thereof.

[0191] In some embodiments, the DNA template sequence comprises a DNA polymerase recruitment portion. The recruitment of DNA polymerase is described herein. Non-limiting examples of DNA polymerase that can be recruited by DNA polymerase recruitment portion include bacterial DNA polymerase such as Pol I (including its Klenow fragment), Pol II, Pol III, Pol IV, or Pol V; eukaryotic DNA polymerase such as Pol α, Pol β, Pol λ, Pol γ, Pol σ, Pol μ, Pol δ, Pol ε, Pol η, Pol ι, Pol κ, Pol ζ, Pol θ, REV1, or REV3; isothermal DNA polymerase such as Bst, T4, or Φ29 (phi29) DNA polymerase; thermostable DNA polymerase such as Taq, Pfu, KOD, Tth, or Pwo DNA polymerase; or variants or homologs thereof.

[0192] In some embodiments, the polynucleotides of the present disclosure can be chemically crosslinked to one or more moieties or conjugates that enhance the activity, cellular distribution, or cellular uptake of the polynucleotide. These moieties or conjugates can include conjugate groups covalently attached to functional groups such as primary or secondary hydroxyl groups. Conjugate groups include, but are not limited to, intercalators, reporter molecules, polyamines, polyamides, polyethylene glycols, polyethers, groups that enhance the pharmacological properties of the oligomer, and groups that enhance the pharmacokinetic properties of the oligomer. Suitable conjugate groups include, but are not limited to, cholesterol, lipids, phospholipids, biotin, phenazine, folic acid, phenanthridine, anthraquinone, acridine, fluorescein, rhodamine, coumarin, and dyes. Groups that enhance pharmacological properties include groups that promote uptake, increase resistance to degradation, and / or enhance sequence-specific hybridization with the target nucleic acid. Groups that enhance pharmacokinetic properties include groups that improve uptake, distribution, metabolism, or excretion of the nucleic acid of interest.

[0193] Conjugate moieties include lipid moieties such as cholesterol moieties (Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86, 6553-6556), cholic acid (Manoharan et al., Bioorg. Med. Chem. Let., 1994, 4, 1053-1060), thioethers such as hexyl-S-tritylthiol (Manoharan et al., Ann. NY Acad. Sci., 1992, 660, 306-309; Manoharan et al., Bioorg. Med. Chem. Let., 1993, 3, 2765-2770), thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20, 533-538), fatty chains such as dodecanediol or undecyl residues (Saison-Behmoaras et al., EMBO J., 1991, 10, 1111-1118; Kabanov et al., FEBS Lett., 1990, 259, 327-330; Svinarchuk et al., Biochimie, 1993, 75, 49-54), phospholipids such as di-hexadecyl-rac-glycerol or triethyl-ammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654; Shea et al., Nucl. Acids, 1997, 20, 533-538), Res., 1990, 18, 3777-3783), polyamine or polyethylene glycol chains (Manoharan et al., Nucleosides & Nucleotides, 1995, 14, 969-973), adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654), palmityl moieties (Mishra et al., Biochim. Biophys. Acta, 1995, 1264, 229-237), or octadecylamine or hexylamino-carbonyl-oxycholesterol moieties (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277, 923-937.

[0194] Conjugates may include "protein transduction domains" or PTDs (also known as CPPs-cell penetrating peptides), which may refer to polypeptides, polynucleotides, carbohydrates, or organic or inorganic compounds that facilitate crossing lipid bilayers, micelles, cell membranes, organelle membranes, or vesicle membranes. PTDs bound to another molecule range from small polar molecules to large macromolecules and / or nanoparticles, and facilitate crossing of the molecule through a membrane, for example, from the extracellular space to the intracellular space, or from the cytosol into an organelle. In some embodiments, the PTD is covalently attached to the amino terminus of an exogenous polypeptide (e.g., a site-directed modifying polypeptide). In some embodiments, the PTD is covalently attached to the carboxyl terminus of an exogenous polypeptide (e.g., a site-directed modifying polypeptide). In some embodiments, the PTD is covalently attached to a nucleic acid (e.g., a DNA-targeting RNA, a polynucleotide encoding a DNA-targeting RNA, a polynucleotide encoding a site-directed modifying polypeptide, etc.).Exemplary PTDs include a minimal undecapeptide protein transduction domain (YGRKKRRQRRR; corresponding to residues 47-57 of HIV-1 TAT, which contains SEQ ID NO:7); a polyarginine sequence containing a sufficient number of arginines to direct cell entry (e.g., 3, 4, 5, 6, 7, 8, 9, 10, or 10-50 arginines); a VP22 domain (Zender et al. (2002) Cancer Gene Ther. 9(6):489-96); the Drosophila antennapedia protein transduction domain (Noguchi et al. (2003) Diabetes 52(7):1732-1737); a truncated human calcitonin peptide (Trehin et al. (2004) Pharm. Research 21:1248-1256); polylysine (Wender et al. (2000) Proc Natl. Acad. Sci. USA 97:13003-13008); RRQRRTSKLMKR (SEQ ID NO: 8); transportan GWTLNSAGYLLGKINLKALAALAKKIL (SEQ ID NO: 9); KALAWEAKLAKALAKALAKHLAKALAKALKCEA (SEQ ID NO: 10); and RQIKIWFQNRRMKWKK (SEQ ID NO: 11). Exemplary PTDs include, but are not limited to, YGRKKRRQRRR (SEQ ID NO: 12), RKKRRQRRR (SEQ ID NO: 13); arginine homopolymers of 3 arginine residues to 50 arginine residues; exemplary PTD domain amino acid sequences include, but are not limited to, any of the following: YGRKKRRQRRR (SEQ ID NO: 14); RKKRRQRR (SEQ ID NO: 15); YARAAARQARA (SEQ ID NO: 16); THRLPRRRRRR (SEQ ID NO: 17); and GGRRARRRRRR (SEQ ID NO: 18). In some embodiments, the PTD is an activatable CPP (ACPP) (Aguilera et al. (2009) Integr Biol (Camb) June; 1(5-6):371-381).ACPPs contain a polycationic CPP (e.g., Arg9 or "R9") linked to a corresponding polyanion (e.g., Glu9 or "E9") by a cleavable linker, which reduces the net charge to near zero, thereby inhibiting adhesion and uptake into cells. Upon cleavage of the linker, the polyanion is released, locally unmasking the polyarginine and its inherent adhesive properties, thereby "activating" the ACPP to cross membranes.

[0195] In some embodiments, the polynucleotides of the present disclosure are codon-optimized for expression in eukaryotic cells. In some embodiments, the polynucleotide sequence encoding stiCas9 is codon-optimized for expression in animal cells. In some embodiments, the polynucleotide sequence encoding the recombinant Cas effector protein is codon-optimized for expression in human cells. In some embodiments, the polynucleotide sequence encoding the recombinant Cas effector protein is codon-optimized for expression in plant cells. Codon optimization is the adjustment of codons to match the host tRNA abundance in order to increase the yield and efficiency of recombinant or heterologous protein expression. Codon optimization is a routine procedure in the art and can be performed using software programs such as the Codon Optimization tool from Integrated DNA Technologies, the Codon Usage Table analysis tool from Entelechon, the Blue Heron software from GENEMAKER, the Gene Forge software from Aptagen, the DNA Builder software, the General Codon Usage Analysis software, the publicly available OPTIMIZER software, and the OptimumGene algorithm from Genscript.

[0196] CRISPR-Cas system In some embodiments, the disclosure encompasses a CRISPR-Cas system comprising a naturally occurring or non-naturally occurring Cas effector protein and a polynucleotide encoding a sequence of interest. In some embodiments, the CRISPR-Cas system comprises a naturally occurring or non-naturally occurring Cas effector protein, a polynucleotide encoding a sequence of interest, and a polynucleotide capable of forming a complex with the Cas effector protein. In some embodiments, the polynucleotide capable of forming a complex with the Cas effector protein comprises a guide sequence, a Cas binding region, and a DNA template region.

[0197] In some embodiments, the CRISPR-Cas system comprises a regulatory element operably linked to a polynucleotide sequence encoding a recombinant Cas effector protein provided herein, and a polynucleotide that forms a complex with the recombinant Cas effector protein and comprises a guide sequence.

[0198] In some embodiments, the regulatory element associated with the polynucleotide sequence encoding the recombinant Cas effector protein is a promoter. In some embodiments, the regulatory element is a eukaryotic promoter. In some embodiments, the regulatory element is a viral promoter. In some embodiments, the regulatory element is a eukaryotic regulatory element, i.e., a eukaryotic promoter. In some embodiments, the eukaryotic regulatory element is a mammalian promoter.

[0199] In some embodiments, the polynucleotide capable of forming a complex with the Cas effector protein of the CRISPR-Cas system is an RNA molecule. The RNA molecule that binds to the CRISPR-Cas components and targets them to a specific location in the target DNA is referred to herein as a "guide RNA", "gRNA" or "small molecule guide RNA", and may also be referred to herein as a "DNA-targeting RNA". A guide polynucleotide, such as a guide RNA, comprises at least two nucleotide segments, namely at least one "DNA-binding segment" and at least one "polypeptide-binding segment". By "segment" is meant a part, section or region of a molecule, such as a contiguous stretch of nucleotides of a guide polynucleotide molecule. The definition of "segment" is not limited to a specific number of total base pairs, unless specifically defined otherwise.

[0200] In some embodiments, the DNA-binding segment of a guide polynucleotide (or "DNA targeting sequence") hybridizes with a target sequence in a cell. In some embodiments, the DNA-binding segment of a guide polynucleotide, e.g., a guide RNA, comprises a polynucleotide sequence that is complementary to a specific sequence in the target DNA.

[0201] In some embodiments, the guide polynucleotide of the present disclosure has a guide sequence that hybridizes to a target sequence in a eukaryotic cell. In some embodiments, the eukaryotic cell is an animal or human cell. In some embodiments, the eukaryotic cell is a human, rodent or bovine cell line or cell line. Examples of such cells, cell lines or cell lines include, but are not limited to, mouse myeloma (NSO) cell lines, Chinese hamster ovary (CHO) cell lines, HT1080, H9, HepG2, MCF7, MDBK Jurkat, NIH3T3, PC12, BHK (baby hamster kidney cells), VERO, SP2 / 0, YB2 / 0, Y0, C127, L cells, COS, e.g., COS1 and COS7, QC1-3, HEK-293, VERO, PER.C6, HeLA, EB1, EB2, EB3, oncolytic or hybridoma cell lines. In some embodiments, the eukaryotic cell is a CHO cell line. In some embodiments, the eukaryotic cell is a CHO cell. In some embodiments, the cell is a CHO-K1 cell, a CHO-K1 SV cell, a DG44 CHO cell, a DUXB11 CHO cell, a CHOS, a CHO GS knockout cell, a CHO FUT8 GS knockout cell, a CHOZN, or a CHO-derived cell. A CHO GS knockout cell (e.g., a GSKO cell) is, for example, a CHO-K1 SV GS knockout cell. A CHO FUT8 knockout cell is, for example, a POTELLIGENT CHOK1 SV (Lonza Biologics, Inc.). The eukaryotic cell can also be an avian cell, cell line, or cell strain, such as an EBX cell, EB14, EB24, EB26, EB66, or EBvl3.

[0202] In some embodiments, the eukaryotic cell is a human cell. In some embodiments, the human cell is a stem cell. The stem cell can be, for example, a pluripotent stem cell, including an embryonic stem cell (ESC), an adult stem cell, an induced pluripotent stem cell (iPSC), a tissue-specific stem cell (e.g., a hematopoietic stem cell), and a mesenchymal stem cell (MSC). In some embodiments, the human cell is any differentiated form of the cells described herein. In some embodiments, the eukaryotic cell is a cell derived from any primary cell in culture.

