Compositions and methods for site-specific modification

Abstract

The present disclosure provides a polynucleotide comprising an RNA guide sequence, a Cas binding region, and a DNA template sequence. The present disclosure also provides a composition comprising a Cas nuclease or Cas nickase and one or more polynucleotides comprising a guide sequence, a Cas binding region, and a DNA template sequence. The present disclosure further provides a fusion protein comprising a Cas nuclease or Cas nickase and a DNA polymerase recruitment portion. Also provided is a method for providing a target insertion in a target DNA of a cell.

Description

[Technical field]

[0001] The present disclosure provides a polynucleotide comprising an RNA guide sequence, a Cas binding region, and a DNA template sequence. The present disclosure also provides a composition comprising a Cas nuclease or Cas nickase and one or more polynucleotides comprising a guide sequence, a Cas binding region, and a DNA template sequence. The present disclosure further provides a fusion protein comprising a Cas nuclease or Cas nickase and a DNA polymerase recruitment portion. Also provided is a method for providing a target insertion in a target DNA of a cell. [Background technology]

[0002] Programmable nucleases such as CRISPR / Cas9 can generate site-specific double-strand breaks (DSBs) that can disrupt genes by inducing combined insertions and deletions (indels) at target sites. However, DSB repair that relies on template-dependent homology-directed repair (HDR) can be infrequent, while highly efficient template-independent non-homologous end joining (NHEJ) can be error-prone and not favor the desired insertion.

[0003] Non-Patent Document 1 describes the development of prime editing, which uses Cas9 nickase-reverse transcriptase fusion enzyme to insert a short sequence at the cleavage site. Prime editing relies on a complex mechanism of RNA removal and single-stranded DNA hybridization to the target site, and also requires the removal of overlapping "flap" sequences by cellular equilibrium. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Anzalone et al.(Nature 576;149-157(2019) Summary of the Invention [Means for solving the problem]

[0005] In some embodiments, the disclosure provides a polynucleotide comprising: (i) an RNA guide sequence, (ii) a Cas binding region, and (iii) a DNA template sequence, wherein the DNA template sequence is at a 3' end of the polynucleotide.

[0006] In some embodiments, the DNA template sequence comprises modified nucleotides, non-B-type DNA structures, DNA polymerase recruiting moieties, DNA ligase recruiting moieties, or combinations thereof.

[0007] In some embodiments, the modified nucleotide comprises an abasic site, a covalent linker, a heterologous nucleic 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. In some embodiments, the covalent linker comprises triethylene glycol (TEG). In some embodiments, the DNA lesion comprises 8-oxoguanine, thymine-glycol, or a combination thereof. In some embodiments, the DNA photoproduct comprises a cyclobutane pyrimidine dimer (CPD), a pyrimidine (6-4) pyrimidone photoproduct, or a combination thereof. In some embodiments, the modified deoxyribonucleoside comprises deoxyuridine. In some embodiments, the methylated nucleotide comprises 5-hydroxymethylcytosine, 5-methylcytosine, or a combination thereof.

[0008] In some embodiments, the non-B-type DNA structure comprises a hairpin, a cruciform, a Z-DNA, an H-DNA (triple helix DNA), a G-quadruplex DNA (quadruplex DNA), a slip DNA, a sticky DNA, or a combination thereof. In some embodiments, the DNA polymerase recruitment portion comprises a DNA polymerase recruitment protein linked to a DNA template sequence. In some embodiments, the DNA polymerase recruitment protein comprises a proliferating cell nuclear antigen (PCNA), a single stranded DNA binding protein (SSBP), a tumor necrosis factor alpha-induced protein (TNFAIP), a polymerase delta interacting protein (PolDIP), an X-ray repair cross-complementing protein (XRCC), a 5-hydroxymethylcytosine-binding embryonic stem cell specific (HMCES) protein, RAD1, RAD9, HUS1, or a combination thereof. In some embodiments, the DNA ligase recruitment portion comprises a 5' adenylation of the DNA template sequence.

[0009] In some embodiments, the disclosure provides a polynucleotide comprising: (i) an RNA guide sequence; (ii) a Cas binding region; and (iii) a DNA template sequence, wherein the DNA template sequence is at the 3' end of the polynucleotide, and wherein the DNA template sequence comprises phosphorothioate linkages. In some embodiments, the DNA template sequence is at the 5' end of the polynucleotide.

[0010] In some embodiments, the DNA template sequence comprises a primer binding sequence and a sequence of interest. In some embodiments, the Cas binding region comprises RNA, or the Cas binding region comprises a combination of RNA and DNA. In some embodiments, the Cas binding region comprises tracrRNA. In some embodiments, the Cas binding region can hybridize to tracrRNA.

[0011] In some embodiments, the tracrRNA can bind to a Cas nuclease. In some embodiments, the Cas nuclease is Cas9 or Cas12a. In some embodiments, the Cas nuclease is a type II-B Cas. In some embodiments, the tracrRNA can bind to a Cas nickase. In some embodiments, the Cas nickase is a Cas9 nickase, a Cas12a nickase, or a type II-B Cas nickase.

[0012] In some embodiments, the DNA template sequence is about 8 to about 10,000 nucleotides in length. In some embodiments, the primer binding sequence is about 4 to about 300 nucleotides in length. In some embodiments, the sequence of interest is about 4 to about 100 nucleotides in length. In some embodiments, the RNA guide sequence is about 15 to about 25 nucleotides in length.

[0013] In some embodiments, the polynucleotide further comprises a spacer located between the Cas binding region and the DNA template sequence. In some embodiments, the spacer comprises a stop sequence for a DNA polymerase. In some embodiments, the spacer comprises two or more stop sequences. In some embodiments, the stop sequence comprises a secondary structure. In some embodiments, the secondary structure comprises a stem loop. In some embodiments, the spacer is about 10 to about 200 nucleotides in length.

[0014] In some embodiments, the disclosure provides a cell comprising a polynucleotide described herein.

[0015] In some embodiments, the disclosure provides a composition comprising a Cas nuclease or Cas nickase and a polynucleotide comprising (i) a guide sequence, (ii) a Cas binding region, and (iii) a DNA template sequence, wherein the DNA template sequence is at the 3' end of the polynucleotide.

[0016] In some embodiments, the disclosure provides a composition comprising a Cas nuclease or Cas nickase; a first polynucleotide comprising (i) a guide sequence, (ii) a Cas binding region, and (iii) a first hybridization region, where the first hybridization region is at a 3' end of the first polynucleotide; and a second polynucleotide comprising (i) a second hybridization region complementary to the first hybridization region, and (ii) a DNA template sequence.

[0017] In some embodiments, the disclosure provides a composition comprising a Cas nuclease or Cas nickase; a first polynucleotide comprising (i) a guide sequence, and (ii) a first hybridization region, where the first hybridization region is at a 3' end of the first polynucleotide; and a second polynucleotide comprising (i) a second hybridization region complementary to the first hybridization region, (ii) a Cas binding region, and (iii) a DNA template sequence.

[0018] In some embodiments, the DNA template sequence comprises a primer binding sequence and a sequence of interest, hi some embodiments, the DNA template sequence comprises modified nucleotides, non-B-type DNA structures, DNA polymerase recruitment moieties, DNA ligase recruitment moieties, or combinations thereof.

[0019] In some embodiments, the modified nucleotide comprises an abasic site, a covalent linker, a heterologous nucleic 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. In some embodiments, the modified nucleotide comprises a phosphorothioate bond. In some embodiments, the covalent linker comprises triethylene glycol (TEG). In some embodiments, the DNA lesion comprises 8-oxoguanine, thymine-glycol, or a combination thereof. In some embodiments, the DNA photoproduct comprises a cyclobutane pyrimidine dimer (CPD), a pyrimidine(6-4) pyrimidone photoproduct, or a combination thereof. In some embodiments, the deoxyribonucleoside comprises a deoxyuridine. In some embodiments, the methylated nucleotide comprises 5-hydroxymethylcytosine, 5-methylcytosine, or a combination thereof.

[0020] In some embodiments, the non-B-type DNA structure comprises a hairpin, a cruciform, a Z-DNA, an H-DNA (triple helix DNA), a G-quadruplex DNA (quadruplex DNA), a slip DNA, a sticky DNA, or a combination thereof. In some embodiments, the DNA polymerase recruitment portion comprises a DNA polymerase recruitment protein linked to a DNA template sequence. In some embodiments, the DNA polymerase recruitment protein comprises a proliferating cell nuclear antigen (PCNA), a single stranded DNA binding protein (SSBP), a tumor necrosis factor alpha-induced protein (TNFAIP), a polymerase delta interacting protein (PolDIP), an X-ray repair cross-complementing protein (XRCC), a 5-hydroxymethylcytosine-binding embryonic stem cell specific (HMCES) protein, RAD1, RAD9, HUS1, or a combination thereof. In some embodiments, the DNA ligase recruitment portion comprises a 5' adenylation of the DNA template sequence.

[0021] In some embodiments, the guide sequence comprises RNA, or the guide sequence comprises a combination of RNA and DNA. In some embodiments, the Cas binding region comprises RNA, or the Cas binding region comprises a combination of RNA and DNA. In some embodiments, the Cas binding region comprises a tracrRNA. In some embodiments, the Cas binding region can hybridize to a tracrRNA, and the composition further comprises a tracrRNA.

[0022] In some embodiments, the tracrRNA can bind to a Cas nuclease or a Cas nickase. In some embodiments, the composition comprises a Cas nuclease. In some embodiments, the Cas nuclease is Cas9 or Cas12a. In some embodiments, the composition comprises a Cas nuclease, and the Cas nuclease is a type II-B Cas. In some embodiments, the composition comprises a Cas nickase. In some embodiments, the Cas nickase is a Cas9 nickase, a Cas12a nickase, or a type II-B Cas nickase.

[0023] In some embodiments, the DNA template sequence is about 8 to about 500 nucleotides in length. In some embodiments, the primer binding sequence is about 4 to about 30 nucleotides in length. In some embodiments, the sequence of interest is about 4 to about 100 nucleotides in length. In some embodiments, the RNA guide sequence is about 15 to about 25 nucleotides in length.

[0024] In some embodiments, the first hybridization region and the second hybridization region are RNA. In some embodiments, the first hybridization region and the second hybridization region are single-stranded DNA. In some embodiments, the first hybridization region is RNA and the second hybridization region is single-stranded DNA, or the first hybridization region is single-stranded DNA and the second hybridization region is RNA. In some embodiments, the first hybridization region is about 4 to about 5000 nucleotides in length. In some embodiments, the second hybridization region is about 4 to about 5000 nucleotides in length.

[0025] In some embodiments, the polynucleotide further comprises a spacer located 5' of the DNA template sequence. In some embodiments, the second polynucleotide further comprises a spacer located 5' of the DNA template sequence. In some embodiments, the spacer comprises a stop sequence for a DNA polymerase. In some embodiments, the spacer comprises two or more stop sequences. In some embodiments, the stop sequence is comprised of a secondary structure. In some embodiments, the secondary structure is comprised of a stem loop. In some embodiments, the spacer is about 10 to about 200 nucleotides in length.

[0026] In some embodiments, the Cas nuclease or Cas nickase is fused to a DNA polymerase recruitment protein, which in some embodiments comprises proliferating cell nuclear antigen (PCNA), single stranded DNA binding protein (SSBP), tumor necrosis factor alpha-induced protein (TNFAIP), polymerase delta interacting protein (PolDIP), X-ray repair cross-complementing protein (XRCC), 5-hydroxymethylcytosine-binding embryonic stem cell-specific (HMCES) protein, RAD1, RAD9, HUS1, or a combination thereof.

[0027] In some embodiments, the disclosure provides a cell comprising the composition described herein. In some embodiments, the cell further comprises an exogenous DNA polymerase, an exogenous DNA ligase, or both.

[0028] In some embodiments, the disclosure provides a fusion protein comprising (i) a Cas nuclease or a Cas nickase, and (ii) a DNA polymerase recruitment protein.

[0029] In some embodiments, the fusion protein comprises a Cas nuclease. In some embodiments, the Cas nuclease is Cas9 or Cas12a. In some embodiments, the Cas nuclease is a type II-B Cas. In some embodiments, the fusion protein comprises a Cas nickase. In some embodiments, the Cas nickase is a Cas9 nickase, a Cas12a nickase, or a type II-B Cas nickase.

[0030] In some embodiments, the DNA polymerase recruitment protein comprises proliferating cell nuclear antigen (PCNA), single stranded DNA binding protein (SSBP), tumor necrosis factor alpha-induced protein (TNFAIP), polymerase delta interacting protein (PolDIP), X-ray repair cross-complementing protein (XRCC), 5-hydroxymethylcytosine-binding embryonic stem cell-specific (HMCES) protein, RAD1, RAD9, HUS1, or a combination thereof.

[0031] In some embodiments, the disclosure provides a polynucleotide encoding a fusion protein described herein. In some embodiments, the disclosure provides a vector comprising a polynucleotide encoding the fusion protein. In some embodiments, the disclosure provides a cell comprising the fusion protein, vector, or polynucleotide. In some embodiments, the cell further comprises a polynucleotide described herein comprising (i) an RNA guide sequence, (ii) a Cas binding region, and (iii) a DNA template sequence, wherein the DNA template sequence is at the 3' end of the polynucleotide.

[0032] In some embodiments, the present disclosure provides a method of providing a targeted insertion in a target DNA in a cell, comprising introducing into the cell a composition described herein, wherein the guide sequence is capable of hybridizing to the target DNA.

[0033] In some embodiments, the method does not include introducing a DNA polymerase into the cell. In some embodiments, the Cas nuclease generates a double-strand break in the target DNA and the cell's endogenous DNA polymerase extends the DNA template sequence.

[0034] In some embodiments, the method further comprises introducing an exogenous DNA polymerase into the cell, hi some embodiments, the Cas nuclease generates a double-stranded break in the target DNA and the exogenous DNA polymerase extends the DNA template sequence.

[0035] In some embodiments, the DNA template sequence comprises a primer binding sequence and a sequence of interest, and a DNA polymerase synthesizes a DNA strand complementary to the sequence of interest to form a double-stranded sequence comprising the sequence of interest. In some embodiments, the double-stranded sequence is inserted into the cleaved target DNA. In some embodiments, the double-stranded sequence is inserted into the cleaved target DNA by non-homologous end joining (NHEJ). In some embodiments, the double-stranded sequence is inserted into the cleaved target DNA by a DNA ligase. In some embodiments, the DNA ligase is an endogenous DNA ligase of the cell. In some embodiments, the DNA ligase is an exogenous DNA ligase.

[0036] In some embodiments, the disclosure relates to a composition comprising: (a) a Cas nuclease or a Cas nickase; (b) a first polynucleotide comprising (i) a guide sequence, (ii) a Cas binding region, (iii) a first hybridization region, and (iv) a primer binding sequence, wherein the primer binding sequence is at a 3' end of the first polynucleotide; and (c) a second polynucleotide comprising (i) a second hybridization region complementary to the first hybridization region, and (ii) a sequence of interest (SOI).

[0037] In some embodiments, the disclosure relates to a composition comprising: (a) a Cas nuclease or a Cas nickase; (b) a first polynucleotide comprising: (i) a guide sequence, (ii) a Cas binding region, and (iii) a first hybridization region, wherein the first hybridization region is at a 3' end of the first polynucleotide; (c) a second polynucleotide comprising: (i) a second hybridization region complementary to the first hybridization region, (ii) a third hybridization region, and (iii) a primer binding sequence, wherein the primer binding sequence is at a 3' end of the second polynucleotide; and (d) a third polynucleotide comprising: (i) a fourth hybridization region complementary to the third hybridization region, and (ii) a sequence of interest (SOI).

[0038] In some embodiments, the disclosure relates to a fusion protein comprising (i) a Cas nuclease or a Cas nickase, and (ii) a DNA polymerase recruitment protein, a DNA ligase, a DNA ligase recruitment moiety, a DNA binding protein, a DNA repair protein, or a combination thereof.

[0039] In some embodiments, the disclosure provides a method comprising introducing a composition as described herein into a cell, wherein the guide sequence is capable of hybridizing to the target DNA, to provide a targeted insertion in the target DNA within the cell.