[0203] In some embodiments, the eukaryotic cell is a hepatocyte, such as a human hepatocyte, an animal hepatocyte, or a non-parenchymal cell.For example, the eukaryotic cell can be an attached metabolic test human hepatocyte, an attached induction test human hepatocyte, an attached human hepatocyte, a suspension test human hepatocyte (including 10 donor and 20 donor pooled hepatocytes), a human hepatic Kupffer cell, a human hepatic stellate cell, a dog hepatocyte (including single and pooled beagle hepatocytes), a mouse hepatocyte (including CD-1 and C57BI / 6 hepatocytes), a rat hepatocyte (including Sprague-Dawley, Wistar Han and Wistar hepatocytes), a monkey hepatocyte (including Cynomolgus or Rhesus hepatocytes), a cat hepatocyte (including domestic shorthair hepatocytes), and a rabbit hepatocyte (including New Zealand White hepatocytes).

[0204] In some embodiments, the eukaryotic cell is a plant cell. For example, the plant cell can be a crop cell, such as cassava, maize, sorghum, wheat, or rice. The plant cell can be an algae, tree, or vegetable cell. The plant cell can be a monocotyledonous or dicotyledonous plant cell, or a crop or cereal plant, a productive plant, a fruit, or a vegetable cell. For example, the plant cell may be a cell of a tree, such as a citrus tree, e.g., an orange, grapefruit or lemon tree; a peach or nectarine tree; an apple or pear tree; a nut tree, e.g., an almond or walnut or pistachio tree; a Solanum plant, e.g., a potato; a Brassica plant, a Lactuca plant, a Spinacia plant; a Capsicum plant; cotton, tobacco, asparagus, carrot, cabbage, broccoli, cauliflower, tomato, eggplant, pepper, lettuce, spinach, strawberry, blueberry, raspberry, blackberry, grape, coffee, cocoa, and the like.

[0205] In some embodiments, the guide sequence of the guide polynucleotide is about 5 to about 50 nucleotides. In some embodiments, the guide sequence of the guide polynucleotide is about 6 to about 45 nucleotides. In some embodiments, the guide sequence of the guide polynucleotide is about 7 to about 40 nucleotides. In some embodiments, the guide sequence of the guide polynucleotide is about 8 to about 35 nucleotides. In some embodiments, the guide sequence of the guide polynucleotide is about 9 to about 30 nucleotides. In some embodiments, the guide sequence of the guide polynucleotide is about 10 to about 20 nucleotides. In some embodiments, the guide sequence of the guide polynucleotide is about 12 to about 20 nucleotides. In some embodiments, the guide sequence of the guide polynucleotide is about 14 to about 20 nucleotides. In some embodiments, the guide sequence of the guide polynucleotide is about 16 to about 20 nucleotides. In some embodiments, the guide sequence of the guide polynucleotide is about 18 to about 20 nucleotides. In some embodiments, the guide sequence of the guide polynucleotide is about 5 to about 10 nucleotides. In some embodiments, the guide sequence of the guide polynucleotide is about 6 to about 10 nucleotides. In some embodiments, the guide sequence of the guide polynucleotide is about 7 to about 10 nucleotides. In some embodiments, the guide sequence of the guide polynucleotide is about 8 to about 10 nucleotides. The length of the guide sequence can be determined by one of skill in the art using a guide sequence design tool such as, for example, CRISPR Design Tool (Hsu et al., Nat Biotechnol 31(9):827-832(2013)), ampliCan (Labun et al., bioRxiv 2018, doi:10.1101 / 249474), CasFinder (Alach et al., bioRxiv 2014, doi:10.1101 / 005074), CHOPCHOP (Labun et al., Nucleic Acids Res 2016, doi:10.1093 / nar / gkw398).

[0206] In some embodiments, a guide polynucleotide, e.g., a guide RNA, of the present disclosure comprises a polypeptide binding sequence / segment. The polypeptide binding segment (or "protein binding sequence") of a guide polynucleotide, e.g., a guide RNA, interacts with a polynucleotide binding domain of a Cas effector protein of the present disclosure. Such polypeptide binding segments or sequences are known to those of skill in the art, for example those disclosed in US Patent Publication Nos. 2014 / 0068797, 2014 / 0273037, 2014 / 0273226, 2014 / 0295556, 2014 / 0295557, 2014 / 0349405, 2015 / 0045546, 2015 / 0071898, 2015 / 0071899, and 2015 / 0071906. In some embodiments, the polypeptide binding segment of the guide polynucleotide binds to Cas9. In some embodiments, the polypeptide binding segment of the guide polynucleotide binds to a recombinant Cas9 protein provided herein.

[0207] In some embodiments, the guide polynucleotide is at least about 10, 15, 20, 25, or 30 nucleotides and at most about 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, or 150 nucleotides. In some embodiments, the guide polynucleotide is about 10 to about 150 nucleotides. In some embodiments, the guide polynucleotide is about 20 to about 120 nucleotides. In some embodiments, the guide polynucleotide is about 30 to about 100 nucleotides. In some embodiments, the guide polynucleotide is about 40 to about 80 nucleotides. In some embodiments, the guide polynucleotide is about 50 to about 60 nucleotides. In some embodiments, the guide polynucleotide is about 10 to about 35 nucleotides. In some embodiments, the guide polynucleotide is about 15 to about 30 nucleotides. In some embodiments, the guide polynucleotide is about 20 to about 25 nucleotides.

[0208] The guide polynucleotide, e.g., guide RNA, can be introduced into the target cell as an isolated molecule, e.g., an RNA molecule, or is introduced into the cell using an expression vector containing DNA encoding the guide polynucleotide, e.g., the guide RNA.

[0209] In some embodiments, the guide polynucleotide of the CRISPR-Cas system is linked to a direct repeat sequence. A direct repeat or DR sequence is an array of repeat sequences in the CRISPR locus that are interleaved with short stretches of non-repetitive sequences (spacers). The spacer sequence targets a protospacer adjacent motif (PAM) on the target sequence. When the non-coding portion of the CRISPR locus (i.e., the guide polynucleotide and the tracrRNA) is transcribed, the transcript is cleaved into short crRNAs that contain individual spacer sequences in the DR sequence, which directs the Cas9 nuclease to the PAM. In some embodiments, the DR sequence is RNA. In some embodiments, the DR sequence is encoded by a nucleic acid. In some embodiments, the DR sequence is linked to the guide polynucleotide. In some embodiments, the DR sequence is linked to the guide sequence of the guide polynucleotide. In some embodiments, the DR sequence comprises a secondary structure. In some embodiments, the DR sequence comprises a stem-loop structure. In some embodiments, the DR sequence is 10-20 nucleotides. In some embodiments, the DR sequence is at least 16 nucleotides. In some embodiments, the DR sequence is at least 16 nucleotides and comprises a single stem loop. In some embodiments, the DR sequence comprises an RNA aptamer. In some embodiments, the secondary structure or stem loop of the DR is recognized by a nuclease for cleavage. In some embodiments, the nuclease is a ribonuclease. In some embodiments, the nuclease is RNase III.

[0210] In some embodiments, the CRISPR-Cas system of the present disclosure further comprises a tracrRNA. The "tracrRNA" or transactivating CRISPR-RNA forms an RNA duplex with the pre-crRNA or pre-CRISPR-RNA, which is then cleaved by the RNA-specific ribonuclease RNase III to form a crRNA / tracrRNA hybrid. In some embodiments, the guide RNA comprises a crRNA / tracrRNA hybrid. In some embodiments, the tracrRNA component of the guide RNA activates the Cas effector protein. In some embodiments, the guide polynucleotide of the CRISPR-Cas system comprises a tracrRNA sequence. In some embodiments, the CRISPR-Cas system comprises a separate polynucleotide comprising a tracrRNA sequence.

[0211] In some embodiments, the polynucleotide encoding the recombinant Cas effector protein and the guide polynucleotide are present on a single vector. In some embodiments, the polynucleotide encoding the recombinant Cas effector protein, the guide polynucleotide (or a nucleotide that can be transcribed into a guide polynucleotide), and the tracrRNA are present on a single vector. In some embodiments, the polynucleotide encoding the recombinant Cas effector protein, the guide polynucleotide (or a nucleotide that can be transcribed into a guide polynucleotide), the tracrRNA, and the direct repeat sequence are present on a single vector. In some embodiments, the vector is an expression vector. In some embodiments, the vector is a mammalian expression vector. In some embodiments, the vector is a human expression vector. In some embodiments, the vector is a plant expression vector.

[0212] In some embodiments, the polynucleotide encoding the recombinant Cas effector protein and the guide polynucleotide are a single nucleic acid molecule. In some embodiments, the polynucleotide encoding the recombinant Cas effector protein, the guide polynucleotide and the tracrRNA are a single nucleic acid molecule. In some embodiments, the polynucleotide encoding the recombinant Cas effector protein, the guide polynucleotide, the tracrRNA and the direct repeat sequence are a single nucleic acid molecule. In some embodiments, the single nucleic acid molecule is an expression vector. In some embodiments, the single nucleic acid molecule is a mammalian expression vector. In some embodiments, the single nucleic acid molecule is a human expression vector. In some embodiments, the single nucleic acid molecule is a plant expression vector.

[0213] In some embodiments, the recombinant Cas effector protein and the guide polynucleotide can form a complex. In some embodiments, the complex between the recombinant Cas effector protein and the guide polynucleotide is not naturally occurring.

[0214] cell In some embodiments of the present disclosure, the eukaryotic cell is a human cell. In some embodiments, the eukaryotic cell is an animal or human cell. In some embodiments, the eukaryotic cell is a human, rodent or bovine cell line or cell line. Examples of such cells, cell lines or cell lines include, but are not limited to, mouse myeloma (NSO) cell lines, Chinese hamster ovary (CHO) cell lines, HT1080, H9, HepG2, MCF7, MDBK Jurkat, NIH3T3, PC12, BHK (baby hamster kidney cells), VERO, SP2 / 0, YB2 / 0, Y0, C127, L cells, COS, e.g., COS1 and COS7, QC1-3, HEK-293, VERO, PER.C6, HeLA, EB1, EB2, EB3, oncolytic or hybridoma cell lines. In some embodiments, the eukaryotic cell is a CHO cell line. In some embodiments, the eukaryotic cell is a CHO cell. In some embodiments, the cell is a CHO-K1 cell, a CHO-K1 SV cell, a DG44 CHO cell, a DUXB11 CHO cell, a CHOS, a CHO GS knockout cell, a CHO FUT8 GS knockout cell, a CHOZN, or a CHO-derived cell. A CHO GS knockout cell (e.g., a GSKO cell) is, for example, a CHO-K1 SV GS knockout cell. A CHO FUT8 knockout cell is, for example, a POTELLIGENT CHOK1 SV (Lonza Biologics, Inc.). The eukaryotic cell can also be an avian cell, cell line, or cell line, such as an EBX cell, EB14, EB24, EB26, EB66, or EBvl3.

[0215] In some embodiments, the eukaryotic cell is a human cell. In some embodiments, the human cell is a stem cell. The stem cell can be a pluripotent stem cell, including, for example, an embryonic stem cell (ESC), an adult stem cell, an induced pluripotent stem cell (iPSC), a tissue-specific stem cell (e.g., a hematopoietic stem cell), and a mesenchymal stem cell (MSC). In some embodiments, the cell is a pluripotent stem cell. In some embodiments, the eukaryotic cell is an induced pluripotent stem cell. In some embodiments, the human cell is any differentiated form of the cells described herein. In some embodiments, the eukaryotic cell is a cell derived from any primary cell in culture.

[0216] In some embodiments, the eukaryotic cell is a hepatocyte, such as a human hepatocyte, an animal hepatocyte, or a non-parenchymal cell.For example, the eukaryotic cell can be an attached metabolic test human hepatocyte, an attached induction test human hepatocyte, an attached human hepatocyte, a suspension test human hepatocyte (including 10 donor and 20 donor pooled hepatocytes), a human hepatic Kupffer cell, a human hepatic stellate cell, a dog hepatocyte (including single and pooled beagle hepatocytes), a mouse hepatocyte (including CD-1 and C57BI / 6 hepatocytes), a rat hepatocyte (including Sprague-Dawley, Wistar Han and Wistar hepatocytes), a monkey hepatocyte (including Cynomolgus or Rhesus hepatocytes), a cat hepatocyte (including domestic shorthair hepatocytes), and a rabbit hepatocyte (including New Zealand White hepatocytes).

[0217] In some embodiments, the eukaryotic cell is a hematopoietic cell. In some embodiments, the hematopoietic cell is a myeloid progenitor cell. In some embodiments, the hematopoietic cell is a lymphoid progenitor cell. In some embodiments, the hematopoietic cell is a mast cell, megakaryocyte, platelet, basophil, neutrophil, eosinophil, dendritic cell, monocyte, or macrophage. In some embodiments, the hematopoietic cell is a natural killer cell (NK cell), T lymphocyte, or B lymphocyte. In some embodiments, the T lymphocyte or B lymphocyte comprises a chimeric antigen receptor (CAR).

[0218] In some embodiments, the eukaryotic cell is a plant cell. For example, the plant cell can be a crop cell, such as cassava, maize, sorghum, wheat, or rice. The plant cell can be an algae, tree, or vegetable cell. The plant cell can be a monocotyledonous or dicotyledonous plant cell, or a crop or cereal plant, a productive plant, a fruit, or a vegetable cell. For example, the plant cell may be a cell of a tree, such as a citrus tree, e.g., an orange, grapefruit or lemon tree; a peach or nectarine tree; an apple or pear tree; a nut tree, e.g., an almond or walnut or pistachio tree; a Solanum plant, e.g., a potato; a Brassica plant, a Lactuca plant, a Spinacia plant; a Capsicum plant; cotton, tobacco, asparagus, carrot, cabbage, broccoli, cauliflower, tomato, eggplant, pepper, lettuce, spinach, strawberry, blueberry, raspberry, blackberry, grape, coffee, cocoa, and the like.

[0219] In some embodiments, the eukaryotic cells are a tissue culture of any of the aforementioned cells. In some embodiments, the eukaryotic cells are in the form of a tissue extract of any of the aforementioned cells.

[0220] In some embodiments, the eukaryotic cell comprises a genomically integrated Cas polynucleotide. In some embodiments, the eukaryotic cell comprises an inducible genomically integrated Cas polynucleotide.

[0221] delivery system Various methods for delivery of the CRISPR-Cas system are known in the art. Suitable delivery systems include microinjection, electroporation, transfection, or hydrodynamic delivery of a polynucleotide encoding a Cas effector protein, a polynucleotide comprising a sequence of interest, and / or a polynucleotide capable of forming a complex with a Cas effector protein. In some embodiments, the delivery system comprises a delivery particle. Examples of such delivery systems, including nanoparticles, cell-penetrating peptides, and DNA nanoclews, are disclosed in Lino et al., Drug Delivery, 25(1):1234-1257(2018)).

[0222] In some embodiments, the CRISPR-Cas system comprising the Cas effector protein of the present disclosure, a polynucleotide encoding the Cas effector protein, a polynucleotide encoding a sequence of interest, and / or a polynucleotide capable of forming a complex with the Cas effector protein is delivered by a delivery particle. The delivery particle is a biological delivery system or formulation comprising a particle. As defined herein, a "particle" is an entity having a maximum diameter of about 100 microns (μm). In some embodiments, the particle has a maximum diameter of about 10 μm. In some embodiments, the particle has a maximum diameter of about 2000 nanometers (nm). In some embodiments, the particle has a maximum diameter of about 1000 nm. In some embodiments, the particle has a maximum diameter of about 900 nm, about 800 nm, about 700 nm, about 600 nm, about 500 nm, about 400 nm, about 300 nm, about 200 nm, or about 100 nm. In some embodiments, the particle has a diameter of about 25 nm to about 200 nm. In some embodiments, the particles have a diameter of about 50 nm to about 150 nm, hi some embodiments, the particles have a diameter of about 75 nm to about 100 nm.

[0223] The delivery particle may be provided in any form, including, but not limited to, a solid, semi-solid, emulsion, or colloid particle. In some embodiments, the delivery particle is a lipid-based system, a liposome, a micelle, a microvesicle, an exosome, or a gene gun. In some embodiments, the delivery particle comprises a CRISPR-Cas system. In some embodiments, the delivery particle comprises a CRISPR-Cas system comprising a recombinant Cas effector protein and a polynucleotide capable of forming a complex with the Cas effector protein, the polynucleotide comprising a guide polynucleotide. In some embodiments, the delivery particle comprises a Cas effector protein, a polynucleotide comprising a sequence of interest, and a polynucleotide capable of forming a complex with the Cas effector protein and comprising a guide polynucleotide. In some embodiments, the delivery particle comprises a CRISPR-Cas system comprising a recombinant Cas effector protein and a polynucleotide complexed with the Cas effector protein and comprising a guide polynucleotide, the recombinant Cas effector protein and the polynucleotide forming a complex. In some embodiments, the delivery particle comprises a CRISPR-Cas system comprising a recombinant Cas effector protein, a polynucleotide complexed with the Cas effector protein and comprising a guide polynucleotide, and a polynucleotide comprising a tracrRNA. In some embodiments, the delivery particle comprises a CRISPR-Cas system comprising a Cas effector protein, a polynucleotide complexed with the Cas effector protein and comprising a guide polynucleotide, and a tracrRNA.

[0224] In some embodiments, the complex of the disclosed Cas effector protein and polynucleotide is a ribonucleoprotein (RNP), and the RNP is delivered by hydrodynamic delivery, nanoparticles, vesicles, cell-penetrating peptides, or DNA nanoclears.

[0225] In some embodiments, the delivery particle further comprises a lipid, a sugar, a metal, or a protein. In some embodiments, the delivery particle is a lipid envelope. Delivery of mRNA using delivery particles comprising a lipid envelope or lipids is described, for example, in Su et al., Molecular Pharmacology 8(3):774-784(2011). In some embodiments, the delivery particle is a sugar-based particle, such as GalNAc. Sugar-based particles are described in WO 2014 / 118272 and Nair et al., J Am Chem. Soc.136(49):16958-16961(2014).

[0226] In some embodiments, the delivery particle is a nanoparticle. The nanoparticles encompassed in the present disclosure can be provided in various forms, such as solid nanoparticles (e.g., metals such as silver, gold, iron, titanium, etc.), non-metallic, lipid-based solids, polymers, suspensions of nanoparticles, or combinations thereof. Metal, dielectric and semiconductor nanoparticles, as well as hybrid structures (e.g., core-shell nanoparticles) can be prepared. Nanoparticles made of semiconductor materials can also be labeled quantum dots, if they are small enough (typically less than 10 nm) that quantization of electronic energy levels occurs. Such nanoscale particles are used as drug carriers or imaging agents in biomedical applications, and can be adapted for similar purposes in the present disclosure.

[0227] The preparation of delivery particles is further described in U.S. Patent Application Publication Nos. 2011 / 0293703, 2012 / 0251560, and 2013 / 0302401, as well as U.S. Pat. Nos. 5,543,158, 5,855,913, 5,895,309, 6,007,845, and 8,709,843.

[0228] In some embodiments, the vesicle comprises the CRISPR-Cas system of the present disclosure. A "vesicle" is a small intracellular structure with fluid enclosed by a lipid bilayer. In some embodiments, the CRISPR-Cas system of the present disclosure is delivered by the vesicle. In some embodiments, the vesicle comprises a recombinant Cas effector protein and a guide polynucleotide. In some embodiments, the vesicle comprises a Cas effector protein and a guide polynucleotide, where the Cas effector protein and the guide polynucleotide are complexed. In some embodiments, the vesicle comprises a CRISPR-Cas system comprising a Cas effector protein, a polynucleotide capable of forming a complex with the Cas effector protein and comprising a guide polynucleotide, and a polynucleotide comprising a tracrRNA. In some embodiments, the vesicle comprises a CRISPR-Cas system comprising a Cas effector protein, a polynucleotide capable of forming a complex with the Cas effector protein and comprising a guide polynucleotide, and a tracrRNA.

[0229] In some embodiments, the vesicle containing the Cas effector protein and the guide polynucleotide that can form a complex with the Cas effector protein is an exosome or liposome. In some embodiments, the vesicle is an exosome. In some embodiments, exosomes are used to deliver the CRISPR-Cas system of the present disclosure. Exosomes are endogenous nanovesicles (i.e., having a diameter of about 30 to about 100 nm) that can transport RNA and proteins and deliver RNA to the brain and other target organs. Engineered exosomes for delivering exogenous biological materials to target organs are described, for example, by Alvarez-Erviti et al., Nature Biotechnology 29:341 (2011), El-Andaloussi et al., Nature Protocols 7:2112-2116 (2012), and Wahlgren et al., Nucleic Acids Research 40(17):e130 (2012).

[0230] In some embodiments, liposomes are used to deliver the CRISPR-Cas system of the present disclosure. Liposomes are spherical vesicular structures with at least one lipid bilayer and can be used as vehicles for the administration of nutrients and pharmaceuticals. Liposomes are often composed of phospholipids, particularly phosphatidylcholine, but also other lipids, such as egg phosphatidylethanolamine. Types of liposomes include, but are not limited to, multilamellar vesicles, small unilamellar vesicles, large unilamellar vesicles, and spiral-wound vesicles. See, for example, Spuch and Navarro, Journal of Drug Delivery, Article ID 469679 (2011). Liposomes for delivery of biological materials, such as CRISPR-Cas components, have been described, for example, by Morrissey et al., Nature Biotechnology 23(8):1002-1007 (2005), Zimmerman et al., Nature Letters 441:111-114 (2006), and Li et al., Gene Therapy 19:775-780 (2012).

[0231] In some embodiments, the Cas effector protein can be delivered using a cell-penetrating peptide fused to the Cas effector protein.

[0232] In some embodiments, the Cas effector proteins and polynucleotides of the present disclosure can be delivered in the form of DNA nanoclews, which are globular structures containing DNA that can be loaded with a payload, such as a Cas effector protein (Sun et al., J. Am. Chem. Soc., 136:14722-14725). DNA nanoclews have been used in vitro for delivery of the Cas9 editing system (Lino et al., Drug Delivery, 25(1):1234-1257).

[0233] In some embodiments, the viral vector comprises the CRISPR-Cas system of the present disclosure. In some embodiments, the CRISPR-Cas system of the present disclosure is delivered by a viral vector. In some embodiments, the viral vector comprises a recombinant Cas9 and a guide polynucleotide. In some embodiments, the viral vector comprises a Cas effector protein and a guide polynucleotide, where the Cas effector protein and the guide polynucleotide are complexed. In some embodiments, the viral vector comprises a CRISPR-Cas system comprising a Cas effector protein, a polynucleotide capable of forming a complex with the Cas effector protein and comprising a guide polynucleotide, and a polynucleotide comprising a tracrRNA. In some embodiments, the viral vector comprises a CRISPR-Cas system comprising a Cas effector protein, a polynucleotide capable of forming a complex with the Cas effector protein and comprising a guide polynucleotide, and a tracrRNA. In some embodiments, the viral vector is a retroviral, lentiviral, adenoviral, or adeno-associated viral vector. Examples of viral vectors are provided herein.

[0234] In some embodiments, viral vectors can be used, including retroviral, lentiviral, adenoviral, and / or adeno-associated viral (AAV) vectors, as well as viral vectors that contain elements of the CRISPR-Cas system described herein. In some embodiments of the present disclosure, Cas effector proteins are expressed intracellularly by cells transduced with the viral vector.

[0235] In some embodiments, the disclosed Cas proteins and methods are used in ex vivo gene editing, e.g., CAR-T type therapy. These embodiments may include modification of cells from human donors. In these examples, viral vectors may be used, but there is also the additional option of directly transfecting Cas9 proteins (along with in vitro transcribed guide RNA and donor DNA) into cultured cells.