[0040] The following drawings form part of the present specification and are included to further demonstrate illustrative embodiments of certain aspects of the present invention. [Brief description of the drawings]

[0041] [Figure 1]1 shows an exemplary method described in the embodiments herein. Cas nuclease is guided to a target DNA in a cell by a guide sequence and cleaves the target DNA. The DNA template sequence includes (i) a primer binding sequence that serves as a primer and hybridizes with the cleaved DNA, and (ii) a target sequence. A DNA polymerase, for example, an endogenous DNA polymerase of the cell, binds to the primer and synthesizes a DNA strand complementary to the target sequence, thereby forming a double-stranded sequence containing the target sequence. The double-stranded sequence can be inserted into the cleaved target DNA by a DNA repair pathway, for example, NHEJ. [Figures 2A-2D] Exemplary polynucleotide constructs ("springRNAs") tested in the in vivo targeted insertion experiments described herein are shown. Figure 2A shows an RNA-only polynucleotide composed of an RNA guide sequence, a tracrRNA, and an RNA template sequence. Figure 2B shows an RNA-DNA hybrid polynucleotide composed of an RNA guide sequence, a tracrRNA, and a DNA template sequence ("DNA") or a DNA template sequence with phosphorothioate linkages ("PS-DNA"). Figure 2C shows an RNA-only polynucleotide composed of an RNA guide sequence, a tracrRNA, and an RNA template sequence with an abasic site. Figure 2D shows an RNA-only polynucleotide composed of an RNA guide sequence, a tracrRNA, and an RNA template sequence with TEG. [Diagram 3] Related to Example 1, results of targeted insertion using SpCas9 alone or SpCas9-reverse transcriptase (SpCas9-RT) fusion protein with various springRNA constructs are shown. Panels A-E of Figure 3 show targeted insertion with SpCas9 and each of the springRNA constructs of Figures 2A-2D. Panels F-J of Figure 3 show targeted insertion with SpCas9-RT ("PE0") and each of the springRNA constructs of Figures 2A-2D. [Figure 4]FIG. 1 shows the relative frequency of insertion by SpCas9 or SpCas9-RT in combination with each of the springRNA constructs of FIGS. 2A-2D, related to Example 1. Each point represents an independent replicate. The mean and standard deviation of triplicate experiments are shown as whisker plots. [Figure 5A-5F]

[0036] Figures 5A-5C show targeted insertion using SpCas9-RT ("PE0") with RNA-only springRNA (Figure 5A), with a DNA tail (Figure 5B), and with PS-DNA (Figure 5C). Figures 5D-5F show targeted insertion with SpCas9 with RNA-only springRNA (Figure 5D), with a DNA tail (Figure 5E), and with PS-DNA (Figure 5F). [Figure 6] An overview of the in vitro assay described herein is shown. Two complementary DNA strands are labeled with different fluorophores (6 FAM-labeled non-target strand and HEX-labeled target strand). The guide sequence is designed such that Cas9 cleavage generates two strands of different lengths. DNA polymerase extends the 6 FAM-labeled non-target strand hybridized to the primer binding sequence of springRNA. The products are denatured and separated by capillary electrophoresis, and the strand bound to the fluorophore is detected. [Figure 7A-7F] FIG. 7 shows the results of the in vitro assay shown in FIG. 6, which relates to Example 3. The blue line corresponds to the non-target strand and the green line corresponds to the target strand. The asterisk indicates the cleaved synthetic target DNA substrate and the black arrow indicates the extension product by DNA polymerase. FIG. 7A shows the results of the assay with Cas9 and springRNA. FIG. 7B shows the results of the assay with Cas9, Klenow fragment, and springRNA. FIG. 7C-7F show the results of the assay with Cas9, Bst 3.0 polymerase, and springRNA (FIGS. 7C and 7D), springRNA with abasic site (FIG. 7E), or springRNA with TEG (FIG. 7F). [Figure 8]1 shows the kinetics of the extension reaction of Bst 3.0 polymerase and Klenow fragment, the DNA polymerases used in the in vitro assay of Example 3. [Figure 9] 23 shows an exemplary composition described in embodiments herein, comprising a Cas protein (e.g., Cas nuclease or Cas nickase), a first polynucleotide comprised of a guide sequence (referred to as a "spacer"), a Cas binding region (referred to as a "gRNA scaffold"), a first hybridization region (referred to as a "landing pad"), and a primer binding sequence (PBS), and a second polynucleotide comprised of a second hybridization region (referred to as a "hybridization sequence"), a SOI (referred to as an "insert"), and a homologous sequence. The terminology for the various components of the composition in FIG. 9 is used throughout FIGS. 9-23. [Figure 10A-10C] An exemplary method described in the embodiments herein is shown. In FIG. 10A, a guide sequence, a Cas binding region, a first hybridization region, and a primer binding site are located on a first polynucleotide. A Cas nuclease guided to the target DNA by the guide sequence generates a double-stranded break in the target DNA. The primer binding site hybridizes to the cleaved DNA, and a second polynucleotide hybridizes to the first polynucleotide through the first and second hybridization regions that contain complementary sequences. The second polynucleotide contains a sequence of interest, which is ligated to the cleaved DNA at both the 5' and 3' ends by a ligase (FIG. 10A). The second polynucleotide can further contain a homologous sequence, the 5' end of which is ligated to the cleaved DNA by the ligase, and the 3' end of which is incorporated by a homology-mediated mechanism (FIG. 10B). The complex is converted into double-stranded DNA after ligation and / or homology-mediated integration, thereby incorporating the sequence of interest into the target DNA (Figure 10C). [Figures 11A-11J]11 shows an exemplary embodiment of the composition described herein. FIG. 11(A) shows a donor containing all three elements: a second hybridization sequence, a sequence of interest, and a homologous sequence. FIG. 11(B) shows a donor without a homologous sequence. FIG. 11(C) shows a donor without a second hybridization sequence. FIG. 11(D) shows a standard donor in which a sequence complementary to the sequence of interest is hybridized to form a double-stranded region of the sequence of interest with overhangs at both 5' and 3'. This configuration is referred to as "overhang". FIG. 11(E) shows a donor like FIG. 11(A), but with the plugRNA composed of two nucleic acids. The sgRNA has a sequence of about 30 nt length added to the 3' end. This 3' sequence can pair with a complementary polynucleotide, followed by a first hybridization sequence and a primer binding sequence. This configuration is referred to as a "split plugRNA" or "split system". FIG. 11(F) shows a configuration like FIG. 11(A), but the hybridization sequence is shorter, creating a gap between the 3' end of the primed target site and the 5' end of the donor. This configuration is referred to as "gap". FIG. 11(G) is the same as FIG. 11(A), but the donor has additional nucleotides at the 5' end, creating a flap that in some embodiments may engage FEN1 and other repair mechanisms. This configuration is referred to as "flap". FIG. 11(H) is the same as FIG. 11(B), but a sequence complementary to the sequence of interest is annealed to create a blunt end at the 3' end of the donor. This configuration is referred to as "blunt". In FIG. 11(I), the donor is split into two polynucleotides. The first donor strand is composed of the second hybridization sequence and the sequence of interest. The second sequence is composed of a homologous sequence and a sequence complementary to the sequence of interest. This configuration is referred to as "bridge". In Figure 11(J), a homologous sequence is embedded in the plugRNA immediately downstream of the gRNA scaffold, which anneals 3' to the Cas-induced cleavage.The donor is composed of a sequence of interest flanked by two hybridization sequences: a second hybridization sequence and an additional hybridization sequence, where the second hybridization sequence is complementary to the first hybridization sequence and the additional hybridization sequence is complementary to the region adjacent to the homologous sequence. This configuration places both the 5' and 3' sides of the donor adjacent to the double-stranded break. This configuration is referred to as "dual hybridization." [Figures 12A-12C] Figure 12A shows the experimental design of an exemplary in vitro assay, as described in Example 8. Figures 12B and 12C show the results of the assay, with Figure 12B showing the ligation production rate and Figure 12C showing the ligation efficiency rate. [Figures 13A-13C] Figure 13A shows the experimental design of an exemplary in vitro ligation assay using HeLa nuclear extract, as described in Example 9. Figures 13B and 13C show heat map results of the assay, with Figure 13B showing the percentage ligation production and Figure 13C showing the percentage ligation efficiency. [Figure 14A-14B] Figure 14A shows the experimental design of an exemplary assay using HeLa nuclear extracts, with the resulting DNA amplified and analyzed for target inserts, as described in Example 10. Figure 14B shows a display of next generation sequencing (NGS) results of the target insert sequence. [Figure 15] 11A-11J show the results of exemplary assays performed with compositions of various configurations shown in FIGS. 11A-11J, as described in Example 11. [Figure 16A-16B] Figure 16A shows an exemplary embodiment of the composition described herein. Figure 16B shows the results of an exemplary assay performed with varying lengths of different components of the composition, as described in Example 12. [Figure 17A-17B] Figure 17A shows an exemplary embodiment of the composition described herein. Figure 17B shows the results of an exemplary assay performed with varying lengths of different components of the composition, as described in Example 13. [Figure 18A-18B]Figure 18A shows an exemplary embodiment of a composition described herein, and Figure 18B shows the results of an exemplary assay performed varying the length and / or modification of different components of the composition, as described in Example 14. [Figure 19A-19B] Figure 19A shows an exemplary embodiment of the composition described herein. Figure 19B shows the results of an exemplary assay performed with varying lengths of different components of the composition, as described in Example 15. [Figure 20A-20B] Figure 20A shows an exemplary embodiment of the compositions described herein. Figure 20B shows the results of an exemplary assay performed with varying lengths and / or modifications of different components of the compositions, in the presence or absence of a DNA-dependent protein kinase (DNA-PK) inhibitor, as described in Example 16. [Figure 21A-21B] Figure 21A shows an exemplary embodiment of the compositions described herein. Figure 21B shows the results of an exemplary assay performed in various composition configurations and in the presence of co-expressed DNA ligase, as described in Example 17. [Figure 22] 1 summarizes the results of assays performed with different configurations of the compositions described herein, as described in Example 18. [Figure 23A-23B] Figure 23A shows an exemplary embodiment of the composition described herein. Figure 23B shows the results of an exemplary assay performed with the composition and different ligase or DNA polymerase recruitment proteins, as described in Example 19. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0042] The present disclosure relates to improved CRISPR systems and their components, as well as methods of using them. In general, CRISPR systems, e.g., CRISPR / Cas systems, contain elements that facilitate 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 in the CRISPR system. The tracrRNA can form a secondary structure, such as a stem-loop, and bind to the Cas9 protein. The crRNA / tracrRNA hybrid associates with Cas9, and the crRNA / tracrRNA / Cas9 complex recognizes and cleaves foreign DNA with a protospacer sequence, thereby conferring immunity against invading viruses or plasmids. CRISPR / Cas systems are further described in, e.g., 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).

[0043] 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.

[0044] 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.

[0045] 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, "a" or "an" when used with the word "comprising" may mean one or more. As used herein, "another" or "further" may mean at least a second or more.

[0046] Throughout this specification, the term "about" is used to indicate that a value includes the variation of error inherent in the method / device utilized to determine the value or the variation that exists between test subjects. Typically, the term "about" is meant to encompass approximately 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.

[0047] 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."

[0048] As used herein, the terms "comprising" (and any variation or form of comprising, e.g., "comprise" and "comprises"), "having" (and any variation or form of having, e.g., "have" and "has"), "including" (and any variation or form of including, e.g., "includes" and "include"), or "containing" (and any variation 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 embodiment described herein can be implemented with respect to any protein, composition, polynucleotide, vector, cell, method, and / or kit of the disclosure. Additionally, the compositions, polynucleotides, vectors, cells, and / or kits of the disclosure can be used to obtain the methods and proteins of the disclosure.

[0049] Use of the term "for example" and its corresponding abbreviation "eg" (whether italicized or not) means that the particular terms recited are representative examples and embodiments of the present disclosure that are not intended to be limited to the specific examples referenced or recited, unless expressly stated otherwise.

[0050] As used herein, "to" refers to a range that includes both ends of the range, for example, a number from x to y explicitly includes the numbers x and y, and any numbers that fall within the range between x and y.

[0051] "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.

[0052] "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.

[0053] A nucleic acid molecule is "hybridizable" or "hybridized" to another nucleic acid molecule, such as cDNA, genomic DNA, or RNA, if the nucleic acid molecule in single-stranded form can anneal to another 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. In general, stringent hybridization conditions are those that meet the thermal melting point T of a particular polynucleotide at a defined ionic strength, concentration of chemical denaturant, pH, and concentration of hybridization partner. m(i.e., the temperature at which 50% of the sequence hybridizes to a substantially complementary sequence). In general, nucleotide sequences containing a higher percentage of G and C bases will hybridize under more stringent conditions than nucleotide sequences 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 the combination of parameters may be more important than the absolute value of any parameter alone.

[0054] 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 nucleoside, 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 nucleoside is hydrogen bonded without any nucleotide mismatches.

[0055] 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 in a chromosome. In some cases, the vector targets a specific chromosomal site for homologous recombination. For specific homologous recombination, the vector typically contains a sufficiently long region that is homologous to a sequence of the chromosome so that the vector can complementarily bind and integrate into the chromosome. A longer complementary region and a greater degree of sequence similarity can increase the efficiency of homologous recombination. In some embodiments, the polynucleotides or compositions described herein promote homologous recombination by generating a break, e.g., a double-strand break, in the nucleic acid sequence.

[0056] 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.

[0057] 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 involved in 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 involved in binding RNA polymerase. Eukaryotic promoters usually contain "TATA" boxes and "CAT" boxes. A variety of promoters, including inducible promoters, may be used to drive expression of the various vectors of the present disclosure.

[0058] A "vector" is any means for cloning and / or introducing a nucleic acid into a host cell. A vector may be a replicon to which another DNA segment may be attached such that replication of the attached segment occurs. 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., capable of replicating 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 a nucleic acid into a cell in vitro, ex vivo, or in vivo. Numerous vectors known in the art can be used to manipulate the nucleic acid, to incorporate response elements and promoters into genes, etc. A vector may 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.).

[0059] 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) to 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.

[0060] 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, adenoviral vectors, adeno-associated virus vectors, pox vectors, baculovirus vectors, vaccinia vectors, herpes simplex vectors, Epstein-Barr vectors, adenovirus vectors, geminivirus vectors, and caulimovirus 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.

[0061] 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).

[0062] 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.

[0063] 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' untranslated sequences into cells. In some embodiments, plasmids are utilized to provide the polynucleotides described herein. In some embodiments, plasmids are utilized to provide polynucleotides encoding the proteins described herein.

[0064] 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 contains an exogenous nucleic acid molecule inside the cell, and a "transformed" cell is one in which the exogenous nucleic acid molecule inside the cell induces a change in the phenotype of 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 contains any of the expression vectors described herein, e.g., an expression vector that includes a polynucleotide encoding a protein described herein.

[0065] 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."

[0066] The terms "peptide," "polypeptide," and "protein" are used interchangeably herein to refer to polymeric forms of amino acids of any length, and can include coded and non-coded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides having modified peptide backbones.

[0067] The beginning of a protein or polypeptide is known as the "N-terminus" (and 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" (and 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.

[0068] As used herein, "amino acid" refers to a compound that contains both a carboxyl (-COOH) group 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 differ 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 typically incorporated into proteins or polypeptides, such as, for example, citrulline (Cit), selenocysteine ​​(Sec), and pyrrolysine (Pyl).

[0069] "Amino acid substitution" refers to a polypeptide or protein that contains one or more substitutions of a wild-type or naturally occurring amino acid at that amino acid residue with an amino acid that is different to the wild-type or naturally occurring amino acid. The substituted amino acid may be a synthetic amino acid or a naturally occurring amino acid. In some embodiments, the substituted amino acid is a naturally occurring 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-naturally occurring or synthetic amino acid. Substitution variants may be described using a shorthand system. For example, a substitution variant in which the fifth amino acid residue has been substituted may be abbreviated as "X5Y," where "X" is the wild-type or naturally occurring amino acid that is replaced, "5" is the amino acid residue position within the amino acid sequence of the protein or polypeptide, and "Y" is the substituted amino acid or a non-wild-type or non-naturally occurring amino acid.

[0070] 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 combined with an excipient, such as a diluent or auxiliary, and still be considered isolated. As used herein, "isolated" does not necessarily imply any particular level of purity of the polypeptide, protein, peptide, or nucleic acid.

[0071] 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 occur 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.

[0072] 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 the host's chromosome as non-chromosomal genetic material, e.g., a plasmid. An "exogenous" protein can be introduced into a host cell via 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 or a homologous encoding nucleic acid, or both.

[0073] The term "domain" when used in reference to a polypeptide or protein refers to a distinct functional and / or structural unit of a protein. A domain may be involved in 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.

[0074] The term "motif" when used in reference to a polypeptide or protein generally refers to a set of 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, such as binding or targeting of proteins to specific subcellular locations, in various proteins. 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, including, for example, PROSITE, PFAM, PRINTS, and MiniMotif Miner.

[0075] As used herein, a "modified" protein refers to a protein that contains one or more modifications to the protein to obtain a desired property. Exemplary modifications include, but are not limited to, insertions, deletions, substitutions, and / or fusion with another domain or protein. A "fusion protein" (also referred to as a "chimeric protein") is a protein that typically contains at least two domains encoded by two separate genes that are linked to be transcribed and translated as a single unit, thereby generating 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.

[0076] 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 the wild-type Cas nuclease.

[0077] As used herein, the term "sequence similarity" or "% similarity" refers to the degree of identity or match between nucleic acid or amino acid sequences. "Sequence similarity" in the context of polynucleotides 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 polynucleotides that do not substantially affect the functional properties of the resulting transcript, such as the 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.

[0078] Additionally, 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.

[0079] "Sequence similarity" in the context of polypeptides 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 according to 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.

[0080] 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.

[0081] 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).

[0082] 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 amended by 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.

[0083] 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.

[0084] As used herein, a "complex" refers to a group of two or more polynucleotides and / or polypeptides that are associated. The term "associated" or "association" in the context of complex formation refers to molecules that are bound to each other by electrostatic interactions, hydrophobic / hydrophilic interactions, and / or hydrogen bonding interactions without covalent bonding. Molecules that include different moieties that are covalently bonded to each other 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 by secondary structure recognition of the polynucleotide by the protein. In some embodiments, the Cas binding region of a polynucleotide provided herein is composed of a secondary structure that is recognized by a Cas nuclease, Cas nickase, or fusion protein provided herein.

[0085] composition In some embodiments, the disclosure provides a composition comprising a Cas nuclease or Cas nickase and a polynucleotide comprising (i) a guide sequence, a Cas binding region, and (iii) a DNA template sequence, wherein the DNA template sequence is at the 3' end of the polynucleotide.