[0236] Inhibitors of the microhomology end joining (MMEJ) pathway As used herein, an inhibitor of the MMEJ pathway is any compound, molecule, or entity that inhibits, antagonizes, blocks, or reduces the activity and / or level of any component of the MMEJ pathway. The MMEJ inhibitor can be an antibody or antigen-binding fragment thereof, a peptide, a soluble protein, an siRNA, an antisense oligonucleotide, an aptamer, or a small molecule compound that inhibits, antagonizes, blocks, or reduces the activity and / or level of any component of the MMEJ pathway. In some embodiments, the MMEJ inhibitor inhibits, antagonizes, blocks, or reduces the activity and / or level of FEN1 (flap endonuclease 1), DNA ligase III, MREII, NBS1 (Nibrin, NBN), XRCC1 (X-ray repair cross-complementing protein 1), PARP1 (poly [ADP-ribose] polymerase 1), or PolQ (DNA polymerase θ). In some embodiments, the inhibitor of the MMEJ pathway is novobiocin. In some embodiments, the inhibitor of the MMEJ pathway is a PolQ inhibitor. In some embodiments, the PolQ inhibitor is ART558 (Zatreanu et al., Nature Communications, 12(1):3636 (2021)). In some embodiments, the PolQ inhibitor is selected from PolQ1 (described in WO2020030925), PolQ2, PolQ3, PolQ4, PolQ5 (all described in WO2021028643), PolQ6, PolQ7 (described in WO2020243549), or a combination thereof, as shown in FIG.

[0237] In some embodiments, the MMEJ pathway inhibitor is added to a composition comprising eukaryotic cells at a concentration of about 0.01 μM to about 1 mM. In some embodiments, the concentration of the MMEJ pathway inhibitor is about 0.01 μM to about 0.75 mM, about 0.01 μM to about 0.5 mM, about 0.01 μM to about 0.25 mM, about 0.01 μM to about 0.1 mM, about 0.01 μM to about 75 μM, about 0.01 μM to about 50 μM, about 0.01 μM to about 25 μM, about 0.01 to about 25 μM, about 0.01 to about 20 μM, about 0.01 μM to about 15 μM, about 0.01 μM to about 10 μM, or about 0.01 μM to about 1 μM. In some embodiments, the concentration of the inhibitor of the MMEJ pathway is about 0.1 μM to about 1 mM, about 1 μM to about 1 mM, about 10 μM to about 1 mM, about 15 μM to about 1 M, about 20 μM to about 1 M, about 25 μM to about 1 mM, about 50 μM to about 1 mM, about 75 μM to about 1 mM, about 0.1 mM to about 1 mM, about 0.25 mM to about 1 mM, about 0.5 mM to about 1 mM, or about 0.75 mM to about 1 mM. In some embodiments, the concentration of the inhibitor of the MMEJ pathway is about 0.1 μM to about 1 mM, 0.1 μM to about 0.75 mM, about 0.1 μM to about 0.5 mM, about 0.1 μM to about 0.25 mM, about 0.1 μM to about 0.1 mM, about 0.1 μM to about 75 μM, about 0.1 μM to about 50 μM, about 0.1 μM to about 25 μM, about 0.1 μM to about 20 μM, about 0.1 μM to about 15 μM, about 0.1 μM to about 10 μM, or about 0.1 μM to about 1 μM. In some embodiments, the concentration of the MMEJ pathway inhibitor is about 1 μM to about 10 μM, about 1 μM to about 15 μM, about 1 μM to about 20 μM, about 1 μM to about 25 μM, about 1 μM to about 50 μM, about 1 μM to about 0.1 mM, about 1 μM to about 0.25 mM, about 1 μM to about 0.5 mM, about 1 μM to about 0.75 mM, or about 1 μM to about 1 mM. In some embodiments, the concentration of the inhibitor of the MMEJ pathway is about 0.01 μM to about 100 μM, about 0.1 μM to about 90 μM, about 0.2 μM to about 80 μM, about 0.3 μM to about 70 μM, about 0.4 μM to about 60 μM, about 0.5 μM to about 50 μM, about 1 μM to about 50 μM, about 2 μM to about 45 μM, about 3 μM to about 40 μM, about 4 μM to about 35 μM, about 5 μM to about 30 μM, about 6 μM to about 25 μM, about 7 μM to about 20 μM, or about 8 μM to about 15 μM.In some embodiments, the concentration of the inhibitor of the MMEJ pathway is about 0.01 μM to about 0.1 μM, about 0.01 to about 1 μM, about 0.05 μM to about 0.1 μM, about 0.5 μM to about 1 μM, about 0.5 μM to about 5 μM, about 0.5 μM to about 10 μM, about 0.1 μM to about 1 μM, about 0.1 μM to about 5 μM, about 0.1 μM to about 10 μM, about 1 μM to about 5 μM, about 1 μM to about 10 μM, about 1 μM to about 15 μM, about 1 μM to about 20 μM, about 1 μM to about 25 μM, about 1 μM to about 50 μM, about 5 μM to about 10 μM, about 5 μM to about 15 μM, about 5 mM to about 20 mM, or about 5 mM to about 25 mM. In some embodiments, the concentration of the inhibitor of the MMEJ pathway is about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.7, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 μM.

[0238] In some embodiments, the concentration of the inhibitor of the MMEJ pathway is from about 0.01 μM to about 1 μM, from about 0.1 μM to about 1 μM, from about 0.1 μM to about 0.5 μM, from about 0.1 μM to about 100 μM, or from about 1 μM to about 50 μM.

[0239] In some embodiments, the inhibitor of the MMEJ pathway is added to the composition comprising the eukaryotic cells about 0 minutes to about 96 hours prior to addition of the Cas effector protein, about 0 minutes to about 72 hours prior to addition of the Cas effector protein, about 0 minutes to about 48 hours prior to addition of the Cas effector protein, about 0 minutes to about 36 hours prior to addition of the Cas effector protein, about 0 minutes to about 24 hours prior to addition of the Cas effector protein, about 0 minutes to about 18 hours prior to addition of the Cas effector protein, about 0 minutes to about 12 hours prior to addition of the Cas effector protein, about 0 minutes to about 6 hours prior to addition of the Cas effector protein, about 0 minutes to about 3 hours prior to addition of the Cas effector protein, about 0 minutes to about 2 hours prior to addition of the Cas effector protein, about 0 minutes to about 1 hour prior to addition of the effector protein, or about 0 minutes to about 30 minutes prior to addition of the Cas effector protein. In some embodiments, an inhibitor of the MMEJ pathway is added to a composition comprising eukaryotic cells about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 hours prior to addition of the Cas effector protein.

[0240] In some embodiments, an inhibitor of the MMEJ pathway is added to a composition comprising eukaryotic cells at the same time as adding the Cas effector protein.

[0241] In some embodiments, the inhibitor of the MMEJ pathway is added to the composition comprising the eukaryotic cells about 0 minutes to about 30 minutes after addition of the Cas effector protein, about 0 minutes to about 1 hour after addition of the Cas effector protein, about 0 minutes to about 3 hours after addition of the Cas effector protein, about 0 minutes to about 6 hours after addition of the Cas effector protein, about 0 minutes to about 12 hours after addition of the Cas effector protein, about 0 minutes to about 18 hours after addition of the Cas effector protein, about 0 minutes to about 24 hours after addition of the Cas effector protein, about 0 minutes to about 36 hours after addition of the Cas effector protein, about 0 minutes to about 48 hours after addition of the Cas effector protein, about 0 minutes to about 72 hours after addition of the Cas effector protein, or about 0 minutes to about 96 hours after addition of the Cas effector protein. In some embodiments, the inhibitor of the MMEJ pathway is added to the composition comprising the eukaryotic cells about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 hours after addition of the Cas effector protein.

[0242] In some embodiments, the inhibitor of the MMEJ pathway is present in the composition comprising the eukaryotic cell for about 1 to about 300 hours, about 10 to about 200 hours, about 10 to about 100 hours, about 20 to about 80 hours, about 30 to about 70 hours, or about 40 to about hours. In some embodiments, the inhibitor of the MMEJ pathway is present in the composition comprising a eukaryotic cell for about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 225, 250, 275, or 300 hours.

[0243] In some embodiments, the inhibitor of the MMEJ pathway is added to the composition comprising the eukaryotic cell at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more times.

[0244] Inhibitors of the non-homologous end joining (NHEJ) pathway As used herein, an inhibitor of the NHEJ pathway is any compound, molecule, or entity that inhibits, antagonizes, blocks, or reduces the activity and / or level of any component of the NHEJ pathway. The NHEJ inhibitor can be an antibody or antigen-binding fragment thereof, a peptide, a soluble protein, an siRNA, an antisense oligonucleotide, an aptamer, or a small molecule compound that inhibits, antagonizes, blocks, or reduces the activity and / or level of any component of the NHEJ pathway. In some embodiments, the NHEJ pathway inhibits, antagonizes, blocks, or reduces the activity and / or level of Ku70, Ku80, DNA ligase IV, XLF (non-homologous end joining factor 1; XRCC4-like factor), or DNA-dependent protein kinase (DNA-PK). In some embodiments, the inhibitor of DNA-PK is M3814, M9831 / VX984, Nu7441, KU0060648, AZD7648, Nu5455, vanillin, wortmannin, or a combination thereof, in some embodiments, the DNA-PK inhibitor is AZD7648.

[0245] In some embodiments, the inhibitor of the NHEJ pathway is added to the composition comprising eukaryotic cells at a concentration of about 0.01 μM to about 1 mM. In some embodiments, the concentration of the inhibitor of the NHEJ pathway is about 0.01 μM to about 0.75 mM, about 0.01 μM to about 0.5 mM, about 0.01 μM to about 0.25 mM, about 0.01 μM to about 0.1 mM, about 0.01 μM to about 75 μM, about 0.01 μM to about 50 μM, about 0.01 μM to about 25 μM, about 0.01 to about 25 μM, about 0.01 to about 20 μM, about 0.01 μM to about 15 μM, about 0.01 μM to about 10 μM, or about 0.01 μM to about 1 μM. In some embodiments, the concentration of the inhibitor of the NHEJ pathway is about 0.1 μM to about 1 mM, about 1 μM to about 1 mM, about 10 μM to about 1 mM, about 15 μM to about 1 M, about 20 μM to about 1 M, about 25 μM to about 1 mM, about 50 μM to about 1 mM, about 75 μM to about 1 mM, about 0.1 mM to about 1 mM, about 0.25 mM to about 1 mM, about 0.5 mM to about 1 mM, or about 0.75 mM to about 1 mM. In some embodiments, the concentration of the inhibitor of the NHEJ pathway is about 0.1 μM to about 1 mM, 0.1 μM to about 0.75 mM, about 0.1 μM to about 0.5 mM, about 0.1 μM to about 0.25 mM, about 0.1 μM to about 0.1 mM, about 0.1 μM to about 75 μM, about 0.1 μM to about 50 μM, about 0.1 μM to about 25 μM, about 0.1 μM to about 20 μM, about 0.1 μM to about 15 μM, about 0.1 μM to about 10 μM, or about 0.1 μM to about 1 μM. In some embodiments, the concentration of the inhibitor of the NHEJ pathway is about 1 μM to about 10 μM, about 1 μM to about 15 μM, about 1 μM to about 20 μM, about 1 μM to about 25 μM, about 1 μM to about 50 μM, about 1 μM to about 0.1 mM, about 1 μM to about 0.25 mM, about 1 μM to about 0.5 mM, about 1 μM to about 0.75 mM, or about 1 μM to about 1 mM. In some embodiments, the concentration of the inhibitor of the NHEJ pathway is about 0.01 μM to about 100 μM, about 0.1 μM to about 90 μM, about 0.2 μM to about 80 μM, about 0.3 μM to about 70 μM, about 0.4 μM to about 60 μM, about 0.5 μM to about 50 μM, about 1 μM to about 50 μM, about 2 μM to about 45 μM, about 3 μM to about 40 μM, about 4 μM to about 35 μM, about 5 μM to about 30 μM, about 6 μM to about 25 μM, about 7 μM to about 20 μM, or about 8 μM to about 15 μM.In some embodiments, the concentration of the inhibitor of the NHEJ pathway is about 0.01 μM to about 0.1 μM, about 0.01 to about 1 μM, about 0.05 μM to about 0.1 μM, about 0.5 μM to about 1 μM, about 0.5 μM to about 5 μM, about 0.5 μM to about 10 μM, about 0.1 μM to about 1 μM, about 0.1 μM to about 5 μM, about 0.1 μM to about 10 μM, about 1 μM to about 5 μM, about 1 μM to about 10 μM, about 1 μM to about 15 μM, about 1 μM to about 20 μM, about 1 μM to about 25 μM, about 1 μM to about 50 μM, about 5 μM to about 10 μM, about 5 μM to about 15 μM, about 5 mM to about 20 mM, or about 5 mM to about 25 mM. In some embodiments, the concentration of the inhibitor of the NHEJ pathway is about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.7, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 μM.

[0246] In some embodiments, the concentration of the inhibitor of the NHEJ pathway is from about 0.01 μM to about 1 μM, from about 0.1 μM to about 1 μM, from about 0.1 μM to about 0.5 μM, from about 0.1 μM to about 100 μM, or from about 1 μM to about 50 μM.

[0247] In some embodiments, the inhibitor of the NHEJ pathway is added to the composition comprising the eukaryotic cells about 0 minutes to about 96 hours prior to addition of the Cas effector protein, about 0 minutes to about 72 hours prior to addition of the Cas effector protein, about 0 minutes to about 48 hours prior to addition of the Cas effector protein, about 0 minutes to about 36 hours prior to addition of the Cas effector protein, about 0 minutes to about 24 hours prior to addition of the Cas effector protein, about 0 minutes to about 18 hours prior to addition of the Cas effector protein, about 0 minutes to about 12 hours prior to addition of the Cas effector protein, about 0 minutes to about 6 hours prior to addition of the Cas effector protein, about 0 minutes to about 3 hours prior to addition of the Cas effector protein, about 0 minutes to about 2 hours prior to addition of the Cas effector protein, about 0 minutes to about 1 hour prior to addition of the effector protein, or about 0 minutes to about 30 minutes prior to addition of the Cas effector protein. In some embodiments, the inhibitor of the NHEJ pathway is added to the composition comprising the eukaryotic cells about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 hours prior to addition of the Cas effector protein.

[0248] In some embodiments, an inhibitor of the NHEJ pathway is added to a composition comprising a eukaryotic cell at the same time as adding the Cas effector protein.

[0249] In some embodiments, the inhibitor of the NHEJ pathway is added to the composition comprising the eukaryotic cell about 0 minutes to about 30 minutes after addition of the Cas effector protein, about 0 minutes to about 1 hour after addition of the Cas effector protein, about 0 minutes to about 3 hours after addition of the Cas effector protein, about 0 minutes to about 6 hours after addition of the Cas effector protein, about 0 minutes to about 12 hours after addition of the Cas effector protein, about 0 minutes to about 18 hours after addition of the Cas effector protein, about 0 minutes to about 24 hours after addition of the Cas effector protein, about 0 minutes to about 36 hours after addition of the Cas effector protein, about 0 minutes to about 48 hours after addition of the Cas effector protein, about 0 minutes to about 72 hours after addition of the Cas effector protein, or about 0 minutes to about 96 hours after addition of the Cas effector protein. In some embodiments, the inhibitor of the NHEJ pathway is added to the composition comprising the eukaryotic cell about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 hours after addition of the Cas effector protein.

[0250] In some embodiments, the inhibitor of the NHEJ pathway is present in the composition comprising the eukaryotic cell for about 1 to about 300 hours, about 10 to about 200 hours, about 10 to about 100 hours, about 20 to about 80 hours, about 30 to about 70 hours, or about 40 to about hours. In some embodiments, the inhibitor of the NHEJ pathway is present in the composition comprising a eukaryotic cell for about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 225, 250, 275, or 300 hours.

[0251] In some embodiments, the inhibitor of the NHEJ pathway is added to the composition comprising the eukaryotic cell at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more times.

[0252] In some embodiments, the inhibitor of the NHEJ pathway is added to the composition comprising the eukaryotic cells before the inhibitor of the MMEJ pathway is added to the composition comprising the eukaryotic cells, or after the inhibitor of the MMEJ pathway is added to the composition, hi some embodiments, the inhibitor of the NHEJ pathway and the inhibitor of the MMEJ pathway are added simultaneously to the composition comprising the eukaryotic cells.

[0253] In some embodiments, the inhibitor of the MMEJ pathway and the inhibitor of the NHEJ pathway are added to the composition comprising the eukaryotic cells before the Cas effector protein is added. In some embodiments, the inhibitor of the MMEJ pathway and the inhibitor of the NHEJ pathway are added to the composition comprising the eukaryotic cells after the Cas effector protein is added. In some embodiments, the inhibitor of the MMEJ pathway and the inhibitor of the NHEJ pathway are added to the composition comprising the eukaryotic cells at the same time as the Cas effector protein is added. In some embodiments, the inhibitor of the MMEJ pathway is added to the composition comprising the eukaryotic cells before the Cas effector protein is added, and the inhibitor of the NHEJ pathway is added after the Cas effector protein is added. In some embodiments, the inhibitor of the MMEJ pathway is added to the composition comprising the eukaryotic cells after the Cas effector protein is added, and the inhibitor of the NHEJ pathway is added before the Cas effector protein is added.

[0254] All references cited herein, e.g., patents, patent applications, articles, textbooks, etc., and the references cited therein, are incorporated by reference in their entirety, unless they have already been cited. EXAMPLES

[0255] Example 1 - Effect of MMEJ and NHEJ inhibitors on double-strand break repair pathways. The effect of inhibitors of MMEJ and NHEJ pathways on the CRISPR-Cas-induced DNA double-strand break repair pathway was investigated using the process outlined in Figure 2A. Briefly, HEK293T cells were seeded in 96-well plates 20 hours before transfection with a plasmid encoding SpCas9 and a guide RNA (sgRNA) targeting CD34 together with a single-stranded oligonucleotide donor (ssDNA). Three hours before transfection, cells were treated with DNA-dependent protein kinase (DNA-PK) inhibitor AZD7648 (NHEJ inhibitor) (final concentration 1 μM) alone and in combination with six different Pol Q inhibitors (MMEJ inhibitors) at the concentrations shown in Figure 4. The Pol Q inhibitors used were PolQ_2, PolQ_3, PolQ_4, PolQ_5, PolQ_6 or PolQ_7. Sixty hours after transfection, genomic DNA was harvested and sequenced using deep targeted amplicon sequencing. Genetic variants of the sequencing data were determined using a bioinformatics workflow, and the percentage of DNA double-strand break repair by MMEJ, NHEJ, and HDR pathways was determined using theoretical indel meta-analysis (RIMA). See, for example, Taheri-Ghafarokhi et al, Nuc. Acids Res., 2018, 46(16):8417-8434. The RIMA results are plotted as shown in Figure 2B, and the deletions associated with microhomology are visualized according to the bars shown in the figure.

[0256] The results of these experiments are shown in Figure 4. In transfected cells not treated with MMEJ or NHEJ inhibitors (DMSO-treated control), approximately 20% of double-strand breaks were repaired by the HDR pathway, with the NHEJ and MMEJ pathways responsible for approximately 40% of double-strand break repair. In contrast, transfected cells treated with an NHEJ inhibitor (AZD7648) alone or in combination with an MMEJ inhibitor (Pol Q 2-7) showed a significant increase in double-strand break repair by the HDR pathway, and a decrease in repair by the MMEJ and NHEJ pathways. When transfected cells were treated with both an NHEJ inhibitor and an MMEJ inhibitor, the majority of double-strand breaks were repaired via the HDR pathway, and in some cases, nearly all of the double-strand break repair was via the HDR pathway.

[0257] To demonstrate the effect of various inhibitors on CRISPR / Cas-editing efficiency, HEK293T cells were treated with the DNA-PK inhibitor AZD7648 (1 μM) alone and in combination with the indicated Pol Q inhibitors, followed by CRISPR / Cas9-mediated gene targeting. As shown in Figure 5, the NHEJ inhibitors and most concentrations of MMEJ inhibitors used in these experiments did not affect CRISPR / Cas-mediated editing efficiency. These studies indicate that inhibition of the NHEJ and / or MMEJ pathways in combination with CRISRP / Cas-gene targeting leads to DNA double-strand break repair by a more accurate HDR pathway, minimizing the contribution of the more error-prone MMEJ and NHEJ pathways.

[0258] Example 2 - Effect of MMEJ and NHEJ inhibitors on CRISPR / Cas-mediated knock-in efficiency. The effect of NHEJ and MMEJ inhibitors on CRISPR / Cas-mediated knock-in efficiency was determined for both mutated and mapped reads. Briefly, HEK293T cells were cultured, transfected, and then treated with NHEJ inhibitors (AZD7648) alone and in combination with MMEJ inhibitors (Pol Q1-7) according to the protocol described in Example 1, followed by isolation of genomic DNA and subsequent analysis of knock-in efficiency for both mutated and mapped reads. Inhibition of the NHEJ and MMEJ pathways increased knock-in events by approximately 3-fold compared to DMSO-treated controls when evaluating both mutated (Figure 6) and mapped (Figure 7) reads. Combining inhibition of the MMEJ pathway with inhibition of the NHEJ pathway increased knock-in efficiency by up to 4.5-fold in the total cell population and up to 5.9-fold in CRISPR / Cas-edited cells.

[0259] Example 3 - Effect of MMEJ inhibition on mutational and mapping reads. HEK293T cells were cultured, transfected, and treated with the DNA-PK inhibitor AZD7648 (1 μM) alone and in combination with the indicated Pol Q inhibitors, followed by CRISPR / Cas9-mediated gene knock-in. The effect of MMEJ pathway inhibition on mutational and mapping reads was assessed. Treatment of CRISPR / Cas edited cells with MMEJ inhibitors resulted in a dose-dependent decrease in MMEJ mutational reads (Figure 8) and MMEJ mapping reads (Figure 9).

[0260] Example 4 - Inhibition of the NHEJ and MMEJ pathways does not affect cell confluence and transfection efficiency in CRISPR / Cas-transfected cells. HEK293T cells were cultured, transfected, and treated with NHEJ and MMEJ inhibitors as described in Example 1. Cell confluence and transfection efficiency were evaluated in transfected cells treated with NHEJ and MMEJ inhibitors. As shown in Figure 10, treatment of transfected cells with NHEJ inhibitor (AZD7648) at a final concentration of 1 mM did not significantly affect cell confluence. Treatment of transfected cells with NHEJ inhibitors in combination with the indicated Pol Q inhibitors did not affect cell confluence, except for the highest concentration of PolQ_1, PolQ_5, and PolQ_7. Similarly, treatment of cells with 1 μM NHEJ inhibitor (AZD7648) prior to transfection did not significantly affect transfection efficiency, as shown in Figure 11. Treatment of the transfected cells with NHEJ inhibitors in combination with the indicated Pol Q inhibitors prior to transfection did not affect transfection efficiency, except at the highest concentrations of PolQ_1 and PolQ_7.

[0261] Example 5 - Effect of NHEJ and MMEJ inhibitors on double-strand break repair pathways in induced pluripotent stem cells (iPSCs). The effect of NHEJ and / or MMEJ pathway inhibition on CRISPR-Cas-induced DNA double-stranded break repair pathway in iPSCs was examined. Briefly, iPSCs containing an inducible Cas9 gene were seeded in a 96-well plate, and after 20 hours, a plasmid encoding a guide RNA (sgRNA) targeting one of three separate target sites was transfected together with a single-stranded oligonucleotide donor (ssDNA), followed by induction of Cas9 expression. Three hours before transfection and induction of Cas9 expression, iPSCs were treated with DNA-dependent protein kinase (DNA-PK) inhibitor AZD7648 (final concentration 1 μM) alone and in combination with 3 μM PolQ2 or PolQ6. Sixty hours after transfection, the percentage of double-stranded break repair by HDR, NHEJ, and MMEJ pathways was determined as described in Example 1.

[0262] The results of these experiments are shown in Figure 12. In transfected cells not treated with MMEJ or NHEJ inhibitors (DMSO-treated control), HDR pathway repair of double-stranded breaks was less than 10%, and NHEJ and MMEJ pathways were responsible for approximately 70% of double-stranded break repair. In contrast, transfected cells treated with NHEJ inhibitors (AZD7648) alone or in combination with MMEJ inhibitors (Pol Q 2 or Pol Q 6) showed a significant increase in double-stranded break repair by the HDR pathway, and a decrease in repair by the MMEJ and NHEJ pathways. Combination treatment with NHEJ and MMEJ inhibitors resulted in an even greater increase in HDR-mediated repair, with a corresponding decrease in NHEJ- and MMEJ-mediated repair.