[0086] In some embodiments, the disclosure provides a composition comprising a Cas nuclease or Cas nickase; a first polynucleotide comprising (i) a guide sequence, (ii) a Cas binding region, and (iii) a first hybridization region, where the first hybridization region is at a 3' end of the first polynucleotide; and a second polynucleotide comprising (i) a second hybridization region complementary to the first hybridization region, and (ii) a DNA template sequence.

[0087] In some embodiments, the disclosure provides a composition comprising a Cas nuclease or Cas nickase; a first polynucleotide comprising (i) a guide sequence, and (ii) a first hybridization region, where the first hybridization region is at a 3' end of the first polynucleotide; and a second polynucleotide comprising (i) a second hybridization region complementary to the first hybridization region, (ii) a Cas binding region, and (iii) a DNA template sequence.

[0088] In some embodiments, the disclosure provides a composition comprising a Cas nuclease or Cas nickase; a first polynucleotide comprising (i) a guide sequence, (ii) a Cas binding region, (iii) a first hybridization region, and (iv) a primer binding sequence, wherein the primer binding sequence is at a 3' end of the first polynucleotide; and a second polynucleotide comprising (i) a second hybridization region complementary to the first hybridization region, and (ii) a sequence of interest (SOI).

[0089] In some embodiments, the disclosure provides a composition comprising a Cas nuclease or Cas nickase; a first polynucleotide comprising (i) a guide sequence, (ii) a Cas binding region, and (iii) a first hybridization region, where the first hybridization region is at a 3' end of the first polynucleotide; a second polynucleotide comprising (i) a second hybridization region complementary to the first hybridization region, (ii) a third hybridization region, and (iii) a primer binding sequence, where the primer binding sequence is at a 3' end of the second polynucleotide; and a third polynucleotide comprising (i) a fourth hybridization region complementary to the third hybridization region, and (ii) a sequence of interest (SOI).

[0090] As used herein, "Cas protein" encompasses both Cas nucleases and Cas nickases. Cas proteins are part of the CRISPR / Cas system described herein. CRISPR / Cas systems, including Cas proteins and polynucleotides (also referred to as "guide polynucleotides"), can be utilized for site-specific genome modification. In some embodiments, the CRISPR / Cas system includes a Cas protein and a guide polynucleotide including a Cas binding region (which binds to and / or activates the Cas protein) and a guide sequence (which hybridizes to a target sequence), where the Cas protein and the guide polynucleotide form a complex as described herein. In some embodiments, the CRISPR / Cas system includes a Cas protein and a first polynucleotide including a guide sequence and a second polynucleotide including a Cas binding region, where the first and second polynucleotides hybridize to each other and form a complex with the Cas protein.

[0091] CRISPR / Cas systems can be classified into types I-VI based on the Cas 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.

[0092] In some embodiments, the composition comprises a Cas nuclease. Generally, the Cas nuclease can produce double-stranded polynucleotide cleavage, e.g., double-stranded DNA cleavage. Generally, the Cas nuclease can comprise one or more nuclease domains, such as RuvC and HNH, and can cleave double-stranded DNA. In some embodiments, the Cas nuclease comprises 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 sticky 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 cleavage of a double-stranded nucleic acid (e.g., DNA). A cohesive or staggered end has a protruding single strand, or "overhang," of unpaired nucleotides, e.g., a 3' or 5' overhang.

[0093] 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. Further exemplary Cas9 nucleases are described, for example, in U.S. Patent Nos. 8,771,945, 9,023,649, 10,000,772, and 10,407,697. In some embodiments, the Cas9 nuclease is derived from S. pyogenes (SpCas9).

[0094] In some embodiments, the Cas nuclease is a Cas12 nuclease. In some embodiments, the Cas nuclease is a Cas12a nuclease (previously known as "Cpf1" or "C2c1"). 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).

[0095] In some embodiments, the Cas nuclease is a type II-B Cas nuclease. Type II-B Cas nucleases can generate 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, the sequenced gut metagenome MH0245_GL0161830.1, and many other bacteria. Additional type II-B Cas9 proteins are described, for example, in WO 2019 / 099943. In some embodiments, the type II-B Cas nuclease is from the sequenced gut metagenome, MH0245_GL0161830.1 (MHCas9).

[0096] In some embodiments, the composition comprises 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 comprises amino acid mutations in the catalytic domain compared to a Cas nuclease. Cas nickases are further described, for example, in 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).

[0097] 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 one of skill in the art that the alignment methods 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) and generate Cas nickases.

[0098] 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.

[0099] In some embodiments, the Cas nuclease or Cas nickase of the composition is fused to a heterologous protein domain. In some embodiments, the Cas nuclease or Cas nickase is fused to a DNA polymerase, a DNA polymerase recruitment moiety, a DNA ligase, a DNA ligase recruitment moiety, a DNA binding protein, a DNA repair protein, or a combination thereof. Fusion proteins comprising a Cas nuclease or Cas nickase are further described herein.

[0100] Polynucleotides In some embodiments, the compositions of the present disclosure comprise a polynucleotide. In some embodiments, the polynucleotide comprises (i) a guide sequence, (ii) a Cas binding region, and (iii) a DNA template sequence, wherein the DNA template sequence is at the 3' end of the polynucleotide. In some embodiments, the guide sequence is an RNA guide sequence. In some embodiments, the polynucleotide comprises, in 5' to 3' order, a guide sequence (e.g., an RNA guide sequence), a Cas binding region, and a DNA template sequence.

[0101] In some embodiments, the compositions of the present disclosure comprise a first and a second polynucleotide. In some embodiments, the first polynucleotide comprises (i) a guide sequence, (ii) a Cas binding region, and (iii) a first hybridization region, the first hybridization region being at the 3' end of the first polynucleotide, and the second polynucleotide comprises (i) a second hybridization region complementary to the first hybridization region, and (ii) a DNA template sequence. In some embodiments, the first polynucleotide comprises, in 5' to 3' order, a guide sequence, a Cas binding region, and a first hybridization region. In some embodiments, the second polynucleotide comprises, in 5' to 3' order, a second hybridization region and a DNA template sequence.

[0102] In some embodiments, the first polynucleotide comprises (i) a guide sequence, and (ii) a first hybridization region, the first hybridization region being at the 3' end of the first polynucleotide, and the second polynucleotide comprises (i) a second hybridization region complementary to the first hybridization region, (ii) a Cas binding region, and (iii) a DNA template sequence. In some embodiments, the first polynucleotide comprises, in 5' to 3' order, a guide sequence and a first hybridization region. In some embodiments, the second polynucleotide comprises, in 5' to 3' order, a second hybridization region, a Cas binding region, and a DNA template sequence.

[0103] In some embodiments, the compositions of the present disclosure comprise a first and a second polynucleotide. In some embodiments, the first polynucleotide comprises (i) a guide sequence, (ii) a Cas binding region, (iii) a first hybridization region, and (iv) a primer binding sequence, where the primer binding sequence is at the 3' end of the first polynucleotide, and the second polynucleotide comprises (i) a second hybridization region complementary to the first hybridization region, and (ii) a sequence of interest (SOI). In some embodiments, the first polynucleotide comprises, in 5' to 3' order, a guide sequence, a Cas binding region, a first hybridization region, and a primer binding sequence. In some embodiments, the second polynucleotide comprises, in 5' to 3' order, a second hybridization region and a SOI. In some embodiments, the first polynucleotide and / or the second polynucleotide comprises RNA, DNA, modified nucleotides, or a combination thereof. Modified nucleotides are described herein. A non-limiting exemplary diagram of a composition comprising a first and second polynucleotide as described herein is shown in FIG. 11(B).

[0104] In some embodiments, the first polynucleotide and / or the second polynucleotide further comprises a homologous sequence. In some embodiments, the first polynucleotide comprises, in 5' to 3' order, a guide sequence, a Cas binding region, a first hybridization region, and a primer binding sequence. In some embodiments, the second polynucleotide comprises, in 5' to 3' order, a second hybridization region, a SOI, and a homologous sequence. In some embodiments, the homologous sequence can hybridize to a sequence adjacent to the cleaved target polynucleotide (e.g., generated by a Cas nuclease or Cas nickase). In some embodiments, the guide sequence hybridizes to a region on one side of the cleavage site and the homologous sequence hybridizes to a region on the other side of the cleavage site, for example, as illustrated in FIG. 9 or FIG. 11(A). In Figures 9 and 11(A), the first polynucleotide is composed of a guide sequence (referred to as the "spacer"), a Cas binding region (referred to as the "gRNA scaffold"), a first hybridization region (referred to as the "landing pad"), and a primer binding sequence (PBS), and the second polynucleotide is composed of a second hybridization region (referred to as the "hybridization sequence"), an SOI (referred to as the "insert"), and a homologous sequence.

[0105] In some embodiments, the first polynucleotide comprises, in 5' to 3' order, a guide sequence, a Cas binding region, a homologous sequence, a first hybridization region, and a primer binding sequence. In some embodiments, the second polynucleotide comprises, in 5' to 3' order, a second hybridization region, an SOI, and a further hybridization region, where the second hybridization region and the further hybridization region hybridize at non-overlapping portions of the first hybridization region. In some embodiments, the second hybridization region and the further hybridization region flank the SOI and hybridize to adjacent portions of the first hybridization region, as illustrated, for example, in FIG. 11(J). In some embodiments, the guide sequence hybridizes to a region on one side of the cleavage site, and the homologous sequence hybridizes to a region on the other side of the cleavage site and to opposing DNA strands of the cleavage site, as illustrated, for example, in FIG. 11(J).

[0106] In some embodiments, the compositions of the present disclosure comprise a first, second and third polynucleotide. In some embodiments, the first polynucleotide comprises (i) a guide sequence, (ii) a Cas binding region, and (iii) a first hybridization region, the first hybridization region being at the 3' end of the first polynucleotide, the second polynucleotide comprises (i) a second hybridization region complementary to the first hybridization region, (ii) a third hybridization region, and (iii) a primer binding sequence, the primer binding sequence being at the 3' end of the second polynucleotide, and the third polynucleotide comprises (i) a fourth hybridization region complementary to the third hybridization region, and (ii) a sequence of interest (SOI). In some embodiments, the first polynucleotide comprises, in order from 5' to 3', a guide sequence, a Cas binding region, and the first hybridization region. In some embodiments, the second polynucleotide comprises, in 5' to 3' order, a second hybridization region, a third hybridization region, and a primer binding sequence. In some embodiments, the third polynucleotide comprises, in 5' to 3' order, a fourth hybridization region and a SOI. In some embodiments, any of the first polynucleotide, the second polynucleotide, and / or the third polynucleotide comprises RNA, DNA, modified nucleotides, or combinations thereof. Modified nucleotides are described herein.

[0107] In some embodiments, any of the first polynucleotide, the second polynucleotide, and / or the third polynucleotide further comprises a homologous sequence as described herein, for example, as depicted in FIG. 11(E). In some embodiments, the first polynucleotide comprises, in 5' to 3' order, a guide sequence, a Cas binding region, and a first hybridization region. In some embodiments, the second polynucleotide comprises, in 5' to 3' order, a second hybridization region, a third hybridization region, and a primer binding sequence. In some embodiments, the third polynucleotide comprises, in 5' to 3' order, a fourth hybridization region, a SOI, and a homologous sequence.

[0108] In some embodiments, the compositions herein, for example comprising a first, second and / or third polynucleotide, further comprise an SOI-complementary oligonucleotide, which is composed of a sequence complementary to the SOI. In some embodiments, the SOI-complementary oligonucleotide is longer than the SOI at one or both of the 5' and 3' ends, as illustrated in FIG. 11(D), thereby generating a double-stranded sequence with an overhanging end. In some embodiments, the SOI-complementary oligonucleotide is the same length as the SOI, as illustrated in FIG. 11(H), thereby generating a double-stranded sequence with a blunt end. In some embodiments, the SOI-complementary oligonucleotide hybridizes with the SOI to form a double-stranded SOI. In some embodiments, the double-stranded SOI has a more accurate insertion efficiency compared to the single-stranded SOI. In some embodiments, the SOI-complementary oligonucleotide further comprises a homologous sequence, as illustrated in FIG. 11(I), for example.

[0109] In some embodiments, the second hybridization region hybridizes over the entire length of the first hybridization region. In some embodiments, the second hybridization region is shorter than the first hybridization region, for example, by about 1 to about 10 nucleotides, or about 2 to about 8 nucleotides, or about 3 to about 6 nucleotides, or about 4 to about 5 nucleotides. In some embodiments, the second hybridization region is shorter than the first hybridization region at the 5' end portion of the second hybridization region, thereby leaving a gap between the second hybridization region and the cleaved DNA, as illustrated, for example, in FIG. 11(F). In some embodiments, the second hybridization region is longer than the first hybridization region, for example, by about 1 to about 10 nucleotides, or about 2 to about 8 nucleotides, or about 3 to about 6 nucleotides, or about 4 to about 5 nucleotides. In some embodiments, the second hybridization region is longer than the first hybridization region at the 5' end portion of the second hybridization region, thereby providing a flap between the first and second hybridization regions after hybridization, as illustrated, for example, in Figure 11(G), In some embodiments, the flap recruits and / or binds repair machinery, such as FEN1, thereby facilitating precise insertion of the sequence of interest.

[0110] In some embodiments, the disclosure provides a polynucleotide comprising (i) an RNA guide sequence, (ii) a Cas binding region, and (iii) a DNA template sequence, wherein the DNA template sequence is at the 3' end of the polynucleotide. In some embodiments, the disclosure provides a polynucleotide comprising (i) an RNA guide sequence, (ii) a Cas binding region, and (iii) a DNA template sequence, wherein the DNA template sequence is at the 3' end of the polynucleotide, and wherein the DNA template sequence comprises phosphorothioate linkages. In some embodiments, the polynucleotide comprises, in 5' to 3' order, a guide sequence (e.g., an RNA guide sequence), a Cas binding region, and a DNA template sequence.

[0111] 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).

[0112] 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.

[0113] In some embodiments, the Cas binding region is capable of binding to a Cas protein (e.g., a Cas nuclease or a Cas nickase) in the composition, 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).

[0114] 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, activating comprises initiating or increasing the cleavage activity of a Cas nuclease or Cas nickase. In some embodiments, activating comprises promoting binding of a Cas nuclease or Cas nickase to a target polynucleotide (e.g., by guiding with a guide sequence). In some embodiments, activating comprises 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).

[0115] In some embodiments, the polynucleotide of the present disclosure 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 sequence of interest to form a double-stranded sequence comprising the sequence of interest. In some embodiments, the double-stranded sequence comprising the sequence of interest is inserted into the cleaved target polynucleotide, for example, by ligation or a DNA repair pathway as described herein. An exemplary, non-limiting overview of Cas-mediated targeted insertion of a sequence of interest is illustrated in FIG. 1. In FIG. 1, Cas nuclease is guided to a target DNA in a cell by a guide sequence and cleaves the target DNA. The DNA template sequence includes (i) a primer binding sequence that serves as a primer and hybridizes with the cleaved DNA, and (ii) a sequence of interest. A DNA polymerase, e.g., an endogenous DNA polymerase of the cell, binds to the primer and synthesizes a DNA strand complementary to the sequence of interest, thereby forming a double-stranded sequence containing the sequence of interest. This double-stranded sequence can be inserted into the cleaved target DNA by a DNA repair pathway, e.g., NHEJ.

[0116] In some embodiments, the components of the DNA template sequence described herein, e.g., the sequence of interest and the primer binding sequence, are located on two separate polynucleotides, e.g., the first and second polynucleotides as described herein. An exemplary, non-limiting embodiment in which the sequence of interest and the primer binding sequence are located on two separate polynucleotides is illustrated in Figures 10A-10C. In Figure 10A, the guide sequence, the Cas binding region, the first hybridization region, and the primer binding site are located on the first polynucleotide. The Cas nuclease guided to the target DNA by the guide sequence creates a double-stranded break in the target DNA. The primer binding site hybridizes to the cleaved DNA, and the second polynucleotide hybridizes to the first polynucleotide through the first and second hybridization regions that contain complementary sequences. The second polynucleotide contains the sequence of interest, which is ligated by a ligase to the cleaved DNA at both the 5' and 3' ends (Figure 10A). The second polynucleotide may further comprise a homologous sequence, the 5' end of which is ligated to the cleaved DNA by a ligase, and the 3' end of which is incorporated by a homology-mediated mechanism (Figure 10B). In some embodiments, the homology-mediated mechanism includes HDR, synthesis-dependent single-strand annealing (SDSA), single-strand annealing (SSA), alternative end joining (alt-EJ), or a combination thereof. The complex is converted to double-stranded DNA after ligation and / or homology-mediated incorporation, thereby incorporating the desired sequence into the target DNA (Figure 10C).

[0117] In some embodiments, the sequence of interest comprises 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 sequence 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 sequence of interest encodes a copy of a native gene of the host cell. In some embodiments, the sequence of interest encodes a copy of a native gene that is defective in the host cell. In some embodiments, the host cell comprises a mutation in a gene and the sequence of interest encodes a wild-type copy of that gene. In some embodiments, the host cell comprises a wild-type gene and the sequence of interest encodes a copy of the gene that comprises a mutation of interest. In some embodiments, the sequence of interest encodes a heterologous gene that does not naturally occur in the host cell.

[0118] 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 sequence of interest 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.

[0119] 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.

[0120] 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 has a length sufficient to hybridize with a region of the cleaved target DNA sequence.

[0121] In some embodiments, the sequence of interest is about 3 to about 500 nucleotides in length. In some embodiments, the sequence of interest is about 4 to about 100 nucleotides in length. In some embodiments, the sequence of interest is about 5 to about 90 nucleotides in length. In some embodiments, the sequence of interest is about 6 to about 80 nucleotides in length. In some embodiments, the sequence of interest is about 7 to about 70 nucleotides in length. In some embodiments, the sequence of interest is about 8 to about 60 nucleotides in length. In some embodiments, the sequence of interest is about 9 to about 50 nucleotides in length. In some embodiments, the sequence of interest is about 10 to about 40 nucleotides in length. In some embodiments, the sequence of interest is about 11 to about 30 nucleotides in length. In some embodiments, the sequence of interest is about 12 to about 20 nucleotides in length. In some embodiments, the sequence of interest 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 sequence of interest 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.

[0122] In some embodiments, the DNA template sequence comprises modified nucleotides, non-B-type DNA structures, DNA polymerase recruiting moieties, DNA ligase recruiting moieties, or combinations thereof.

[0123] In some embodiments, the DNA template sequence comprises modified nucleotides, hi some embodiments, the modified nucleotides comprise an abasic site, a covalent linker, a heterologous nucleic 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.

[0124] 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).

[0125] 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 is composed of 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 contains a sulfur atom instead 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.

[0126] 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 strand of the cleaved target polynucleotide and extends the sequence of interest on the DNA template sequence, as described herein.

[0127] 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.

[0128] In some embodiments, the modified nucleotide comprises a DNA photoproduct. The DNA photoproduct is an ultraviolet (UV)-induced DNA lesion, 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.

[0129] In some embodiments, the modified nucleotide comprises a modified deoxyribonucleoside. In some embodiments, the modified deoxyribonucleoside is capable of recruiting a 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.

[0130] In some embodiments, the modified nucleotide comprises a methylated nucleotide. In some embodiments, the methylated nucleotide, e.g., a methylated cytosine, can recruit a DNA polymerase. In some embodiments, the methylated nucleotide comprises 5-hydroxymethylcytosine, 5-methylcytosine, or a combination thereof.

[0131] 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-quadruplex, triplex DNA (H-DNA), Z-DNA, cruciform, slipped DNA strand, 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, Z-DNA, H-DNA (triple helix DNA), G-quadruplex DNA (quadruplex DNA), slipped DNA, sticky DNA, or a combination thereof.

[0132] 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 mutant or homolog thereof.

[0133] In some embodiments, the DNA polymerase recruitment moiety comprises modified nucleotides, such as DNA lesions, DNA photoproducts, modified deoxyribonucleosides, and / or methylated nucleotides described herein. In some embodiments, the modified nucleotides recruit a translesion DNA synthesis (TLS) polymerase. The TLS polymerase can extend DNA comprising modified nucleotides described herein. Exemplary TLS polymerases include, but are not limited to, Pol II, Pol IV, and Pol V from E. coli; Rev1p, Rev3p, and Pol η from S. cerevisiae; and human REV1, REV3, Pol η, Pol ι, and Pol κ. Additional TLS polymerases are described, for example, in Goodman et al., Cold Spring Harb Perspect Biol 5(10);a010363(2013) and Waters et al., Microbiol Mol Biol Rev 73(1):134-154(2009). In some embodiments, the DNA polymerase recruitment portion comprises a non-B DNA structure described herein. In some embodiments, a DNA polymerase recruited by the non-B DNA structure can extend DNA through the non-B DNA structure. In some embodiments, the non-B DNA structure recruits a DNA polymerase selected from PrimPol, REV1, REV3, Pol δ, Pol η, Pol ι, Pol κ, and Pol θ.

[0134] In some embodiments, the DNA polymerase recruitment portion is comprised of a DNA polymerase recruitment protein. In some embodiments, the DNA polymerase recruitment protein is capable of recognizing and binding to a DNA polymerase. In some embodiments, the DNA polymerase recruitment protein is linked to a DNA template sequence. In some embodiments, the DNA polymerase recruitment protein is crosslinked to a primer binding sequence. Methods for linking proteins to polynucleotides are known to those skilled in the art, and include, for example, covalent methods such as copper-catalyzed cycloaddition, strain-promoted azide-alkyne cycloaddition, and inverse electron demand Diels-Alder reaction (e.g., reaction between cyclopropene and tetrazine); or affinity-based methods, such as linking a polynucleotide containing a biotin moiety to a protein containing an avidin or streptavidin moiety.

[0135] In some embodiments, the DNA polymerase recruitment protein comprises proliferating cell nuclear antigen (PCNA), single stranded DNA binding protein (SSBP), tumor necrosis factor alpha-induced protein (TNFAIP), polymerase delta interacting protein (PolDIP), X-ray repair cross-complementing protein (XRCC), 5-hydroxymethylcytosine-binding embryonic stem cell-specific (HMCES) protein, RAD1, RAD9, HUS1, or a combination thereof.

[0136] In some embodiments, the DNA template sequence comprises a DNA ligase recruitment moiety. In some embodiments, the presence of a DNA ligase recruitment moiety in a polynucleotide (e.g., a polynucleotide of a composition provided herein) can recruit a DNA ligase to the polynucleotide. In some embodiments, recruiting a DNA ligase includes, for example, increasing the likelihood that a DNA ligase will recognize a polynucleotide due to the presence of a DNA ligase recruitment moiety in the polynucleotide; promoting the binding of a DNA ligase to a polynucleotide; and / or activating a DNA ligase, for example, initiating or increasing the activity of a DNA ligase. In some embodiments, the recruited DNA ligase binds to a double-stranded sequence containing a sequence of interest generated by a DNA polymerase as described herein and ligates the double-stranded sequence to the cleaved target polynucleotide.

[0137] In some embodiments, the DNA ligase recruitment portion comprises 5' adenylation of the DNA template sequence. In some embodiments, the DNA ligase recruitment portion comprises a DNA ligase recruitment protein. Exemplary DNA ligase recruitment proteins include, but are not limited to, DNA-dependent protein kinase (DNA-PK), proliferating cell nuclear antigen (PCNA), or X-ray repair cross-complementing protein 1 (XRCC1). Non-limiting examples of DNA ligases that can be recruited by the DNA ligase portion include bacterial DNA ligases such as E. coli DNA ligase, T4 DNA ligase, T7 DNA ligase, mammalian DNA ligases such as DNA ligase I, DNA ligase II, DNA ligase III, or DNA ligase IV, thermostable DNA ligases such as Taq DNA ligase, or mutants or homologs thereof.

[0138] In some embodiments, the guide sequence, Cas binding region, and DNA template sequence described herein are present on a single polynucleotide in the composition. In some embodiments, the DNA template sequence is located 3' of the guide sequence and Cas binding region. In some embodiments, the DNA template sequence is at the 3' end of the polynucleotide. In some embodiments, the DNA template sequence located at the 3' end of the polynucleotide facilitates binding and / or extension of the cleaved target polynucleotide by a DNA polymerase as described herein to form a double-stranded sequence that includes a sequence of interest. In some embodiments, the DNA template sequence located at the 3' end of the polynucleotide facilitates ligation of the double-stranded sequence to the cleaved target polynucleotide by a DNA ligase as described herein.

[0139] In some embodiments, the guide sequence, Cas binding region, and DNA template sequence described herein are present on two or more polynucleotides in the composition. In some embodiments, the guide sequence and Cas binding region are on a first polynucleotide and the DNA template sequence is on a second polynucleotide. In some embodiments, the guide sequence is on a first polynucleotide and the Cas binding region and the DNA template sequence are on a second polynucleotide. In some embodiments, the first polynucleotide comprises a first hybridization region. In some embodiments, the second polynucleotide comprises a second hybridization region that is complementary to the first hybridization region. In some embodiments, the first hybridization region and the second hybridization region are hybridizable. In some embodiments, the first hybridization region and the second hybridization region are comprised of RNA. In some embodiments, the first hybridization region and the second hybridization region are comprised of single-stranded DNA. In some embodiments, the first hybridization region is comprised of RNA and the second hybridization region is comprised of single-stranded DNA. In some embodiments, the first hybridization region is comprised of single-stranded DNA and the second hybridization region is comprised of RNA. In some embodiments, the RNA and the single-stranded DNA are hybridizable.

[0140] In some embodiments, the first hybridization region is at the 3' end of the first polynucleotide. In some embodiments, the second hybridization region is at the 5' end of the second polynucleotide. In some embodiments, when the first hybridization region hybridizes to the second hybridization region, the DNA template sequence is located 3' of both the guide sequence and the Cas binding region. In some embodiments, the DNA template sequence is at the 3' end of the second polynucleotide. In some embodiments, the DNA template sequence located at the 3' end of the second polynucleotide facilitates binding and / or extension of the cleaved target polynucleotide by a DNA polymerase to form a double-stranded sequence comprising a sequence of interest, as described herein. In some embodiments, the DNA template sequence located at the 3' end of the second polynucleotide facilitates ligation of the double-stranded sequence to the cleaved target polynucleotide by a DNA ligase, as described herein.

[0141] In some embodiments, the guide sequence, Cas binding region, sequence of interest (SOI), and primer binding sequence are present on two or more polynucleotides, as described herein. In some embodiments, the guide sequence, Cas binding region, and primer binding sequence are on a first polynucleotide, and the SOI is on a second polynucleotide, and the first and second polynucleotides each comprise a first and second hybridization region capable of hybridizing to one another. In some embodiments, the primer binding sequence is located at the 3' end of the first polynucleotide. In some embodiments, the primer binding sequence located at the 3' end of the first polynucleotide facilitates binding and / or ligation of the SOI to the cleaved target polynucleotide by a DNA ligase, as described herein. In some embodiments, the primer binding sequence located at the 3' end of the first polynucleotide facilitates binding and / or extension of the cleaved target polynucleotide by a DNA polymerase, as described herein, to form a double-stranded sequence comprising the SOI.

[0142] In some embodiments, the first hybridization region is about 3 to about 10,000 nucleotides in length. In some embodiments, the first hybridization region is about 4 to about 5,000 nucleotides in length. In some embodiments, the first hybridization region is about 5 to about 1,000 nucleotides in length. In some embodiments, the first hybridization region is about 10 to about 800 nucleotides in length. In some embodiments, the first hybridization region is about 20 to about 600 nucleotides in length. In some embodiments, the first hybridization region is about 30 to about 500 nucleotides in length. In some embodiments, the first hybridization region is about 40 to about 400 nucleotides in length. In some embodiments, the first hybridization region is about 50 to about 300 nucleotides in length. In some embodiments, the first hybridization region is about 100 to about 200 nucleotides in length. In some embodiments, the first hybridization region 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, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2500, 3000, 3500, 4000, 4500, or 5000 nucleotides in length.

[0143] In some embodiments, the second hybridization region is about 3 to about 10,000 nucleotides in length. In some embodiments, the second hybridization region is about 4 to about 5,000 nucleotides in length. In some embodiments, the second hybridization region is about 5 to about 1,000 nucleotides in length. In some embodiments, the second hybridization region is about 10 to about 800 nucleotides in length. In some embodiments, the second hybridization region is about 20 to about 600 nucleotides in length. In some embodiments, the second hybridization region is about 30 to about 500 nucleotides in length. In some embodiments, the second hybridization region is about 40 to about 400 nucleotides in length. In some embodiments, the second hybridization region is about 50 to about 300 nucleotides in length. In some embodiments, the second hybridization region is about 100 to about 200 nucleotides in length. In some embodiments, the second hybridization region 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, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2500, 3000, 3500, 4000, 4500, or 5000 nucleotides in length.

[0144] In some embodiments, the first and second hybridization regions are substantially the same length. In some embodiments, the first and second hybridization regions differ in length by less than about 1 nucleotide, less than about 2 nucleotides, less than about 3 nucleotides, less than about 4 nucleotides, less than about 5 nucleotides, less than about 6 nucleotides, less than about 7 nucleotides, less than about 8 nucleotides, less than about 9 nucleotides, less than about 10 nucleotides, less than about 15 nucleotides, less than about 20 nucleotides, less than about 25 nucleotides, less than about 30 nucleotides, less than about 35 nucleotides, less than about 40 nucleotides, less than about 45 nucleotides, less than about 50 nucleotides, less than about 55 nucleotides, less than about 60 nucleotides, less than about 65 nucleotides, less than about 70 nucleotides, less than about 75 nucleotides, less than about 80 nucleotides, less than about 85 nucleotides, less than about 90 nucleotides, less than about 95 nucleotides, or less than about 100 nucleotides. In some embodiments, the first and second hybridization regions differ in length by less than about 30%, less than about 25%, less than about 20%, less than about 15%, less than about 10%, less than about 5%, less than about 2%, or less than about 1% of the number of nucleotides in the first hybridization region or the second hybridization region. In some embodiments, the first and second hybridization regions are substantially identical in length. In some embodiments, the first and second hybridization regions are completely complementary. In some embodiments, the first and second hybridization regions are at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% complementary.In some embodiments, the first and second hybridization regions are capable of hybridizing under standard nucleic acid hybridization conditions, e.g., as described in Herzer and Englert, "Chapter 14. Nucleic Acid Hybridization," in Molecular Biology Problem Solver; A Laboratory Guide, (2001) ed. Alan S. Gersterin; Wiley-Liss, Inc.

[0145] In some embodiments, the guide sequence is on a first polynucleotide, the Cas binding region is on a second polynucleotide, and the DNA template sequence is on a third polynucleotide. In some embodiments, the first polynucleotide comprises a first hybridization region at its 3' end, the second polynucleotide comprises (i) a second hybridization region at its 5' end that is complementary to the first hybridization region, and (ii) a third hybridization region at its 3' end that is complementary to the fourth hybridization region, and the third polynucleotide comprises a fourth hybridization region at its 5' end. In some embodiments, the second polynucleotide comprises a first hybridization region at its 3' end, the first polynucleotide comprises (i) a second hybridization region at its 5' end that is complementary to the first hybridization region, and (ii) a third hybridization region at its 3' end that is complementary to the fourth hybridization region, and the third polynucleotide comprises a fourth hybridization region at its 5' end. In some embodiments, when the first, second, third, and fourth hybridization regions hybridize, the DNA template sequence is located 3' of both the guide sequence and the Cas binding region. In some embodiments, each of the first, second, third, and fourth hybridization regions is about 3 to about 10,000 nucleotides in length, or about 4 to about 5,000 nucleotides in length, or about 5 to about 1,000 nucleotides in length, or about 10 to about 800 nucleotides in length, or about 20 to about 600 nucleotides in length, or about 30 to about 500 nucleotides in length, or about 40 to about 400 nucleotides in length, or about 50 to about 300 nucleotides in length, or about 100 to about 200 nucleotides in length. In some embodiments, the first and second hybridization regions are substantially the same length, and the third and fourth hybridization regions are substantially the same length. In some embodiments, the first and second hybridization regions are at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% complementary.In some embodiments, the third and fourth hybridization regions are at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% complementary.

[0146] In some embodiments, the guide sequence, Cas binding region, SOI, and primer binding sequence are present on a first, second, and third polynucleotide, as described herein. In some embodiments, the guide sequence and Cas binding region are present on a first polynucleotide, the primer binding sequence is present on a second polynucleotide, and the SOI is present on a third polynucleotide, the first and second polynucleotides comprising first and second hybridization regions, respectively, capable of hybridizing to each other, and the second and third polynucleotides comprising third and fourth hybridization regions, respectively, capable of hybridizing to each other. In some embodiments, the primer binding sequence is located at the 3' end of the second polynucleotide. In some embodiments, the primer binding sequence located at the 3' end of the second polynucleotide facilitates binding and / or ligation of the SOI to the cleaved target polynucleotide by a DNA ligase, as described herein. In some embodiments, the primer binding sequence located at the 3' end of the first polynucleotide facilitates binding and / or extension of the cleaved target polynucleotide by a DNA polymerase to form a double-stranded sequence comprising an SOI, as described herein. In some embodiments, each of the first, second, third, and fourth hybridization regions is about 3 to about 10,000 nucleotides in length, or about 4 to about 5,000 nucleotides in length, or about 5 to about 1,000 nucleotides in length, or about 10 to about 800 nucleotides in length, or about 20 to about 600 nucleotides in length, or about 30 to about 500 nucleotides in length, or about 40 to about 400 nucleotides in length, or about 50 to about 300 nucleotides in length, or about 100 to about 200 nucleotides in length. In some embodiments, the first and second hybridization regions are substantially the same length, and the third and fourth hybridization regions are substantially the same length. In some embodiments, the first and second hybridization regions are at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% complementary.In some embodiments, the third and fourth hybridization regions are at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% complementary.

[0147] In some embodiments, the polynucleotide of the disclosure further comprises a spacer located 5' of the DNA template sequence. In some embodiments, the polynucleotide comprises a guide sequence, a Cas binding region, and a DNA template sequence, wherein the spacer is located between the Cas binding region and the DNA template sequence. In some embodiments, the second polynucleotide of the disclosure further comprises a spacer located 5' of the DNA template sequence. In some embodiments, the second polynucleotide comprises a second hybridization region, a Cas binding region, and a DNA template sequence, wherein the spacer is located between the Cas binding region and the DNA template sequence. In some embodiments, the second polynucleotide comprises a second hybridization region and a DNA template sequence, wherein the spacer is located between the second hybridization region and the DNA template sequence.