[0263] Example 6 - Effect of NHEJ and MMEJ inhibitors on single stranded template repair (SSTR) gene insertion in iPSCs. The effect of NHEJ and MMEJ pathway inhibition on gene knock-in efficiency mediated by the SSTR pathway in iPSCs was examined. Briefly, Cas9-induced iPSCs were cultured and transfected with sgRNA and ssDNA polynucleotides as described in Example 5. As shown in Figure 13, in transfected cells not treated with NHEJ or MMEJ inhibitors (DMSO-treated control), the contribution of the SSTR pathway in gene knock-in mapping reads at three separate target sites was less than 5%. Addition of NHEJ inhibitor AZD7648 increased SSTR-mediated gene knock-in at all three target sites. Similarly, addition of MMEJ inhibitor PolQ2 or PolQ6 also increased SSTR-mediated gene knock-in at all three target sites. When NHEJ inhibitor and MMEJ inhibitor were added in combination, SSTR-mediated gene knock-in was greatly increased at all three target sites.

[0264] Example 7 - Effect of NHEJ and MMEJ inhibitors on gene insertion in primary human T cells. The effect of NHEJ and MMEJ pathway inhibition on gene insertion in human primary T cells was examined. Briefly, human T cells were treated with NHEJ inhibitor AZD7648 (1 μM) alone or in combination with MMEJ inhibitor PolQ2 or PolQ6 (3 μM). After 3 hours, cells were transfected with ribonucleoprotein (RNP) containing sgRNA targeting Cas9 and TRAC, and polynucleotide encoding green fluorescent protein (GFP). 60 hours after transfection, GFP knock-in efficiency was measured as described in Example 1.

[0265] The results of these experiments are shown in Figures 14A-C. Transfection of primary T cells and treatment with NHEJ and MMEJ inhibitors did not affect cell viability (Figure 14A) and resulted in a moderate decrease in cell number (Figure 14B). Transfected primary human T cells that were not treated with either NHEJ or MMEJ inhibitors showed a GFP knock-in efficiency of approximately 5%. However, treatment with NHEJ inhibitors, either alone or in combination with MMEJ inhibitors, significantly increased the GFP knock-in efficiency (Figure 14C). Knock-in efficiency was greatly enhanced, especially by combined inhibition of the NHEJ and MMEJ pathways.

[0266] Example 8 - Effect of various DNAPK inhibitors on precise gene editing outcomes HEK293T cells were seeded in 96-well plates containing media and the following conditions: a) DMSO b) 0.3125, 0.625, 1.25, 2.5, 10 μM of DNAPK inhibitor TLR1 (ISAC: (4-fluoro-3-(7-morpholinoquinazolin-4-yl)phenyl)(3-methylazin-2-yl)methanol surechembl: SCHEMBL16235486) c) 0.3125, 0.625, 1.25, 2.5, 10 μM of DNAPK inhibitor TLR2 (ISAC: 5-methyl-2-((7-methyl-[1,2,4]triazolo[1,5-a]pyridin-6-yl)amino)-8-(tetrahydro-2H-pyran-4-yl)-7,8-dihydropteridin-6(5H)-one; MedChem ELN: ELNC025305144) d) 0.3125, 0.625, 1.25, 2.5, 10 μM DNAPK inhibitor M9831 / VX-984 e) 0.3125, 0.625, 1.25, 2.5, 10 μM DNAPK inhibitor AZD7648. Cells were allowed to attach for 12 hours prior to transfection. Cells were transfected with a DNA plasmid encoding SpCas9-EGFP and an sgRNA targeting CD34 (gINS) in the presence of a single-stranded oligonucleotide donor (ssDNA). 70 hours after transfection, cell confluence and transfection efficiency based on EGFP were determined using an Incucyte S3. Genomic DNA was extracted and editing results were analyzed by deep targeted amplicon sequencing using bioinformatics analysis.

[0267] As shown in the data in Table 2 below, all DNAPK inhibitors tested increased the frequency of precise knock-in of the provided single-stranded oligonucleotide donor and reduced imprecise DNA repair events from NHEJ in a concentration-dependent manner and with similar efficiency.

[0268] [Table 6]

[0269] Example 9 - Effect of PolQ inhibitors PolQ2 and PolQ6 on precise gene editing outcomes at various target sites To assess whether PolQ2 and PolQ6 increase precise gene editing at different genomic loci, inhibitors were tested with different sgRNAs using the conditions specified in the following experiments.

[0270] HEK293T cells were seeded in 96-well plates and allowed to attach for 20 hours. Two hours prior to transfection, cells were subjected to inhibitor treatment including the following conditions: a) DMSO control b) 1 μM DNAPK inhibitor AZD7648 c) 1 μM DNAPK inhibitor AZD7648 in combination with 3 μM PolQ inhibitor (PolQ2) d) 1 μM DNAPK inhibitor AZD7648 in combination with 3 μM PolQ inhibitor (PolQ6). Cells were transfected with DNA plasmids encoding SpCas9-EGFP and sgRNAs targeting CD34 (gMEJ, gINS) and STAT1 (gDel) in the presence of single-stranded oligonucleotide donors (ssDNA). 70 hours after transfection, cell confluence and transfection efficiency based on EGFP were determined using an Incucyte S3. Genomic DNA was extracted and editing results were analyzed by deep targeted amplicon sequencing using bioinformatics analysis.

[0271] As shown below in Table 3, both PolQ inhibitors, PolQ2 and PolQ6, increase the frequency of precise knock-in of the provided single-stranded oligonucleotide donor at all target sites tested in DNAPK-inhibited cells. Furthermore, the combination of inhibitors tested reduces incorrect DNA repair events.

[0272] [Table 7]

[0273] Example 10 - Effect of PolQ inhibitor ART558 on precise gene editing outcomes To test the efficacy of the PolQ inhibitor ART558 on precise gene editing, the inhibitor was titrated using the conditions specified in the following experiment.

[0274] HEK293T cells were seeded in 96-well plates and allowed to attach for 20 h. Two hours prior to transfection, cells were subjected to inhibitor treatment including the following conditions: a) 1 μM DNAPK inhibitor AZD7648 b) 1 μM DNAPK inhibitor AZD7648 in combination with 0.1, 0.3, 1, 3 10 μM PolQ inhibitor (ART558). Cells were transfected with a DNA plasmid encoding SpCas9-EGFP and an sgRNA targeting CD34 (gMEJ) in the presence of single-stranded oligonucleotide donor (ssDNA). Seventy hours after transfection, cell confluence and transfection efficiency based on EGFP were determined using an Incucyte S3. Genomic DNA was extracted and editing results were analyzed by deep targeted amplicon sequencing using Crispresso2 bioinformatics analysis. As shown in Table 4 below, ART558 concentration-dependently increases the frequency of precise knock-in of provided single-stranded oligonucleotide donors and reduces incorrect DNA repair events with increasing inhibitor concentrations.

[0275] [Table 8]

[0276] Example 11 - Combination treatment with PolQ inhibitors targeting two functional enzyme domains To maintain genome integrity upon DNA double-strand breaks, cells have evolved different mechanisms to repair broken DNA ends. In addition to non-homologous end joining (NHEJ) and homologous recombination (HR), cells have evolved the error-prone microhomology-mediated end joining (MMEJ) DNA repair pathway. DNA polymerase theta (PolQ) is the key enzyme mediating MMEJ repair. PolQ multi-domain enzymes contain an N-terminal helicase-like function, a non-structural central domain, and a C-terminal polymerase domain. Both functional protein units are involved in PolQ-mediated DNA repair and can be inhibited using domain-specific inhibitors. This experiment addresses the question of whether simultaneous inhibition of both functional PolQ domains enhances the effect on gene editing outcomes compared to targeting of individual domains.

[0277] HEK293T cells were seeded in 96-well plates and allowed to attach for 20 hours. Two hours prior to transfection, cells were subjected to inhibitor treatment including the following conditions: a) DMSO control, b) 1 μM DNAPK inhibitor AZD7648 in combination with 1 μM and 2 μM polymerase domain-targeted PolQ inhibitor (PolQ2), c) 1 μM DNAPK inhibitor AZD7648 in combination with 1 μM and 2 μM helicase domain-targeted PolQ inhibitor (PolQ6), and d) 0.5 μM polymerase- and helicase domain-targeted PolQ inhibitors (PolQ2 and PolQ6), and 1 μM DNAPK inhibitor AZD7648 in combination with 1 μM polymerase- and helicase domain-targeted PolQ inhibitors (PolQ2 and PolQ6). Cells were transfected with a DNA plasmid encoding SpCas9-EGFP and sgRNA targeting CD34 (gMEJ) together with a single-stranded oligonucleotide donor (ssDNA). 70 hours after transfection, cell confluence and transfection efficiency based on EGFP were determined using an Incucyte S3. Genomic DNA was extracted and editing results were analyzed by deep targeted amplicon sequencing using KI RIMA for bioinformatics analysis.

[0278] As demonstrated by the data presented in Table 5, treatment with a combination PolQ inhibitor targeting both functional PolQ domains shows a significant increase in targeted knock-in and a concomitant significant decrease in incorrect DNA repair products when compared to individual PolQ inhibitors targeting only one functional enzyme domain at the same concentration.

[0279] [Table 9]

[0280] Example 12 - Assessment of off-target editing in the presence of DNAPK and PolQ inhibitors To test the effect of DNAPK / PolQ inhibitor combinations on established on- and off-target sites in HEK3 and HEK4 in off-target editing, the following experiments were analyzed.

[0281] HEK293T cells were seeded in 96-well plates and allowed to attach for 20 hours. Two hours prior to transfection, cells were subjected to inhibitor treatment including the following conditions: a) DMSO control, b) 1 μM DNAPK inhibitor AZD7648, c) 1 μM DNAPK inhibitor AZD7648 in combination with 3 μM polymerase domain-targeted PolQ inhibitor (PolQ2), and d) 1 μM DNAPK inhibitor AZD7648 in combination with 3 μM helicase domain-targeted PolQ inhibitor (PolQ6). Cells were transfected with a DNA plasmid encoding SpCas9-EGFP and sgRNAs targeting established HEK3 and HEK4 off-target sites in the absence and presence of single-stranded oligonucleotide donor (ssDNA). Seventy hours after transfection, cell confluence and transfection efficiency based on EGFP were determined using an Incucyte S3. Genomic DNA was extracted and editing results were analyzed by deep targeted amplicon sequencing using Crispresso2 bioinformatics analysis.

[0282] As shown in Table 6 below, the reduction in on- and off-target editing with DNAPK inhibitors is even more pronounced when DNAPK inhibitors are combined with PolQ inhibitors. In the presence of single-stranded oligonucleotide donors, on- and off-target editing is reduced by about 20% compared to no DNA donor samples. The reduction in on-target editing in the presence of DNAPK and PolQ inhibitors is partially restored in the presence of single-stranded oligonucleotide donors, while off-target is reduced.

[0283] [Table 10]

Claims

1. 1. A method for inserting a polynucleotide of interest into the genome of a eukaryotic cell, comprising: a. adding an inhibitor of the microhomology-mediated end joining (MMEJ) pathway to the composition comprising the eukaryotic cells; b. adding a Cas effector protein to the composition; c. adding the polynucleotide of interest to the composition wherein the polynucleotide of interest is inserted into the genome by homology directed repair (HDR) or single-stranded template repair (SSTR).

2. 2. The method of claim 1, wherein (a) comprises adding an inhibitor of the non-homologous end joining (NHEJ) pathway.

3. (d) adding a polynucleotide comprising an RNA guide sequence, a Cas binding region, a DNA template sequence, or a combination thereof to the composition. The method of claim 1 further comprising:

4. 2. The method of claim 1, wherein the Cas effector protein is added in (b) by adding a Cas polynucleotide encoding the Cas effector protein.