[0148] In some embodiments, the spacer comprises a stop sequence for DNA polymerase such that the DNA polymerase stops after synthesizing a complementary strand of the target sequence. In some embodiments, the spacer comprises two or more stop sequences. In some embodiments, the spacer comprises one, two, three, four, five, or more than five stop sequences. In some embodiments, the presence of multiple stop sequences provides redundancy in stopping the DNA polymerase. In some embodiments, the stop sequence inhibits activation of the DNA polymerase. In some embodiments, the stop sequence promotes dissociation of the DNA polymerase from the DNA template sequence.

[0149] In some embodiments, the termination sequence is comprised of a secondary structure. In some embodiments, the secondary structure is an inhibitor of DNA polymerase activity. In some embodiments, the secondary structure promotes dissociation of the DNA polymerase from the DNA template sequence. In some embodiments, the secondary structure is a hairpin loop (also known as a stem loop). In some embodiments, the secondary structure is a pseudoknot.

[0150] In some embodiments, the spacer is about 5 to about 500 nucleotides in length. In some embodiments, the spacer is about 10 to about 400 nucleotides in length. In some embodiments, the spacer is about 10 to about 300 nucleotides in length. In some embodiments, the spacer is about 10 to about 200 nucleotides in length. In some embodiments, the spacer is about 20 to about 150 nucleotides in length. In some embodiments, the spacer is about 30 to about 100 nucleotides in length. In some embodiments, the spacer is about 50 to about 100 nucleotides in length. In some embodiments, the spacer is about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 75, about 100, about 110, about 120, about 130, about 140, about 150, about 160, about 170, about 180, about 190, or about 200 nucleotides in length.

[0151] The disclosure herein contemplates that the elements and features of the polynucleotides as described herein can be combined in any combination. For exemplary purposes only, a polynucleotide can include features as shown in Table 1 (5' to 3'):

[0152] [Table 1]

[0153] [Table 2]

[0154] [Table 3]

[0155] [Table 4]

[0156] [Table 5]

[0157] [Table 6]

[0158] In some embodiments, any of the Cas nuclease binding regions binds to a Cas9 or Cas12a nuclease. In some embodiments, any of the Cas nickase binding regions binds to a Cas9 nickase or a Cas12a nickase. In some embodiments, the polynucleotide comprises a spacer between any of the guide sequences described above (e.g., an RNA guide sequence or an RNA / DNA guide sequence), the Cas binding region (e.g., a Cas nuclease binding region or a Cas nickase binding region), and the DNA template sequence.

[0159] The disclosure herein contemplates that the compositions described herein may comprise more than one polynucleotide, for example, two polynucleotides. For illustrative purposes only, a composition may comprise two polynucleotides that may comprise the characteristics as shown in Table or Table 3 (5' to 3'):

[0160] [Table 7]

[0161] [Table 8]

[0162]

Table 9

[0163]

Table 10

[0164]

Table 11

[0165]

Table 12

[0166]

Table 13

[0167]

Table 14

[0168]

Table 15

[0169]

Table 16

[0170]

Table 17

[0171] In some embodiments, the DNA template sequence of any of the second polynucleotides described above comprises a primer binding sequence and a sequence of interest. In some embodiments, the DNA template sequence of any of the second polynucleotides described above comprises a primer binding sequence, a sequence of interest, and one or more of modified nucleotides, a DNA polymerase recruitment moiety, and a DNA ligase recruitment moiety.

[0172] In some embodiments, any of the Cas nuclease binding regions binds to a Cas9 or Cas12a nuclease. In some embodiments, any of the Cas nickase binding regions binds to a Cas9 nickase or a Cas12a nickase. In some embodiments, the first and / or second polynucleotide comprises a spacer between any of the guide sequences described above (e.g., an RNA guide sequence or an RNA / DNA guide sequence), the Cas binding region (e.g., a Cas nuclease binding region or a Cas nickase binding region), and the DNA template sequence.

[0173] Fusion proteins In some embodiments, the compositions of the present disclosure include a Cas nuclease or Cas nickase, and the Cas nuclease or Cas nickase is fused to a DNA polymerase recruitment protein. In some embodiments, the present disclosure provides a fusion protein comprising (i) a Cas nuclease or Cas nickase, and (ii) a DNA polymerase recruitment protein. In some embodiments, the present disclosure provides a fusion protein comprising (i) a Cas nuclease or Cas nickase, and (ii) a DNA polymerase recruitment protein, a DNA ligase, a DNA ligase recruitment moiety, a DNA binding protein, a DNA repair protein, or a combination thereof.

[0174] As described herein, a fusion protein typically comprises at least two domains with distinct functions. In some embodiments, the fusion protein comprises a Cas nuclease. Cas nucleases are described herein. In some embodiments, the Cas nuclease is a Cas9 nuclease. In some embodiments, the Cas nuclease is a Cas12a nuclease. In some embodiments, the Cas nuclease is a type II-B Cas nuclease. In some embodiments, the fusion protein comprises a Cas nickase. Cas nickases are further described herein. 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.

[0175] In some embodiments, the fusion protein comprises a DNA polymerase recruitment protein. DNA polymerase recruitment proteins are further described herein. In some embodiments, the DNA polymerase recruitment protein comprises a proliferating cell nuclear antigen (PCNA), a single stranded DNA binding protein (SSBP), a tumor necrosis factor alpha-induced protein (TNFAIP), a polymerase delta interacting protein (PolDIP), an X-ray repair cross-complementing protein (XRCC), a 5-hydroxymethylcytosine-binding embryonic stem cell specific (HMCES) protein, RAD1, RAD9, HUS1, or a combination thereof.

[0176] In some embodiments, the DNA polymerase recruitment protein is capable of recruiting a DNA polymerase, the DNA polymerase comprising Pol I or the Klenow fragment thereof, Pol II, Pol III, Pol IV, Pol V, 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, REV3, Bst DNA polymerase, T4 DNA polymerase, Φ29 (phi29) DNA polymerase, Taq DNA polymerase, Pfu DNA polymerase, KOD DNA polymerase, Tth DNA polymerase, Pwo DNA polymerase, or a mutant or homologue thereof.

[0177] In some embodiments, the DNA ligase comprises T4 DNA ligase, Paramecium bursaria Chlorella virus 1 (PCBV-1) DNA ligase, Mycobacterium Ligase D (LigD), Ligase 1 (Lig1), Ligase 3 (Lig3α), Ligase 4 (Lig4), or a combination thereof. In some embodiments, the DNA ligase recruitment moiety comprises a 5'-adenyl pyrophosphoryl cap.

[0178] In some embodiments, the DNA binding protein comprises replication protein A (RPA) or its subunits, such as RPA1, RPA2, and RPA3, single-stranded DNA binding proteins (SSBPs), such as SSBP1, SSBP2, SSBP3, and SSBP4, or a combination thereof. In some embodiments, the DNA repair protein comprises tyrosyl-DNA phosphodiesterase 1 (TDP1), aprataxin, topoisomerase I, or a combination thereof.

[0179] In some embodiments, one or more polynucleotides comprising a guide sequence, a Cas binding region, and a DNA template sequence as described herein are provided in a fusion protein. In some embodiments, the Cas nuclease or Cas nickase of the fusion protein can bind and cleave a target polynucleotide. In some embodiments, the DNA polymerase recruitment portion of the fusion protein recruits a DNA polymerase to the cleaved target polynucleotide. In some embodiments, the recruited DNA polymerase synthesizes a DNA strand complementary to the sequence of interest on the DNA template sequence, thereby generating a double-stranded sequence comprising the sequence of interest that can be inserted into the cleaved target polynucleotide.

[0180] In some embodiments, the fusion protein recruits a DNA polymerase, a DNA ligase, and / or a DNA repair protein to increase the efficiency of inserting double-stranded sequences into the cleaved target polynucleotide, thereby increasing the efficiency of generating double-stranded sequences. In some embodiments, a fusion protein comprising a DNA polymerase recruitment moiety, a DNA ligase, a DNA ligase recruitment moiety, a DNA binding protein, a DNA repair protein, or a combination thereof, has a higher insertion efficiency compared to a Cas nuclease or Cas nickase that is not fused to a DNA polymerase recruitment moiety, a DNA ligase, a DNA ligase recruitment moiety, a DNA binding protein, a DNA repair protein, or a combination thereof.

[0181] In some embodiments, the fusion protein further comprises a nuclear localization signal (NLS). As used herein, a "nuclear localization signal" or "nuclear localization sequence" (NLS) refers to a polypeptide that "tags" a protein for transport to the cell nucleus by nuclear transport, i.e., a protein having an NLS is transported to the cell nucleus. Typically, an NLS comprises a positively charged Lys or Arg residue exposed on the protein surface. Exemplary NLSs include, but are not limited to, NLSs from SV40 large T antigen, nucleoplasmin, EGL-13, c-Myc, and TUS-proteins.

[0182] In some embodiments, the fusion protein further comprises a linker linking the Cas nuclease or Cas nickase and the DNA polymerase recruitment protein. In some embodiments, the linker is of sufficient length and / or flexibility such that the Cas nuclease or Cas nickase can be positioned without steric hindrance from the DNA polymerase recruitment protein. In some embodiments, the linker comprises about 3 to about 100 amino acids in length. In some embodiments, the linker comprises about 5 to about 80 amino acids in length. In some embodiments, the linker comprises about 10 to about 60 amino acids in length. In some embodiments, the linker comprises about 20 to about 50 amino acids in length. In some embodiments, the linker comprises about 25 to about 40 amino acids in length.

[0183] In some embodiments, the disclosure provides a polynucleotide encoding a fusion protein described herein. In some embodiments, the polynucleotide is a polynucleotide that is codon-optimized for expression in a bacterial cell. In some embodiments, the polynucleotide is a polynucleotide that is codon-optimized for expression in a eukaryotic cell. In some embodiments, the polynucleotide is a polynucleotide that is codon-optimized for expression in a mammalian cell. In some embodiments, the polynucleotide is a polynucleotide that is codon-optimized for expression in a human cell. As used herein, "codon optimization" refers to adjusting codons to match the abundance of host tRNAs to improve the yield and efficiency of recombinant or heterologous protein expression. Codon optimization methods are known in the art and can be performed using software programs such as, for example, Integrated DNA Technologies' Codon Optimization Tool, Entelechon's Codon Usage Table Analysis Tool, and the like.

[0184] In some embodiments, the present disclosure provides a vector comprising a polynucleotide encoding a fusion protein described herein. Various types of vectors are provided herein, e.g., viral and non-viral vectors. In some embodiments, the vector is an expression vector. In some embodiments, the vector is a bacterial 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.

[0185] In some embodiments, the vector is a viral vector. In some embodiments, the viral vector is a retroviral vector, an adeno-associated viral vector, a pox vector, a baculoviral vector, a vaccinia vector, a herpes simplex vector, an Epstein-Barr viral vector, an adenoviral vector, a geminiviral vector, or a caulimoviral vector. In some embodiments, the viral vector is an adenoviral vector, a lentiviral vector, or an adeno-associated viral vector. Viral transduction with adenoviral vectors, adeno-associated viral (AAV) vectors, and lentiviral vectors (where administration can be local, targeted, or systemic) has been used as a delivery method for in vivo gene therapy. Methods for introducing vectors, such as viral vectors, into cells (e.g., transfection) are described herein.

[0186] In some embodiments, the vector further comprises a regulatory element operably linked to the polynucleotide encoding the fusion protein. In some embodiments, the regulatory element comprises a promoter, an enhancer, a terminator, a 5'UTR, a 3'UTR, or a combination thereof. Regulatory elements are further described herein. In some embodiments, the regulatory element comprises a promoter. In some embodiments, the promoter is a bacterial promoter. In some embodiments, the promoter is a viral promoter. In some embodiments, the promoter is a mammalian promoter.

[0187] In some embodiments, the present disclosure provides a kit comprising a fusion protein described herein. In some embodiments, the fusion protein in the kit is provided as a polynucleotide encoding the fusion protein. In some embodiments, the polynucleotide encoding the fusion protein is provided on a vector, such as a vector described herein.

[0188] In some embodiments, the kit further comprises a polynucleotide, wherein the polynucleotide comprises a Cas binding region. In some embodiments, the Cas binding region is capable of binding to a Cas nuclease or a Cas nickase of the fusion protein. In some embodiments, the Cas binding region comprises a tracrRNA. In some embodiments, the Cas binding region is capable of hybridizing to a tracrRNA. In some embodiments, the kit further comprises a tracrRNA. Polynucleotides comprising a Cas binding region are further described herein.

[0189] In some embodiments, the kit further comprises a DNA polymerase. In some embodiments, the DNA polymerase comprises phi29 DNA polymerase, DNA polymerase μ, DNA polymerase δ, or DNA polymerase ε. In some embodiments, the kit further comprises a DNA ligase. In some embodiments, the DNA ligase comprises T4 DNA ligase, PCBV-1 DNA ligase, LigD, human Lig1, human Lig3α, or human Lig4.

[0190] In some embodiments, the kit further comprises a reaction buffer and / or a storage buffer for the fusion protein, the DNA polymerase, and / or the DNA ligase. In some embodiments, the kit further comprises a reagent for performing a DNA cleavage reaction, a DNA polymerase reaction, and / or a DNA ligase reaction. In some embodiments, the reagent comprises ATP, dNTPs, MgCl2, oligo(dT), and / or an RNase inhibitor. In some embodiments, the kit comprises one or more controls, e.g., a control target DNA. For example, the control target DNA can be designed to be specifically cleaved with a certain efficiency by the Cas nuclease or Cas nickase of the fusion protein, thereby allowing the activity of the Cas nuclease or Cas nickase to be calibrated.

[0191] cell In some embodiments, the disclosure provides a cell comprising a fusion protein described herein. In some embodiments, the disclosure provides a cell comprising a polynucleotide encoding a fusion protein described herein. In some embodiments, the disclosure provides a cell comprising a vector comprising a polynucleotide encoding a fusion protein provided herein. In some embodiments, the cell further comprises a polynucleotide described herein, the polynucleotide comprising a guide sequence (e.g., an RNA guide sequence), a Cas binding protein, and a DNA template sequence.

[0192] In some embodiments, the disclosure provides a cell comprising a polynucleotide described herein, wherein the polynucleotide comprises an RNA guide sequence, a Cas binding region, and a DNA template sequence. In some embodiments, the disclosure provides a cell comprising a composition described herein, wherein the composition comprises a Cas nuclease or Cas nickase and one or more polynucleotides comprising a guide sequence, a Cas binding region, and a DNA template sequence. In some embodiments, the disclosure provides a cell comprising a composition described herein, wherein the composition comprises (a) a Cas nuclease or Cas nickase and (b) a polynucleotide comprising a guide sequence, a Cas binding region, and a DNA template sequence, wherein the DNA template sequence is at the 3' end of the polynucleotide. In some embodiments, the disclosure provides a cell comprising a composition described herein, wherein the composition comprises: (a) a Cas nuclease or a Cas nickase; (b) a first polynucleotide comprising (i) a guide sequence, (ii) a Cas binding region, and (iii) a first hybridization region, wherein the first hybridization region is at a 3' end of the first polynucleotide; and (c) a second polynucleotide comprising (i) a second hybridization region complementary to the first hybridization region, and (ii) a DNA template sequence. In some embodiments, the disclosure provides a cell comprising a composition described herein, the composition comprising: (a) a Cas nuclease or Cas nickase; (b) a first polynucleotide comprising (i) a guide sequence and (ii) a first hybridization region, the first hybridization region being at a 3' end of the first polynucleotide; and (c) a second polynucleotide comprising (i) a second hybridization region complementary to the first hybridization region, (ii) a Cas binding region, and (iii) a DNA template sequence. The components of the polynucleotides and compositions are further described herein. In some embodiments, the Cas nuclease or Cas nickase is fused to a DNA polymerase recruitment protein.Fusion proteins comprising a Cas nuclease or a Cas nickase and a DNA polymerase recruitment protein are further described herein.

[0193] In some embodiments, the disclosure provides a cell comprising a composition described herein, wherein the composition comprises: (a) a Cas nuclease or a Cas nickase; (b) a first polynucleotide comprising (i) a guide sequence, (ii) a Cas binding region, (iii) a first hybridization region, and (iv) a primer binding sequence, wherein the primer binding sequence is at a 3' end of the first polynucleotide; and (c) a second polynucleotide comprising (i) a second hybridization region complementary to the first hybridization region, and (ii) a sequence of interest (SOI). In some embodiments, the disclosure provides a cell comprising a composition described herein, the composition comprising: (a) a Cas nuclease or Cas nickase; (b) a first polynucleotide comprising (i) a guide sequence, (ii) a Cas binding region, and (iii) a first hybridization region, the first hybridization region being at a 3' end of the first polynucleotide; (c) a second polynucleotide comprising (i) a second hybridization region complementary to the first hybridization region, (ii) a third hybridization region, and (iii) a primer binding sequence, the second polynucleotide comprising (i) a second hybridization region complementary to the first hybridization region, (ii) a third hybridization region, and (iii) a primer binding sequence, the primer binding sequence being at a 3' end of the second polynucleotide; and (d) a third polynucleotide comprising (i) a fourth hybridization region complementary to the third hybridization region, and (ii) a sequence of interest (SOI). The components of the composition are further described herein. In some embodiments, the Cas nuclease or Cas nickase is fused to a DNA polymerase recruitment moiety, a DNA ligase, a DNA ligase recruitment moiety, a DNA binding protein, a DNA repair protein, or a combination thereof. Fusion proteins are further described herein.

[0194] In some embodiments, the cells comprise an endogenous DNA polymerase, an endogenous DNA ligase, or both. In some embodiments, the cells do not comprise an exogenous DNA polymerase. In some embodiments, the cells do not comprise an exogenous DNA ligase.

[0195] In some embodiments, the cell further comprises an exogenous DNA polymerase, an exogenous DNA ligase, or both. In some embodiments, the exogenous DNA polymerase is homologous to the cell. In some embodiments, the exogenous DNA polymerase is heterologous to the cell. In some embodiments, the exogenous DNA polymerase is from a different organism than the cell. For example, the cell can be an E. coli cell and the DNA polymerase can be T4 DNA polymerase. In some embodiments, the exogenous DNA polymerase comprises Pol I or the Klenow fragment thereof, Pol II, Pol III, Pol IV, Pol V, 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, REV3, Bst DNA polymerase, T4 DNA polymerase, Φ29 (phi29) DNA polymerase, Taq DNA polymerase, Pfu DNA polymerase, KOD DNA polymerase, Tth DNA polymerase, Pwo DNA polymerase, or a mutant or homolog thereof. In some embodiments, the exogenous DNA ligase is homologous to the cell. In some embodiments, the exogenous DNA ligase is heterologous to the cell. In some embodiments, the exogenous DNA ligase is from a different organism than the cell. In some embodiments, the exogenous DNA ligase comprises E. coli DNA ligase, T4 DNA ligase, T7 DNA ligase, DNA ligase I, DNA ligase II, DNA ligase III, DNA ligase IV, Taq DNA ligase, or a mutant or homologue thereof. For example, the cell can be an E. coli cell and the DNA ligase can be T4 ligase.

[0196] In some embodiments, the cell is a bacterial cell. In some embodiments, the bacterial cell is a laboratory strain. Examples of such bacterial cells include, but are not limited to, E. coli, S. aureus, V. cholerae, S. pneumoniae, B. subtilis, C. crescentus, M. genitalium, A. fischeri, Synechocystis, P. fluorescens, A. vinelandii, S. coelicolor. In some embodiments, the bacterial cell is a bacterial cell used in the preparation of food and / or beverages.Non-limiting exemplary genera of such cells include Acetobacter, Arthrobacter, Bacillus, Bifidobacterium, Brachybacterium, Brevibacterium, Carnobacterium, Corynebacterium, Enterococcus, Gluconacetobacter, Hafnia, Halomonas, Kocuria, Lactobacillus, (L. acetotolerans, L. acidipiscis, L. acidophilus, L.alimentarius, L.brevis, L.bucheri, L.casei, L.curvatus, L.fermentum, L.hilgardii, L.jensenii, L.kimchii, L.lactis, L.paracasei, L. Examples of suitable bactericidal bacteria include, but are not limited to, L. paracasei, L. plantarum and L. sakei, Leuconostoc, Microbacterium, Pediococcus, Propionibacterium, Weissella, and Zymomonas.

[0197] In some embodiments, the cell is a eukaryotic cell. In some embodiments, the eukaryotic cell is a mammalian cell. In some embodiments, the eukaryotic cell is an animal cell. In some embodiments, the eukaryotic cell is a mammalian cell. In some embodiments, the eukaryotic cell is an animal or human cell, cell line, or cell strain. Examples of animal or mammalian cells, cell lines, or cell strains include, but are not limited to, mouse myeloma (NS0) cells, Chinese hamster ovary (CHO) cells, HT1080 cells, H9 cells, HepG2 cells, MCF7 cells, MDBK cells, Jurkat cells, NIH3T3 cells, PC12 cells, BHK (baby hamster kidney) cells, EBX cells, EB14 cells, EB24 cells, EB26 cells, EB66 cells, or Ebvl3 cells, VERO cells, SP2 / 0 cells, YB2 / 0 cells, Y0 cells, C127 cells, L cells, COS (e.g., COS1 and COS7) cells, QC1-3 cells, HEK293 cells, VERO cells, PER.C6 cells, HeLA cells, EB1 cells, EB2 cells, EB3 cells, oncolytic cells, or hybridoma cells. In some embodiments, the eukaryotic cells are CHO cells. 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) can be, for example, a CHO-K1 SV GS knockout cell.

[0198] In some embodiments, the eukaryotic cells are human stem cells. The stem cells may be pluripotent stem cells, including, for example, embryonic stem cells (ESCs), adult stem cells, induced pluripotent stem cells (iPSCs), tissue-specific stem cells (e.g., hematopoietic stem cells), and mesenchymal stem cells (MSCs). In some embodiments, the cells are differentiated forms of any of the cells described herein. In some embodiments, the eukaryotic cells are cells derived from any primary cell in culture.

[0199] 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 adherent metabolic test human hepatocyte, an adherent induction test human hepatocyte, an adherent 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).

[0200] In some embodiments, the eukaryotic cell is a plant cell. For example, the plant cell may be a crop cell, such as cassava, maize, sorghum, wheat, or rice. The plant cell may be an algae, tree, or vegetable cell. The plant cell may 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, e.g., a citrus tree such as an orange, grapefruit or lemon tree; a peach or nectarine tree; an apple or pear tree; a nut tree such as an almond or walnut or pistachio tree; a plant of the Solanum genus, e.g., potato, tomato, eggplant, pepper, paprika; a plant of the Brassica genus, a plant of the Lactuca genus, a plant of the Spinacia genus; a plant of the Capsicum genus; cotton, tobacco, asparagus, carrot, cabbage, broccoli, cauliflower, lettuce, spinach, strawberry, blueberry, raspberry, blackberry, grape, coffee, cocoa, etc.

[0201] method In some embodiments, the present disclosure provides a method for providing a target insertion in a target polynucleotide in a cell, comprising introducing a composition of the present disclosure into the cell. In some embodiments, the target polynucleotide is a target DNA. In some embodiments, the composition comprises a Cas nuclease or Cas nickase and one or more polynucleotides comprising a guide sequence, a Cas binding region, and a DNA template sequence. In some embodiments, the composition comprises (a) a Cas nuclease or Cas nickase and (b) a polynucleotide comprising a guide sequence, a Cas binding region, and a DNA template sequence, wherein the DNA template sequence is at the 3' end of the polynucleotide. In some embodiments, a composition comprises: (a) a Cas nuclease or a Cas nickase; (b) a first polynucleotide comprising (i) a guide sequence, (ii) a Cas binding region, and (iii) a first hybridization region, wherein the first hybridization region is at a 3' end of the first polynucleotide; and (c) a second polynucleotide comprising (i) a second hybridization region complementary to the first hybridization region, and (ii) a DNA template sequence. In some embodiments, the composition comprises: (a) a Cas nuclease or a Cas nickase; (b) a first polynucleotide comprising (i) a guide sequence, and (ii) a first hybridization region, where the first hybridization region is at a 3' end of the first polynucleotide; and (c) a second polynucleotide comprising (i) a second hybridization region complementary to the first hybridization region, (ii) a Cas binding region, and (iii) a DNA template sequence. The components of the polynucleotides and compositions are further described herein.

[0202] An exemplary, non-limiting overview of the method is illustrated in Figure 1. In Figure 1, Cas nuclease is guided to a target DNA in a cell by a guide sequence and cleaves the target DNA. The DNA template sequence includes (i) a primer binding sequence that serves as a primer and hybridizes with the cleaved DNA, and (ii) a target sequence. A DNA polymerase, e.g., an endogenous DNA polymerase of the cell, binds to the primer and synthesizes a DNA strand complementary to the target sequence, thereby forming a double-stranded sequence that includes the target sequence. The double-stranded sequence can be inserted into the cleaved target DNA by a DNA repair pathway, e.g., NHEJ.

[0203] In some embodiments, a method of providing a target insertion in a target DNA in a cell comprises introducing a composition described herein into a cell, wherein the guide sequence is capable of hybridizing to the target DNA. In some embodiments, the DNA template sequence comprises single-stranded DNA. In some embodiments, the guide sequence is capable of hybridizing to the target DNA. In some embodiments, a Cas nuclease or Cas nickase is directed to the target DNA by hybridizing the guide sequence and the target DNA. In some embodiments, the method is performed under conditions sufficient to allow the Cas nuclease or Cas nickase to cause cleavage in the target DNA.

[0204] In some embodiments, one strand of the cleaved target DNA serves as a primer for a DNA polymerase. In some embodiments, the DNA template sequence comprises a primer binding sequence and a sequence of interest. In some embodiments, the primer binding sequence can bind to a primer. In some embodiments, the method does not include introducing an exogenous DNA polymerase into the cell. In some embodiments, the method is performed under conditions sufficient to allow an endogenous DNA polymerase of the cell to bind to the primer and extend the DNA template sequence. In some embodiments, the extending includes synthesizing a DNA strand complementary to the sequence of interest to form a double-stranded sequence that includes the sequence of interest.

[0205] In some embodiments, the method includes introducing an exogenous DNA polymerase into the cell. In some embodiments, the exogenous DNA polymerase is introduced into the cell by a polynucleotide encoding the exogenous DNA polymerase. In some embodiments, the exogenous DNA polymerase is introduced into the cell by a vector comprising the polynucleotide. Methods for introducing an exogenous component, such as an exogenous DNA polymerase, into a cell are described herein. In some embodiments, the exogenous DNA polymerase is homologous to the cell. In some embodiments, the exogenous DNA polymerase is heterologous to the cell. In some embodiments, the exogenous DNA polymerase is from a different organism than the cell. In some embodiments, the exogenous DNA polymerase comprises Pol I or Klenow fragment thereof, Pol II, Pol III, Pol IV, Pol V, 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, REV3, Bst DNA polymerase, T4 DNA polymerase, Φ29 (phi29) DNA polymerase, Taq DNA polymerase, Pfu DNA polymerase, KOD DNA polymerase, Tth DNA polymerase, Pwo DNA polymerase, or a mutant or homologue thereof. In some embodiments, the method is performed under conditions sufficient for the exogenous DNA polymerase to bind to the primer and extend the DNA template sequence. In some embodiments, extending comprises synthesizing a DNA strand complementary to the sequence of interest to form a double-stranded sequence comprising the sequence of interest.

[0206] In some embodiments, the double-stranded sequence is inserted into the cleaved target DNA. In some embodiments, the double-stranded sequence containing the sequence of interest is inserted into the cleaved target sequence by a DNA repair pathway. DNA repair pathways include the non-homologous end joining (NHEJ) pathway, the microhomology-mediated end joining (MMEJ) pathway, the homology-directed repair (HDR) pathway, synthesis-dependent single-strand annealing (SDSA), single-strand annealing (SSA), and alternative end joining (Alt-EJ). NHEJ does not require a homologous template. In general, NHEJ has a high repair efficiency but low fidelity when compared with HDR. However, errors are reduced when there is a sticky end or overhang that can be accommodated at the double-strand break. MMEJ is one that has microhomology (e.g., about 2 to about 10 base pairs) on both sides of the double-strand break. HDR requires a homologous template to guide repair, and HDR repair is typically more fidelity but less efficient than NHEJ and MMEJ. SDSA, SSA, and Alt-EJ are HDR-based repair pathways. SDSA is further described, for example, in Sung et al., Nat Rev Mol Cell Biol 7:741 (2006). SSA and Alt-EJ are further described, for example, in Bhargava et al., Trends Genet 32(9):566-575 (2016). In some embodiments, the method is performed under conditions sufficient for non-homologous end joining (NHEJ). In some embodiments, NHEJ inserts a double-stranded sequence containing a sequence of interest into the cleaved target sequence. In some embodiments, the method is performed under conditions sufficient for HDR, SDSA, SSA, Alt-EJ, or a combination thereof.

[0207] In some embodiments, the ligation inserts a double-stranded sequence into the cleaved target DNA. In some embodiments, the DNA ligase inserts a double-stranded sequence containing a sequence of interest into the cleaved target sequence. In some embodiments, the DNA ligase is an endogenous DNA ligase of the cell. In some embodiments, the method does not include introducing an exogenous DNA ligase into the cell.

[0208] In some embodiments, the method further comprises introducing an exogenous DNA ligase into the cell, where the exogenous DNA ligase inserts a double-stranded sequence into the cleaved target DNA. In some embodiments, the exogenous DNA ligase is introduced into the cell by a polynucleotide encoding the exogenous DNA ligase. In some embodiments, the exogenous DNA ligase is introduced into the cell by a vector comprising the polynucleotide. Methods for introducing an exogenous component, e.g., an exogenous DNA ligase, into a cell are described herein. In some embodiments, the exogenous DNA ligase is homologous to the cell. In some embodiments, the exogenous DNA ligase is heterologous to the cell. In some embodiments, the exogenous DNA ligase is from a different organism than the cell. In some embodiments, the exogenous DNA ligase comprises E. coli DNA ligase, T4 DNA ligase, T7 DNA ligase, DNA ligase I, DNA ligase II, DNA ligase III, DNA ligase IV, Taq DNA ligase, or variants or homologs thereof.

[0209] In some embodiments, the double-stranded sequence further comprises a recognition site for an endonuclease, a transposase, or a recombinase. In some embodiments, the double-stranded sequence is integrated into the target DNA by an endonuclease, a transposase, or a recombinase. In some embodiments, the endonuclease, the transposase, or the recombinase is endogenous to the cell. The mechanism of integration of sequences by endonucleases, transposases, and recombinases is known to those skilled in the art and is further described, for example, in Carlson et al., Mol Microbiol 27(4):671-676(1998), Nesmelova et al., Adv Drug Deliv Rev 62:1187-1195(2010), and Hallet et al., FEMS Microbiol Rev 21(2):157-178(1997).

[0210] In some embodiments, the DNA template sequence is on the same polynucleotide as the guide sequence and the Cas binding region, and thus is adjacent to the cleavage site of the target DNA. In some embodiments, one or more polynucleotides comprising the DNA template sequence, the guide sequence, and the Cas binding region are hybridized, and thus the DNA template sequence is adjacent to the cleavage site of the target DNA. In some embodiments, the proximity of the DNA template sequence to the cleavage site facilitates insertion of the double-stranded sequence formed by the DNA polymerase into the cleaved target sequence.

[0211] In some embodiments, the method provides a double-stranded sequence adjacent to the cleaved target DNA, thereby increasing the efficiency of inserting the double-stranded sequence into the cleaved target DNA. In some embodiments, the method provides a double-stranded sequence adjacent to the cleaved target DNA, thereby increasing the efficiency of incorporating the double-stranded sequence into the cleaved target DNA. In some embodiments, the method has improved efficiency compared to methods that do not provide a DNA template sequence adjacent to the cleaved target DNA. In some embodiments, the method has at least 2-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 30-fold, at least 40-fold, at least 50-fold, at least 60-fold, at least 70-fold, at least 80-fold, at least 90-fold, at least 100-fold, at least 150-fold, or at least 200-fold or more efficiency compared to methods that do not provide a DNA template sequence adjacent to the cleaved target DNA.

[0212] In some embodiments, the present disclosure provides a method of providing a target insertion in a target DNA comprising contacting the target DNA with a composition described herein, wherein the guide sequence can hybridize to the target DNA. In some embodiments, the composition comprises a Cas nuclease or Cas nickase and one or more polynucleotides comprising a guide sequence, a Cas binding region, a primer binding sequence, and a sequence of interest. In some embodiments, the composition comprises (a) a Cas nuclease or Cas nickase, (b) a first polynucleotide comprising (i) a guide sequence, (ii) a Cas binding region, (iii) a first hybridization region, and (iv) a primer binding sequence, wherein the primer binding sequence is at the 3' end of the first polynucleotide, and (c) a second polynucleotide comprising (i) a second hybridization region complementary to the first hybridization region, and (ii) a sequence of interest (SOI). In some embodiments, the composition comprises: (a) a Cas nuclease or a Cas nickase; (b) a first polynucleotide comprising (i) a guide sequence, (ii) a Cas binding region, and (iii) a first hybridization region, the first hybridization region being at a 3' end of the first polynucleotide; (c) a second polynucleotide comprising (i) a second hybridization region complementary to the first hybridization region, (ii) a third hybridization region, and (iii) a primer binding sequence, the second polynucleotide comprising (i) a fourth hybridization region complementary to the third hybridization region, and (ii) a sequence of interest (SOI). The components of the composition are further described herein. In some embodiments, the Cas nuclease or Cas nickase is fused to a DNA polymerase recruitment moiety, a DNA ligase, a DNA ligase recruitment moiety, a DNA binding protein, a DNA repair protein, or a combination thereof. Fusion proteins are further described herein.

[0213] In some embodiments, the Cas nuclease or Cas nickase is guided to the target DNA by hybridizing the guide sequence and the target DNA. In some embodiments, the method is performed under conditions sufficient to cause the Cas nuclease or Cas nickase to cleave the target DNA. In some embodiments, one strand of the cleaved target DNA serves as a primer for a DNA polymerase. In some embodiments, the primer binding sequence of the first and / or second polynucleotide can bind to the primer.

[0214] In some embodiments, the method further comprises contacting the target DNA with a DNA ligase. DNA ligases are further described herein. In some embodiments, the DNA ligase is T4 ligase, PCBV-1 DNA ligase, LigD, a hitoriase protein, or a combination thereof. In some embodiments, the DNA ligase ligates a sequence of interest to the cleaved target DNA.

[0215] In some embodiments, the method further comprises contacting the target DNA with a DNA polymerase, a protein in a DNA repair pathway, or a combination thereof. In some embodiments, the sequence of interest is a single-stranded sequence that is converted into a double-stranded sequence by a DNA polymerase, a protein in a DNA repair pathway, or a combination thereof. DNA polymerases and DNA repair pathways are described herein.