5. 5. The method of any one of claims 1 to 4, wherein one or more of (i) the polynucleotide of interest, (ii) the polynucleotide of (d), or (iii) the Cas polynucleotide are encoded on a vector.

6. 6. The method of claim 5, wherein (i) the polynucleotide of interest, (ii) the polynucleotide of step (d), and (iii) the Cas polynucleotide are encoded on a single vector.

7. The method according to any one of claims 1 to 4, wherein the polynucleotide of interest is added as DNA.

8. 4. The method of claim 3, wherein the polynucleotide in step (d) is added as DNA.

9. 4. The method of claim 3, wherein the polynucleotide of step (d) is added as RNA.

10. 5. The method of claim 4, wherein the Cas polynucleotide is added as DNA.

11. 5. The method of claim 4, wherein the Cas polynucleotide is added as RNA.

12. 5. The method of claim 4, wherein the Cas polynucleotide is added as mRNA.

13. The method of claim 5 , wherein the vector is a viral vector.

14. 14. The method of claim 13, wherein the viral vector is a retrovirus, lentivirus, adenovirus, or adeno-associated virus (AAV).

15. 4. The method of claim 3, wherein the Cas effector protein and the polynucleotide of (d) are added in the form of a ribonucleoprotein (RNP).

16. 4. The method of claim 3, wherein the Cas effector protein, the polynucleotide of interest, and the polynucleotide of (d) are added to the cells by microinjection, electroporation, or by lipid nanoparticles, liposomes, exosomes, gold nanoparticles, or DNA nanoclews.

17. The method of claim 5 , wherein the vector is added to the composition by transfecting the eukaryotic cell.

18. The method of any one of claims 1 to 4, wherein the Cas effector protein is a Cas9 nuclease, a Cas12a nuclease, or a Cas12f nuclease.

19. 19. The method of claim 18, wherein the Cas effector protein is a Cas9 nuclease.

20. 20. The method of claim 19, wherein the Cas9 nuclease is a Cas9 nuclease fused to a reverse transcriptase, a Cas9 nuclease fused to a DNA polymerase, a Cas9 nuclease fused to DN1S, a Cas9 nickase, a Cas9 fused to a geminin degron domain, or a Cas9 nuclease fused to CTIP.

21. The method according to any one of claims 1 to 4, wherein the polynucleotide of interest is added by a vector.

22. 22. The method of claim 21, wherein the vector is a viral vector.

23. 23. The method of claim 22, wherein the viral vector is a retrovirus, lentivirus, adenovirus, or adeno-associated virus (AAV).

24. The method according to any one of claims 1 to 4, wherein the polynucleotide of interest comprises a gene of interest.

25. The method of any one of claims 1 to 4, wherein the polynucleotide of interest is 1 to 50 base pairs in length.

26. The method of any one of claims 1 to 4, wherein the polynucleotide of interest is 50 to 5000 base pairs in length.

27. The method according to any one of claims 1 to 4, wherein the polynucleotide of interest is single-stranded.

28. The method according to any one of claims 1 to 4, wherein the polynucleotide of interest is double-stranded.

29. The method according to any one of claims 1 to 4, wherein the polynucleotide of interest is a hybrid polynucleotide comprising a single-stranded region and a double-stranded region.

30. 30. The method of claim 29, wherein the hybrid polynucleotide comprises double-stranded sequences at the 5' and 3' ends and an internal single-stranded sequence.

31. The method according to any one of claims 1 to 4, wherein the polynucleotide of interest is double-stranded and has blunt ends.

32. The method of any one of claims 1 to 4, wherein the polynucleotide of interest is double-stranded with a 3' overhang.

33. The method of any one of claims 1 to 4, wherein the polynucleotide of interest is double-stranded with a 5' overhang.

34. The method according to any one of claims 1 to 4, wherein the polynucleotide of interest is a circular polynucleotide.

35. The method of any one of claims 1 to 4, wherein the polynucleotide of interest comprises a chemical modification that enhances the stability, activity, distribution, or uptake of the polynucleotide.

36. The method of any one of claims 1 to 4, wherein the inhibitor of the MMEJ pathway is an inhibitor of POLQ.

37. 37. The method of claim 36, wherein the inhibitor of PolQ is PolQ_1, PolQ_2, PolQ_3, PolQ_4, PolQ_5, PolQ_6, PolQ_7, or a combination thereof.

38. 37. The method of claim 36, wherein the inhibitor of PolQ is a peptide.

39. The method of any one of claims 1 to 4, wherein the concentration of the inhibitor of the MMEJ pathway in the composition is from about 0.01 µM to about 1 mM.

40. The method of any one of claims 1 to 4, wherein the concentration of the inhibitor of the MMEJ pathway in the composition is from about 0.1 μM to about 100 μM.

41. The method of claim 2, wherein the inhibitor of the NHEJ pathway is an inhibitor of DNA-dependent protein kinase (DNA-PK).

42. 42. The method of claim 41, wherein the inhibitor of DNA-PK is M3814, M9831 / VX984, Nu7441, Nu7026, KU0060648, AZD7648, or a combination thereof.

43. 43. The method of claim 42, wherein the inhibitor of DNA-PK is AZD7648.

44. The method of claim 41, wherein the inhibitor of DNA-PK is a peptide.

45. 3. The method of claim 2, wherein the concentration of the inhibitor of the NHEJ pathway in the composition is from about 0.01 μM to about 1 mM.

46. 3. The method of claim 2, wherein the concentration of the inhibitor of the NHEJ pathway in the composition is from about 0.1 μM to about 100 μM.

47. 5. The method of any one of claims 1 to 4, wherein the inhibitor of the MMEJ pathway is added to the composition from 0 minutes to about 48 hours before adding the Cas effector protein to the composition.

48. 5. The method of any one of claims 1 to 4, wherein the inhibitor of the MMEJ pathway is added to the composition from 0 minutes to about 24 hours before adding the Cas effector protein to the composition.

49. 5. The method of any one of claims 1 to 4, wherein the inhibitor of the MMEJ pathway is added to the composition from 0 minutes to about 6 hours before adding the Cas effector protein to the composition.

50. 5. The method of any one of claims 1-4, wherein the inhibitor of the MMEJ pathway is added to the composition 0 minutes to about 1 hour after adding the Cas effector protein to the composition.

51. 3. The method of claim 2, wherein the inhibitor of the NHEJ pathway is added to the composition from 0 minutes to about 48 hours prior to adding the Cas effector protein to the composition.

52. 3. The method of claim 2, wherein the inhibitor of the NHEJ pathway is added to the composition from 0 minutes to about 24 hours prior to adding the Cas effector protein to the composition.

53. 3. The method of claim 2, wherein the inhibitor of the NHEJ pathway is added to the composition from 0 minutes to about 6 hours prior to adding the Cas effector protein to the composition.

54. 3. The method of claim 2, wherein the inhibitor of the NHEJ pathway is added to the composition 0 minutes to about 1 hour after adding the Cas effector protein to the composition.

55. The method of claim 2 , wherein the inhibitor of the MMEJ pathway and the inhibitor of the NHEJ pathway are added to the composition simultaneously.

56. The method of claim 2 , wherein the inhibitor of the MMEJ pathway and the inhibitor of the NHEJ pathway are added to the composition at different times.

57. 3. The method of claim 2, wherein the inhibitor of the MMEJ pathway, the inhibitor of the NHEJ pathway, and the Cas effector protein are added to the composition simultaneously.

58. 5. The method of any one of claims 1 to 4, wherein the inhibitor of the MMEJ pathway is present in the composition for about 1 to about 300 hours.

59. 5. The method of any one of claims 1 to 4, wherein the inhibitor of the MMEJ pathway is present in the composition for about 10 to about 100 hours.

60. The inhibitor of the MMEJ pathway is added at least once, at least twice, or at least three times. The method according to any one of claims 1 to 4.

61. 3. The method of claim 2, wherein the inhibitor of the NHEJ pathway is present in the composition for about 1 to about 300 hours.

62. 3. The method of claim 2, wherein the inhibitor of the NHEJ pathway is present in the composition for about 10 to about 100 hours.

63. 3. The method of claim 2, wherein the inhibitor of the NHEJ pathway is added at least once, at least twice, or at least three times.

64. The method of any one of claims 1 to 4, wherein the composition comprising the eukaryotic cells is a cell culture.

65. 65. The method of claim 64, wherein the cell culture is an in vitro cell culture or an ex vivo cell culture.

66. 65. The method of claim 64, wherein the cell culture comprises a cell extract.

67. The method according to any one of claims 1 to 4, wherein the eukaryotic cells are lymphocytes.

68. 68. The method of claim 67, wherein the lymphocyte comprises a chimeric antigen receptor (CAR) or a T cell receptor (TCR).

69. The method according to any one of claims 1 to 4, wherein the eukaryotic cells are pluripotent stem cells.

70. 70. The method of claim 69, wherein the pluripotent stem cells are induced pluripotent stem cells.

71. 65. The method of claim 64, wherein the cell culture is a mammalian cell culture.

72. 1. A method for inserting a polynucleotide of interest into the genome of a eukaryotic cell, comprising: a. adding an inhibitor of the microhomology-mediated end-joining (MMEJ) pathway to a composition comprising said eukaryotic cells; b. adding the polynucleotide of interest to the composition Including, The method, wherein the genome comprises a Cas polynucleotide integrated into the genome, and the polynucleotide of interest is inserted into the genome by homology directed repair (HDR) or single-stranded template repair (SSTR).

73. 73. The method of Claim 72, wherein (a) further comprises adding an inhibitor of the non-homologous end joining (NHEJ) pathway to the composition.

74. (c) adding a polynucleotide comprising an RNA guide sequence, a Cas binding region, a DNA template sequence, or a combination thereof to the composition.

73. The method of claim 72, further comprising:

75. 75. The method of claim 74, wherein (i) the polynucleotide of interest and (ii) the polynucleotide of (c) are encoded on a vector.

76. 76. The method of any one of claims 72 to 75, wherein the polynucleotide of interest is added as DNA.

77. 76. The method of claim 74 or 75, wherein the polynucleotide of (c) is added as DNA.

78. 76. The method of claim 74 or 75, wherein the polynucleotide of (c) is added as RNA.

79. 76. The method of claim 75, wherein the vector is a viral vector.

80. 80. The method of claim 79, wherein the viral vector is a retrovirus, lentivirus, adenovirus, or adeno-associated virus (AAV).

81. 76. The method of claim 75, wherein the vector is added to the composition by transfecting the eukaryotic cell.

82. 76. The method of any one of claims 72 to 75, wherein the genomically integrated Cas polynucleotide is inducible.

83. 76. The method of any one of claims 72 to 75, wherein the Cas effector protein is a Cas9 nuclease, a Cas12a nuclease, or a Cas12f nuclease.

84. 84. The method of Claim 83, wherein the Cas effector protein is a Cas9 nuclease.

85. 85. The method of claim 84, wherein the Cas9 nuclease is a Cas9 nuclease fused to a reverse transcriptase, a Cas9 nuclease fused to a DNA polymerase, a Cas9 nuclease fused to DN1S, a Cas9 nickase, a Cas9 fused to a geminin degron domain, or a Cas9 nuclease fused to CTIP.

86. The method of any one of claims 72 to 75, wherein the polynucleotide of interest is added by a vector.

87. 87. The method of claim 86, wherein the vector is a viral vector.

88. 88. The method of claim 87, wherein the viral vector is a retrovirus, lentivirus, adenovirus, or adeno-associated virus (AAV).

89. 76. The method of any one of claims 72 to 75, wherein the polynucleotide of interest comprises a gene of interest.

90. 76. The method of any one of claims 72 to 75, wherein the polynucleotide of interest is 1 to 50 base pairs in length.

91. 76. The method of any one of claims 72 to 75, wherein the polynucleotide of interest is 50 to 5000 base pairs in length.

92. 76. The method of any one of claims 72 to 75, wherein the polynucleotide of interest is single-stranded.

93. 76. The method of any one of claims 72 to 75, wherein the polynucleotide of interest is double-stranded.