[0216] In some embodiments, the method includes contacting the target DNA with a cell extract, the cell extract including a DNA ligase, a DNA polymerase, and / or a DNA repair pathway protein described herein. In some embodiments, the target DNA is contacted with a recombinant DNA ligase, a DNA polymerase, and / or a DNA repair pathway protein.

[0217] In some embodiments, the method is performed in vivo. In some embodiments, the method is performed in vitro. In some embodiments, the method is performed ex vivo. In some embodiments, the target DNA comprises a plasmid, a PCR product, a cosmid, a bacterial artificial chromosome (BAC), a yeast artificial chromosome (YAC), genomic DNA, mitochondrial DNA, chloroplast DNA, or a combination thereof.

[0218] In some embodiments, the present disclosure provides a method of providing a target insertion in a target polynucleotide in a cell, comprising introducing a composition of the present disclosure into the cell. In some embodiments, the target polynucleotide is a target DNA. In some embodiments, the composition comprises a Cas nuclease or Cas nickase and one or more polynucleotides comprising a guide sequence, a Cas binding region, a primer binding sequence, and a sequence of interest. In some embodiments, the composition comprises (a) a Cas nuclease or Cas nickase, (b) a first polynucleotide comprising (i) a guide sequence, (ii) a Cas binding region, (iii) a first hybridization region, and (iv) a primer binding sequence, wherein the primer binding sequence is at a 3' end of the first polynucleotide, and (c) a second polynucleotide comprising (i) a second hybridization region complementary to the first hybridization region, and (ii) a sequence of interest (SOI). In some embodiments, the composition comprises: (a) a Cas nuclease or a Cas nickase; (b) a first polynucleotide comprising (i) a guide sequence, (ii) a Cas binding region, and (iii) a first hybridization region, the first hybridization region being at a 3' end of the first polynucleotide; (c) a second polynucleotide comprising (i) a second hybridization region complementary to the first hybridization region, (ii) a third hybridization region, and (iii) a primer binding sequence, the second polynucleotide comprising (i) a second hybridization region complementary to the first hybridization region, (ii) a third hybridization region, and (iii) a primer binding sequence, the primer binding sequence being at a 3' end of the second polynucleotide; and (d) a third polynucleotide comprising (i) a fourth hybridization region complementary to the third hybridization region, and (ii) a sequence of interest (SOI). Components of the composition are further described herein. In some embodiments, the Cas nuclease or Cas nickase is fused to a DNA polymerase recruitment moiety, a DNA ligase, a DNA ligase recruitment moiety, a DNA binding protein, a DNA repair protein, or a combination thereof. Fusion proteins are further described herein.

[0219] An exemplary, non-limiting overview of the method is illustrated in Figures 10A-10C. In Figure 10A, a guide sequence, a Cas binding region, a first hybridization region, and a primer binding site are located on a first polynucleotide. A Cas nuclease guided to the target DNA by the guide sequence creates a double-stranded break in the target DNA. The primer binding site hybridizes to the cleaved DNA, and a second polynucleotide hybridizes to the first polynucleotide through the first and second hybridization regions that contain complementary sequences. The second polynucleotide contains a sequence of interest, which is ligated to the cleaved DNA at both the 5' and 3' ends by a ligase (Figure 10A). The second polynucleotide may further contain a homologous sequence, the 5' end of which is ligated to the cleaved DNA by the ligase, and the 3' end of which is incorporated by a homology-mediated mechanism. In some embodiments, the homology-mediated mechanism includes HDR, synthesis-dependent single-strand annealing (SDSA), single-strand annealing (SSA), alternative end joining (alt-EJ), or a combination thereof (Figure 10B). The complex is converted into double-stranded DNA after ligation and / or homology-mediated integration, thereby incorporating the desired sequence into the target DNA (Figure 10C).

[0220] In some embodiments, a method of providing a targeted insertion in a target DNA in a cell comprises introducing a composition described herein into a cell, wherein the guide sequence is capable of hybridizing to the target DNA. In some embodiments, a Cas nuclease or Cas nickase is directed to the target DNA by hybridizing the guide sequence and the target DNA. In some embodiments, the method is performed under conditions sufficient to allow the Cas nuclease or Cas nickase to effect cleavage in the target DNA.

[0221] In some embodiments, one strand of the cleaved target DNA serves as a primer for a DNA polymerase. In some embodiments, the primer binding sequence of the first and / or second polynucleotide can bind to the primer. In some embodiments, the method does not include introducing an exogenous DNA polymerase into the cell. In some embodiments, the method is performed under conditions sufficient for the endogenous DNA polymerase of the cell to bind to the primer and extend the sequence of interest. In some embodiments, the extending includes synthesizing a DNA strand complementary to the sequence of interest to form a double-stranded sequence that includes the sequence of interest. In some embodiments, the method does not include introducing an exogenous DNA ligase into the cell. In some embodiments, the method is performed under conditions sufficient for the endogenous DNA ligase of the cell to ligate the sequence of interest to the cleaved target DNA.

[0222] In some embodiments, the method includes introducing an exogenous DNA polymerase into the cell. The exogenous DNA polymerase is described herein. In some embodiments, the exogenous DNA polymerase synthesizes a DNA strand complementary to the sequence of interest. In some embodiments, the method includes introducing an exogenous DNA ligase into the cell. In some embodiments, the exogenous DNA ligase ligates the sequence of interest to the cleaved target DNA. In some embodiments, the sequence of interest is a single-stranded sequence that is converted to a double-stranded sequence by the cell's DNA repair pathway.

[0223] In some embodiments, the method further comprises contacting the cell with a DNA-dependent protein kinase (DNA-PK) inhibitor. In some embodiments, inhibiting DNA-PK reduces NHEJ and promotes HDR. In some embodiments, the cell is contacted with a DNA-PK inhibitor before contacting with the composition described herein. In some embodiments, the DNA-PK inhibitor is AZD7648.

[0224] In some embodiments, the compositions, polynucleotides, and / or fusion proteins described herein are introduced into cells by transfection. Transfection methods are further described herein. In some embodiments, a polynucleotide of the present disclosure, including, for example, a guide sequence, a Cas binding region, a DNA template sequence, or any combination thereof, is transfected into a cell. In some embodiments, the polynucleotide is on one or more vectors.

[0225] In some embodiments, the proteins of the disclosure, e.g., fusion proteins, Cas nucleases, Cas nickases, DNA polymerases, and / or DNA ligases, are introduced into the cell by one or more polynucleotides encoding the proteins. In some embodiments, the one or more polynucleotides are on one or more vectors. In some embodiments, the method comprises introducing multiple proteins, e.g., any combination of fusion proteins, Cas nucleases, Cas nickases, DNA polymerases, and DNA ligases, into the cell. In some embodiments, the method comprises introducing Cas nucleases, Cas nickases, or fusion proteins and DNA polymerases into the cell. In some embodiments, the method comprises introducing Cas nucleases, Cas nickases, or fusion proteins and DNA ligases into the cell. In some embodiments, the method comprises introducing Cas nucleases, Cas nickases, or fusion proteins, DNA polymerases, and DNA ligases into the cell. In some embodiments, the method comprises introducing Cas nucleases, Cas nickases, or fusion proteins, DNA polymerases, and DNA ligases into the cell. In some embodiments, the polynucleotides encoding multiple proteins are on a single vector. In some embodiments, polynucleotides encoding multiple proteins are on two or more vectors.

[0226] In some embodiments, the components of the compositions described herein are on a single vector. In some embodiments, the components of the compositions described herein are on two or more vectors. In some embodiments, the method includes transfecting one or more vectors into a cell, the one or more vectors including one or more polynucleotides encoding a fusion protein, a Cas nuclease, or a Cas nickase, and one or more polynucleotides including a guide sequence, a Cas binding region, or a DNA template sequence. In some embodiments, the method includes transfecting a first vector and a second vector into a cell, the first vector including a polynucleotide encoding a fusion protein, a Cas nuclease, or a Cas nickase, and the second vector including one or more polynucleotides including a guide sequence, a Cas binding region, or a DNA template sequence. In some embodiments, the method includes transfecting a single vector into a cell, the single vector including (i) one or more polynucleotides encoding a fusion protein, a Cas nuclease, or a Cas nickase, and (ii) one or more polynucleotides including a guide sequence, a Cas binding region, or a DNA template sequence.

[0227] In some embodiments, the compositions, polynucleotides, and / or fusion proteins described herein are introduced into cells by delivery particles. In some embodiments, the components of the composition are delivered in a single delivery particle. In some embodiments, the components of the composition are delivered in multiple delivery particles. The delivery particles can be used to deliver exogenous biological materials, such as, for example, the polynucleotides and proteins described herein. In some embodiments, the delivery particles are solids, semi-solids, emulsions, or colloids. In some embodiments, the delivery particles are lipid-based particles, liposomes, micelles, vesicles, or exosomes. In some embodiments, the delivery particles are nanoparticles. Delivery particles are further described in, for example, U.S. Patent Application Publication Nos. 2011 / 0293703, 2012 / 0251560, 2013 / 0302401, U.S. Patent No. 5,543,158, U.S. Patent No. 5,855,913, U.S. Patent No. 5,895,309, U.S. Patent No. 6,007,845, and U.S. Patent No. 8,709,843.

[0228] In some embodiments, the compositions, polynucleotides, and / or fusion proteins described herein are introduced into cells by vesicles. In some embodiments, the components of the composition are delivered in a single vesicle. In some embodiments, the components of the composition are delivered in multiple vesicles. In some embodiments, the vesicle is comprised of an exosome or a liposome. Vesicles modified to deliver exogenous biomaterials to target cells are described, for example, in U.S. Patent Application Publication Nos. 2008 / 0234183; 2019 / 0167810; 2020 / 0207833; and Alvarez-Erviti et al., Nat Biotechnol 29:341 (2011).

[0229] 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

[0230] Example 1. Evaluation of targeted insertion of polynucleotides S. pyogenes Cas9 protein (SpCas9) alone or SpCas9 fused to reverse transcriptase (SpCas9-RT) was tested for in vivo targeted insertion with a guide polynucleotide (referred to herein as "springRNA") consisting of an RNA guide sequence targeting the AAVS1 locus, a tracrRNA for binding to SpCas9, and a template sequence at the 3' end. Five springRNA constructs were prepared:

[0231] (1) RNA-only springRNA - a polynucleotide consisting of RNA nucleotides and consisting of an RNA guide sequence, a tracrRNA, a 6-bp insertion sequence (AATATG), and a primer binding sequence (see Figure 2A);

[0232] (2) DNA-tailed springRNA ("DNA") - a sequence identical to the RNA-only springRNA, containing all RNA nucleotides except for the insertion sequence and primer binding sequence, which are made up of DNA nucleotides (see Figure 2B);

[0233] (3) springRNA with a DNA tail and phosphorothioate bonds ("PS-DNA") - identical to springRNA with a DNA insert, except that the insert sequence and primer binding sequence contain phosphorothioate bonds instead of phosphodiester bonds (see Figure 2B);

[0234] (4) springRNA with abasic site - identical to RNA-only springRNA and composed of RNA nucleotides, except that the third base of the insertion sequence is replaced by an abasic site, 1'2'-dideoxyribose, a dSpacer nucleotide (see Figure 2C);

[0235] (5) springRNA with TEG linker - identical to RNA-only springRNA and composed of RNA nucleotides, except that the third base of the insert sequence is covalently linked to triethylene glycol (TEG) (see Figure 2D).

[0236] HEK293T cells were transfected with plasmids expressing either SpCas9 or SpCas9-RT using FUGENE HD®. After 24 hours, LIPOFECTAMINE (商標) Cells were further transfected with 2 pmol of the springRNA constructs listed above using RNAiMAX.

[0237] Cells were harvested 48 hours after transfection with springRNA. The AAVS1 locus was amplified by PCR and sequenced using an Illumina sequencing platform. The results are shown in Figures 3 and 4. Panels A-E of Figure 3 show targeted insertions by SpCas9 and each of the above springRNA constructs. Panels F-J of Figure 3 show targeted insertions by SpCas9-RT ("PE0") and each of the above springRNA constructs. Figure 4 shows the relative frequency of insertions by SpCas9 alone or SpCas9-RT in combination with each of the above springRNA constructs.

[0238] The results in Figures 3 and 4 show that co-transfection of springRNAs containing DNA tails or DNA tails with phosphorothioate bonds (i.e., insert sequences and primer binding sequences containing DNA nucleotides as described for constructs (2) and (3) above) with SpCas9 expression plasmids resulted in targeted insertion of the insert sequence even in the absence of reverse transcriptase fused to SpCas9. Thus, endogenous cellular factors, such as endogenous cellular DNA polymerases, can use the insert sequence as a template for DNA synthesis. Thus, targeted insertion can be achieved using Cas9 alone as well as springRNAs containing DNA inserts, i.e., DNA insert sequences and DNA primer binding sequences.

[0239] Example 2. Further evaluation of targeted insertion of polynucleotides SpringRNA with only RNA, springRNA with DNA tail, and springRNA with DNA tail and phosphorothioate linkage (PS-DNA) were further tested with SpCas9 alone or in SpCas9-RT fusion. The springRNA construct contained a guide sequence targeting AAVS1 and the tracrRNA sequence of SpCas9 as in Example 1. The sequences of springRNA are shown in Table 4. The insert and primer binding sequences are underlined, with double underlines representing DNA nucleotides. SpringRNA was synthesized by Agilent.

[0240] [Table 18]

[0241] Cells were transfected with SpCas9 or SpCas9-RT and springRNA, cells were harvested, and the AAVS1 locus was analyzed as described in Example 1. The results are shown in Figures 5A-5F. Figures 5A-5C show targeted insertion by SpCas9-RT ("PE0") and RNA-only springRNA (Figure 5A), DNA-tailed springRNA (Figure 5B), and PS-DNA-containing springRNA (Figure 5C). Figures 5D-5F show targeted insertion by SpCas9 and RNA-only springRNA (Figure 5D), DNA-tailed springRNA (Figure 5E), and PS-DNA-containing springRNA (Figure 5F).

[0242] This result indicates that the combination of SpCas9 and springRNA containing a DNA tail or a PS-DNA tail with PS-DNA, i.e., an insertion sequence containing DNA nucleotides and a primer binding sequence, achieved an insertion pattern similar to that of the combination of SpCas9-RT and springRNA.

[0243] Example 3. In vitro assay To evaluate the role of DNA polymerase in target insertion using Cas9, an in vitro assay was performed. A schematic of the assay is shown in Figure 6. In the in vitro assay, two complementary DNA strands are labeled with different fluorophores (6 FAM-labeled non-target strand and HEX-labeled target strand). The guide sequence is designed such that Cas9 cleavage generates two strands of different lengths. DNA polymerase extends the 6 FAM-labeled non-target strand hybridized to the primer binding sequence of springRNA. The products are denatured and separated by capillary electrophoresis, and the strand bound to the fluorophore is detected.

[0244] Synthetic target DNA substrates were prepared by annealing two complementary strands, a 6 FAM-labeled non-target strand and a HEX-labeled target strand. Cas9 and springRNA were mixed in an equimolar ratio to form a ribonucleoprotein complex at room temperature. Cas9:springRNA complexes were added to the synthetic target DNA substrate at 15-fold molar excess. DNA polymerase (either Bst 3.0 DNA polymerase or Klenow fragment of E. coli DNA Pol I in optimal buffer) was added to the reaction mixture and incubated at 37°C for 1 h. The reaction was stopped by proteinase K digestion. 1 μL of the reaction mixture was transferred to HIDI (商標) The DNA was added to formamide and denatured at 95° C. for 3 minutes, and the denatured products were separated and analyzed by capillary electrophoresis on a 3730 DNA analyzer (Applied Biosystems).

[0245] The results are shown in Figures 7A-7F. The blue line corresponds to the non-target strand, and the green line corresponds to the target strand. The asterisk indicates the cleaved synthetic target DNA substrate, and the black arrow indicates the extension product by DNA polymerase. Figure 7A shows the assay results with Cas9 and springRNA. Figure 7B shows the assay results with Cas9, Klenow fragment, and springRNA. Figures 7C-7F show the assay results with Cas9, Bst 3.0 polymerase, and springRNA (Figures 7C-7D), springRNA with abasic site (Figure 7E), or springRNA with TEG (Figure 7F). The in vitro assay results show that DNA polymerase can perform target insertion without fusing with Cas9. Figure 8 shows the kinetics of the extension reaction by Klenow fragment or Bst 3.0 polymerase.

[0246] Example 4. Proximity ligation upstream of gRNA Examples 5-19 below relate to proximity ligation upstream of a gRNA ("plugRNA").

[0247] As described throughout this disclosure and the Examples, plugRNA comprises a gRNA molecule with a polynucleotide attached to its 3' end. The polynucleotide is composed of two elements: a first hybridization region (also referred to as "landing pad" in the Examples and Figures) and a primer binding sequence (PBS) that hybridizes to a sequence upstream of the cleavage site induced by the Cas protein. The plugRNA may be a single molecule or may be composed of multiple polynucleotides that anneal to each other to form a binary, ternary or quaternary complex. Any element of the plugRNA may comprise DNA, RNA, LNA, PNA, or chemically modified nucleotides.