94. 76. The method of any one of claims 72 to 75, wherein the polynucleotide of interest is a hybrid polynucleotide comprising a single-stranded region and a double-stranded region.

95. 95. The method of claim 94, wherein the hybrid polynucleotide comprises double-stranded sequences at the 5' and 3' ends and an internal single-stranded sequence.

96. 76. The method of any one of claims 72 to 75, wherein the polynucleotide of interest is double-stranded with blunt ends.

97. 76. The method of any one of claims 72 to 75, wherein the polynucleotide of interest is double-stranded with a 3' overhang.

98. 76. The method of any one of claims 72 to 75, wherein the polynucleotide of interest is double-stranded with a 5' overhang.

99. The method of any one of claims 72 to 75, wherein the polynucleotide of interest is a circular polynucleotide.

100. 76. The method of any one of claims 72 to 75, wherein the polynucleotide of interest comprises a chemical modification that enhances the stability, activity, distribution, or uptake of the polynucleotide.

101. The method of any one of claims 72 to 75, wherein the inhibitor of the MMEJ pathway is an inhibitor of POLQ.

102. 102. The method of claim 101, wherein the inhibitor of PolQ is PolQ_1, PolQ_2, PolQ_3, PolQ_4, PolQ_5, PolQ_6, PolQ_7, or a combination thereof.

103. 102. The method of claim 101, wherein the inhibitor of PolQ is a peptide.

104. 76. The method of any one of claims 72 to 75, wherein the concentration of the inhibitor of the MMEJ pathway in the composition is from about 0.01 μM to about 1 mM.

105. 76. The method of any one of claims 72 to 75, wherein the concentration of the inhibitor of the MMEJ pathway in the composition is from about 0.1 μM to about 100 μM.

106. The method of any one of claims 73 to 75, wherein the inhibitor of the NHEJ pathway is an inhibitor of DNA-dependent protein kinase (DNA-PK).

107. 107. The method of claim 106, wherein the inhibitor of DNA-PK is M3814, M9831 / VX984, Nu7441, Nu7026, KU0060648, AZD7648, or a combination thereof.

108. 107. The method of claim 106, wherein the inhibitor of DNA-PK is a peptide.

109. 109. The method of claim 108, wherein the inhibitor of DNA-PK is AZD7648.

110. 76. The method of any one of claims 73 to 75, wherein the concentration of the inhibitor of the NHEJ pathway in the composition is from about 0.01 μM to about 1 mM.

111. 76. The method of any one of claims 73 to 75, wherein the concentration of the inhibitor of the NHEJ pathway in the composition is from about 0.1 μM to about 100 μM.

112. 76. The method of any one of claims 72-75, wherein the inhibitor of the MMEJ pathway is added to the composition from 0 minutes to about 48 hours prior to induction of the genomically integrated Cas polynucleotide.

113. 76. The method of any one of claims 72-75, wherein the inhibitor of the MMEJ pathway is added to the composition from 0 minutes to about 24 hours prior to induction of the genomically integrated Cas polynucleotide.

114. 76. The method of any one of claims 72-75, wherein the inhibitor of the MMEJ pathway is added to the composition from 0 minutes to about 6 hours prior to induction of the genomically integrated Cas polynucleotide.

115. 76. The method of any one of claims 73-75, wherein the inhibitor of the NHEJ pathway is added to the composition from 0 minutes to about 24 hours prior to induction of the genomic integrated Cas polynucleotide.

116. 76. The method of any one of claims 73-75, wherein the inhibitor of the NHEJ pathway is added to the composition from 0 minutes to about 24 hours prior to induction of the genomic integrated Cas polynucleotide.

117. 76. The method of any one of claims 73-75, wherein the inhibitor of the NHEJ pathway is added to the composition from 0 minutes to about 6 hours prior to induction of the genomic integrated Cas polynucleotide.

118. 76. The method of any one of claims 73 to 75, wherein the inhibitor of the MMEJ pathway and the inhibitor of the NHEJ pathway are added to the composition simultaneously.

119. 76. The method of any one of claims 73 to 75, wherein the inhibitor of the MMEJ pathway and the inhibitor of the NHEJ pathway are added to the composition at different times.

120. 76. The method of any one of claims 73 to 75, wherein the inhibitor of the MMEJ pathway and the inhibitor of the NHEJ pathway are added to the composition simultaneously with induction of the genomic integrated Cas polynucleotide.

121. 76. The method of any one of claims 72-75, wherein the inhibitor of the MMEJ pathway is present in the composition for about 1 to about 300 hours.

122. 76. The method of any one of claims 72-75, wherein the inhibitor of the MMEJ pathway is present in the composition for about 10 to about 100 hours.

123. 76. The method of any one of claims 72 to 75, wherein the inhibitor of the MMEJ pathway is added at least once, at least twice, or at least three times.

124. 76. The method of any one of claims 73 to 75, wherein the inhibitor of the NHEJ pathway is present in the composition for about 1 to about 300 hours.

125. 76. The method of any one of claims 73 to 75, wherein the inhibitor of the NHEJ pathway is present in the composition for about 10 to about 100 hours.

126. 76. The method of any one of claims 73 to 75, wherein the inhibitor of the NHEJ pathway is added at least once, at least twice, or at least three times.

127. 76. The method of any one of claims 72 to 75, wherein the composition comprising the eukaryotic cells is a cell culture.

128. 128. The method of claim 127, wherein the cell culture is an in vitro cell culture or an ex vivo cell culture.

129. 128. The method of claim 127, wherein the cell culture comprises a cell extract.

130. 76. The method of any one of claims 72 to 75, wherein the eukaryotic cell is a lymphocyte.

131. 131. The method of claim 130, wherein the lymphocyte comprises a chimeric antigen receptor or a T cell receptor (TCR).

132. 76. The method of any one of claims 72 to 75, wherein the eukaryotic cell is a pluripotent stem cell.

133. 133. The method of claim 132, wherein the pluripotent stem cells are induced pluripotent stem cells.

134. 130. The method of claim 129, wherein the cell culture is a mammalian cell culture.

135. 1. A method for inserting a polynucleotide into the genome of a eukaryotic cell, comprising: a. adding an inhibitor of the microhomology-mediated end-joining (MMEJ) pathway to a composition comprising said eukaryotic cells; b. the eukaryotic cell i. a vector encoding a Cas effector protein; ii. a vector encoding a polynucleotide of interest; iii. A vector comprising a polynucleotide comprising an RNA guide sequence, a Cas binding region, a DNA template sequence, or a combination thereof. Transfecting Including, The vectors (i), (ii) and (iii) can be on the same vector or different vectors, and the polynucleotide of interest is inserted into the genome by homology directed repair (HDR) or single-stranded template repair (SSTR).

136. 136. The method of claim 135, further comprising adding an inhibitor of the non-homologous end joining (NHEJ) pathway to the composition comprising the eukaryotic cell.

137. 136. The method of claim 135, wherein the Cas effector protein is encoded by a Cas polynucleotide.

138. 138. The method of any one of claims 135 to 137, wherein (i) the Cas effector protein and (ii) the polynucleotide of interest are encoded on a single vector.

139. 138. The method of any one of claims 135 to 137, wherein the Cas effector protein and the polynucleotide of (iii) are encoded on a single vector.

140. 138. The method of any one of claims 135 to 137, wherein the Cas effector protein, the polynucleotide of interest, and the polynucleotide of (iii) are encoded on a single vector.

141. 137. The method of claim 135 or 136, wherein the Cas effector protein and the polynucleotide of (iii) are added in the form of a ribonucleoprotein (RNP).

142. 1. A method for increasing the gene insertion efficiency of homology-directed repair (HDR) and single-stranded template repair (SSTR) in eukaryotic cells, comprising adding an inhibitor of the microhomology-mediated end-joining (MMEJ) pathway when performing CRISPR / Cas-mediated gene insertion in the eukaryotic cells.

143. 143. The method of claim 142, comprising adding an inhibitor of the non-homologous end joining (NHEJ) pathway.

144. 144. The method of claim 142 or 143, wherein the CRISPR / Cas-mediated gene insertion is CRISPR / Cas9-mediated gene insertion.

145. A method for reducing microhomology-mediated end-joining (MMEJ) pathway recombination during CRISPR / Cas-mediated gene insertion in a cell, the method comprising adding an inhibitor of the microhomology-mediated end-joining (MMEJ) pathway to the cell when performing Cas-mediated gene insertion.

146. 146. The method of claim 145, further comprising reducing non-homologous end joining (NHEJ) recombination during CRISPR / Cas-mediated gene insertion in the cell, comprising adding an inhibitor of the non-homologous end joining (NHEJ) pathway to the cell.

147. 147. The method of claim 145 or 146, wherein the CRISPR / Cas-mediated gene insertion is CRISPR / Cas9-mediated gene insertion.

148. a. a Cas effector protein or a vector encoding a Cas effector protein; and b. Inhibitors of the microhomology-mediated end-joining (MMEJ) pathway A composition comprising:

149. 149. The composition of claim 148, further comprising an inhibitor of the non-homologous end joining (NHEJ) pathway.

150. 149. The composition of Claim 148, further comprising a polynucleotide comprising at least one RNA guide sequence, Cas binding region, DNA template sequence, or a combination thereof.

151. 151. The composition of any one of claims 148 to 150, wherein the Cas effector protein is a Cas9 nuclease, a Cas12a nuclease, or a Cas12f nuclease.

152. 152. The composition of claim 151, wherein the Cas effector protein is a Cas9 nuclease.

153. 153. The composition of claim 152, wherein the Cas9 nuclease is a Cas9 nuclease fused to a reverse transcriptase, a Cas9 nuclease fused to a DNA polymerase, a Cas9 nuclease fused to DN1S, a Cas9 nickase, a Cas9 fused to a geminin degron domain, or a Cas9 nuclease fused to CTIP.

154. 151. The composition of any one of claims 148 to 150, wherein the vector encoding a Cas effector protein is a viral vector.

155. 151. The composition of Claim 150, wherein the polynucleotide comprising at least one RNA guide sequence, Cas binding region, DNA template sequence, or combinations thereof is encoded on a vector.

156. 156. The composition of Claim 155, wherein the vector encoding the polynucleotide comprising at least one RNA guide sequence, Cas binding region, DNA template sequence, or combinations thereof is a viral vector.

157. 151. The composition of Claim 150, wherein the Cas effector protein and the polynucleotide comprising at least one RNA guide sequence, Cas binding region, DNA template sequence, or combinations thereof are in the form of a ribonucleoprotein (RNP).

158. 151. The composition of any one of claims 148 to 150, further comprising a pharmaceutically acceptable carrier, diluent, or excipient.

159. a. a Cas effector protein or a vector encoding a Cas effector protein; and b. Inhibitors of the microhomology-mediated end-joining (MMEJ) pathway Kit including:

160. 160. The kit of claim 159, further comprising an inhibitor of the non-homologous end joining (NHEJ) pathway.

161. The kit described in claim 159, further comprising a polynucleotide comprising at least one RNA guide sequence, Cas binding region, DNA template sequence, or a combination thereof.

162. 162. The kit of any one of claims 159 to 161, wherein the Cas effector protein is a Cas9 nuclease, a Cas12a nuclease, or a Cas12f nuclease.

163. 163. The kit of claim 162, wherein the Cas effector protein is a Cas9 nuclease.

164. 164. The kit of claim 163, wherein the Cas9 nuclease is a Cas9 nuclease fused to a reverse transcriptase, a Cas9 nuclease fused to a DNA polymerase, a Cas9 nuclease fused to DN1S, a Cas9 nickase, a Cas9 fused to a geminin degron domain, or a Cas9 nuclease fused to CTIP.

165. 162. The kit of any one of claims 159 to 161, wherein the vector encoding a Cas effector protein is a viral vector.

166. 162. The kit of claim 161, wherein the polynucleotide is encoded on a vector.

167. 167. The kit of claim 166, wherein the vector encoding the polynucleotide is a viral vector.

168. 162. The kit of claim 161, wherein the Cas effector protein and the polynucleotide are in the form of a ribonucleoprotein (RNP).

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