[0248] As described throughout this disclosure and the Examples, the Cas protein may be any Cas protein capable of binding to the plugRNA. The Cas protein may be any type I or type II Cas protein, including but not limited to Cas9, Cas12, and the Cascade complex. The Cas protein can be fused to a fluorescent tag, a DNA polymerase, a reverse transcriptase, a DNA ligase (such as, but not limited to, T4 DNA ligase, Paramecium bursaria Chlorella virus 1 (PBCV-1) ligase, Mycobacterium Ligase D (LigD), firefly ligase 1, firefly ligase 3, firefly ligase 4), a ligase adaptor protein (such as, but not limited to, XRCC1, XRCC4, PCNA), a single-stranded DNA binding protein (RPA1 / 2 / 3, SSBP1-4, etc.), a DNA repair protein (TDP1, aprataxin, topoisomerase I), or a combination thereof.

[0249] The examples herein further refer to a "donor" polynucleotide, also referred to as a "second polynucleotide" in this disclosure. The donor polynucleotide is composed of a second hybridization region complementary to the first hybridization region of the plugRNA, a sequence of interest (also referred to as the "insert" in the examples and figures), and a homologous sequence complementary or substantially complementary to a sequence adjacent to the target cleavage site. The donor design may omit any of these elements, may be composed of two or more polynucleotides that hybridize to each other, or may pair with another polynucleotide to form a double-stranded region. Any portion of the donor may comprise DNA, RNA, PNA, LNA, or chemically modified nucleotides. The 5' and 3' ends of the donor may be phosphorylated, adenylated, phosphorothioated, etc.

[0250] The present disclosure provides a method for site-specific modification, also referred to herein as knock-in nucleotide guided ("KING") editing. A general scheme for KING editing is shown in FIG. 10. In summary, Cas9 forms a ternary (or higher) complex with the plugRNA and the donor as described herein. Cas9 induces a double-strand break. The PBS of the plugRNA anneals to the target site, juxtaposing the donor to the cut site. The donor can be directly ligated by endogenous or exogenously provided DNA ligase (FIGS. 10A and 10B). Alternatively, the donor can be incorporated by the cooperative action of DNA ligase and homology-based mechanisms (homologous recombination, single-strand annealing [SSA], microhomology-mediated end joining [MMEJ], synthesis-dependent single-strand annealing [SDSA], DNA- or RNA-templated DNA repair). Once the donor is incorporated into the target strand, endogenous cellular machinery can degrade the intermediate, resulting in the insertion of the desired sequence into the target DNA.

[0251] Example 5: Design of plugRNA and donor FIG. 9 shows the general design of a plugRNA, including the donor and associated elements. The plugRNA consists of a guide sequence ("specific sequence-targeting spacer RNA") (1), followed by a gRNA scaffold to which the Cas9 protein can bind and activate. The 3' end of the spacer-gRNA scaffold extends into a polynucleotide. This polynucleotide (made from DNA, RNA, and combinations thereof modified by various chemical methods) consists of two elements: a first hybridization sequence ("landing pad") (2) and a primer binding sequence (3) that hybridizes to a sequence upstream of the cleavage induced by the Cas protein. The nucleotides immediately proximal to the cleavage (i.e., the junction between the first hybridization sequence and the primer binding sequence) consist of DNA.

[0252] The first hybridization sequence hybridizes to the second hybridization sequence ("hybridization sequence") (4) of the donor, juxtaposing the 5' end of the donor to the cleavage site. In this configuration, the three polynucleotides become nicked nucleic acids that are excellent substrates for DNA ligase.

[0253] The hybridization sequence can be phosphorylated, adenylated, covalently attached to a protein, or chemically modified in different ways. The donor further comprises a sequence of interest (the "insertion sequence") (5) and a homologous sequence (6) that is homologous to the sequence adjacent to the cleavage site. The homologous sequence is used to participate in homology-based mechanisms (HR, SSA, or MMEJ), potentially improving the editing efficiency.

[0254] Both the donor and the plugRNA can contain additional elements, can omit some elements, or can be split into multiple oligonucleotides (see FIG. 11 and Example 7). For clarity, base pairs between the donor and the plugRNA are not shown.

[0255] Example 6: A method for generating precise insertions by ligase activity FIG. 10 shows a general scheme for knock-in nucleotide-guided ("KING") editing. Cas proteins guided by plugRNA can induce cleavage at a specific DNA sequence. A primer binding sequence ("PBS") hybridizes to the cleavage site, juxtaposing the donor with the cleavage site. The donor can be directly ligated at both the 5' and 3' ends, or ligated at one end (shown here at 5') and incorporated at the other end by a homology-mediated mechanism (HR, SDSA, SSA, alt-EJ, etc.). The resulting chimeric molecule is converted to dsDNA, and the desired sequence is incorporated at the target site.

[0256] Example 7: Examples of different plugRNA:donor configurations FIG. 11 shows part of a scheme for performing KING editing. FIG. 11(A) shows a donor containing all three elements: a second hybridization sequence, a sequence of interest, and a homologous sequence. FIG. 11(B) shows a donor without a homologous sequence. FIG. 11(C) shows a donor without a second hybridization sequence. FIG. 11(D) shows a standard donor in which a sequence complementary to the sequence of interest is hybridized to form a double-stranded region of the sequence of interest with overhangs at both 5' and 3'. This configuration is referred to as an "overhang". FIG. 11(E) shows a donor like FIG. 11(A), but with the plugRNA composed of two nucleic acids. The sgRNA has a sequence of about 30 nt in length added to the 3' end. This 3' sequence can pair with a complementary polynucleotide, followed by a first hybridization sequence and a primer binding sequence. This configuration is referred to as a "split plugRNA" or "split system." Figure 11(F) shows a configuration like Figure 11(A), but the hybridization sequence is shorter, creating a gap between the 3' end of the primed target site and the 5' end of the donor. This configuration is referred to as "gap." Figure 11(G) is the same as Figure 11(A), but the donor has an additional nucleotide at the 5' end, creating a flap that in some embodiments may engage FEN1 and other repair mechanisms. This configuration is referred to as "flap." Figure 11(H) is the same as Figure 11(B), but a sequence complementary to the sequence of interest is annealed to create a blunt end at the 3' end of the donor. This configuration is referred to as "blunt." In Figure 11(I), the donor is split into two polynucleotides. The first donor strand is composed of the second hybridization sequence and the sequence of interest. The second sequence is composed of a homologous sequence and a sequence complementary to the sequence of interest. This configuration is called a "bridge." In Figure 11(J), a homologous sequence is embedded in the plugRNA immediately downstream of the gRNA scaffold. This homologous sequence anneals to the 3' side of the Cas-induced cut.The donor is composed of a sequence of interest flanked by two hybridization sequences: a second hybridization sequence and an additional hybridization sequence, where the second hybridization sequence is complementary to the first hybridization sequence and the additional hybridization sequence is complementary to the region adjacent to the homologous sequence. This configuration places both the 5' and 3' sides of the donor adjacent to the double-stranded break. This configuration is referred to as "dual hybridization."

[0257] Example 8: Proof of concept in vitro assay Materials and Methods HEX-labeled DNA oligos were annealed with 200 nM of complementary sequences in annealing buffer (10 mM Tris, 50 mM NaCl at pH 7.5) by denaturing at 95 °C for 2 min, then ramping steadily to 4 °C at 0.1 °C / sec. The plugRNA was mixed with the donor oligos (final concentration 1 μM in annealing buffer) in an equimolar ratio and annealed as described above. Cas9 nuclease was dissolved in reaction buffer (1X T4 DNA ligase buffer, NEB) in equimolar amounts and mixed (final concentration 150 nM). The Cas9-plugRNA-donor complex was assembled by incubating at ambient temperature for 10 min. The fluorescent double-stranded DNA substrate was added to the assembled RNP (final concentration of 10 nM). The reaction was incubated at 37 °C for 10 min to cleave the target DNA. After incubation, 200 U of T4 DNA ligase was added and the reaction was allowed to continue for 1 hour at 37° C. The reaction was terminated by adding stop solution (final concentrations: 0.1 mg / ml proteinase K, 0.1% SDS, 12.5 mM EDTA) and incubated for 30 minutes at 56° C. The reaction products were dissolved in HiDi formamide using a GeneScan LIZ500, denatured at 95° C., and then separated by capillary electrophoresis.

[0258] Figure 12A shows the layout of the assay. Figures 12B and 12C show the quantification of the in vitro assay in which plugRNA was combined with donors containing first and second hybridization sequences of different lengths, respectively. Two parameters were calculated: % ligation product, calculated as the percentage of total DNA detected in the assay that corresponds to ligated DNA; and % ligation efficiency, calculated as the percentage of cleaved DNA that was ligated. Figure 12B shows that ligation products are generated frequently and reach maximum amounts when the plugRNA contains a 20 bp landing pad and a 15 bp hybridization sequence. When the plugRNA does not contain a landing pad, no ligation is observed, indicating the need to anneal the plugRNA to the donor DNA. Figure 12C shows that ligation is highly efficient, regardless of the combination, reaching a ligation efficiency of >90%.

[0259] Example 9. In vitro ligation using HeLa nuclear extract Materials and Methods HEX-labeled DNA oligos were annealed with 200 nM of complementary sequences in annealing buffer (10 mM Tris, 50 mM NaCl, pH 7.5) by denaturing at 95°C for 2 min, followed by a constant ramp down to 4°C at 0.1°C / sec. The plugRNA was mixed with the donor oligos (final concentration 1 μM in annealing buffer) in an equimolar ratio and annealed as described above. Cas9 nuclease was dissolved in equimolar amounts in reaction buffer (50 mM Tris-HCl, pH 7.5, 0.5 mM magnesium acetate, 60 mM potassium acetate, and 0.1 mg / ml BSA) and mixed (final concentration 150 nM). The Cas9-plugRNA-donor complex was assembled by incubation at ambient temperature for 10 min. A fluorescent double-stranded DNA substrate was added to the assembled RNP (final concentration of 10 nM). The reaction was incubated at 37°C for 10 minutes to cleave the target DNA. During the incubation, HeLa nuclear extract (Ipracell) was premixed with the dNTP and rNTP mix and incubated at room temperature for 10 minutes. After incubation, the premixed HeLa nuclear extract was added to the RNP / substrate mix (2.5 mg / ml HeLa nuclear extract, 5 mM ATP, 0.2 mM CTP, 0.2 mM GTP, 0.2 mM UTP, 0.2 mM dATP, 0.2 mM dCTP, 0.2 mM dGTP, 0.2 mM dUTP). The reaction was incubated for an additional hour at 37°C. The reaction was terminated by adding stop solution (final concentrations: 0.1 mg / ml proteinase K, 0.1% SDS, 12.5 mM EDTA) and incubated at 37°C for 30 minutes. Reaction products were dissolved in HiDi formamide using a GeneScan LIZ500, denatured at 95°C, and then separated by capillary electrophoresis.

[0260] Figure 13A represents the layout of the experiment. HeLa nuclear extracts are widely used in biochemical studies because they retain the activity of most proteins, and can be used to reconstitute a variety of complex processes, including replication, repair, and transcription. In addition to using viral recombinant ligase, HeLa nuclear extracts can provide insight into the feasibility of KING editing using enzyme activities present in human cells.

[0261] Figures 13B and 13C show the quantification of the in vitro assay with HeLa nuclear extract. As in Figure 12 and Example 8, both the abundance of the product (as a percentage of the total DNA corresponding to the ligation product) and the efficiency of ligation (as a percentage of the cleaved DNA converted to the ligated product) were quantified. The heatmap of the results in Figures 13B and 13C demonstrates that KING editing is efficient. It is observed that as the size of the first hybridization region ("landing pad") and the second hybridization region ("hybridization sequence") increases, more ligation products are obtained. It is also observed that ligation is exceptionally efficient when using HeLa nuclear extract.

[0262] Example 10. Reconstitution of KING editing using nuclear extracts Materials and Methods The plugRNA was mixed with the donor oligo (final concentration 1 μM in annealing buffer) in an equimolar ratio and annealed by denaturing at 95 °C for 2 min and then decreasing steadily to 4 °C at 0.1 °C / s. Cas9 nuclease was dissolved in equimolar amounts in reaction buffer (50 mM Tris-HCl (pH 7.5), 0.5 mM magnesium acetate, 60 mM potassium acetate, and 0.1 mg / ml BSA) and mixed (final concentration 150 nM). The Cas9-plugRNA-donor complex was assembled by incubating at ambient temperature for 10 min. To the assembled RNP, a plasmid containing the target AAVS1 sequence was added (final concentration of 10 nM). To cleave the target DNA, the reaction was incubated at 37 °C for 10 min. During the incubation, HeLa nuclear extract (Ipracell) was premixed with the dNTP and rNTP mix and incubated at room temperature for 10 min. After incubation, the premixed HeLa nuclear extract was added to the RNP / substrate mixture (2.5 mg / ml HeLa nuclear extract, 5 mM ATP, 0.2 mM CTP, 0.2 mM GTP, 0.2 mM UTP, 0.2 mM dATP, 0.2 mM dCTP, 0.2 mM dGTP, 0.2 mM dUTP). The reaction was incubated for an additional hour at 37°C. Half a microliter of the reaction was used as a template in a PCR reaction to amplify the target AAVS1 sequence. PCR products were separated using capillary electrophoresis or subjected to next generation sequencing using a NextSeq500. Sequencing results were aligned to a reference sequence and editing was analyzed by RIMA (see, e.g., Taheri-Ghahfarokhi et al., PMID;30032200).

[0263] Figure 14A shows the layout of the procedure. Figure 14B shows a representative result after NGS. The top 10 insertion variants detected at the recovered AAVS1 target site are shown. The top variants with >75% edited reads corresponded to the predicted desired sequence (hybridization sequence [dot] and insertion [crosshatch]). Truncated insertions were also detected at a much lower frequency. Insertions of the desired sequence with errors were also detected at a frequency lower than 0.5%. These results indicate that the entire KING reaction can be reconstituted and results in the insertion of the desired sequence at the target site with high accuracy and high fidelity.

[0264] Example 11. Insertion efficiency in KING assays reconstituted with various substrates Materials and Methods The plugRNA was mixed with the donor oligo (final concentration 1 μM in annealing buffer) in an equimolar ratio and annealed by denaturing at 95 °C for 2 min and then decreasing steadily to 4 °C at 0.1 °C / s. Cas9 nuclease was dissolved in equimolar amounts in reaction buffer (50 mM Tris-HCl (pH 7.5), 0.5 mM magnesium acetate, 60 mM potassium acetate, and 0.1 mg / ml BSA) and mixed (final concentration 150 nM). The Cas9-plugRNA-donor complex was assembled by incubating at ambient temperature for 10 min. To the assembled RNP, a plasmid containing the target AAVS1 sequence was added (final concentration of 10 nM). To cleave the target DNA, the reaction was incubated at 37 °C for 10 min. During the incubation, HeLa nuclear extract (Ipracell) was premixed with the dNTP and rNTP mix and incubated at room temperature for 10 min. After incubation, the premixed HeLa nuclear extract was added to the RNP / substrate mixture (2.5 mg / ml HeLa nuclear extract, 5 mM ATP, 0.2 mM CTP, 0.2 mM GTP, 0.2 mM UTP, 0.2 mM dATP, 0.2 mM dCTP, 0.2 mM dGTP, 0.2 mM dUTP). The reaction was incubated for an additional hour at 37°C. Half a microliter of the reaction was used as a template in a PCR reaction to amplify the target AAVS1 sequence. PCR products were separated using capillary electrophoresis or subjected to next generation sequencing using a NextSeq500. Sequencing results were aligned to a reference sequence and editing was analyzed by RIMA (PMID; 30032200).

[0265] Figure 15 shows the frequency of desired insertion (assayed by NGS) using different formats (see Figure 11). As shown in Figure 15, the presence of hybridization and homologous sequences and / or double-stranded donors increased the frequency of accurate KING editing. PlugRNA:donor complexes with different compositions of these elements could produce insertions at various levels.

[0266] Example 12. Optimization of KING editing components for precise insertion In this example, various KING editing components were tested for their ability to generate more precise insertions (see Figure 16A).

[0267] method: FugeneHD was used to transfect HEK293T cells with a plasmid inducing the expression of Cas9. After 24 hours, plugRNA and ssDNA donors were assembled using the following conditions: denaturation at 95°C for 2 minutes, followed by a constant ramp down to 4°C at a rate of 0.1°C / sec. RNAiMAX was used to transfect cells with 2 pmol of assembled plugRNA and ssDNA donors. Different lengths of the first hybridization region for the plugRNA ("landing pad") and different lengths of the second hybridization region for the ssDNA donor ("hybridization sequence") and homologous sequence of the ssDNA donor were tested, targeting various AAVS sites. Genomic DNA was harvested from transfected cells after 48 hours, amplified, and analyzed by ILLUMINA next-generation sequencing.

[0268] result: It was observed that the landing pad, homology sequence, and hybridization sequence were used to introduce precise insertions by KING editing using ssDNA donors (see Figure 16B). The intended precise insertions were observed using plugRNA1.2 (+AAVS6), plugRNA1.4 (+AAVS7), and plugRNA1.5 (+AAVS7 or +AAVS8).

[0269] Example 13. KING editing with ssDNA donor In this example, ssDNA donors and homologous sequences of various lengths for KING editing were tested.

[0270] method: FugeneHD was used to transfect HEK293T cells with a plasmid that induces the expression of Cas9. After 24 hours, plugRNA and ssDNA donors were assembled using the following conditions: denaturation at 95°C for 2 minutes, followed by a constant ramp to 4°C at a rate of 0.1°C / sec. RNAiMAX was used to transfect cells with 2 pmol of assembled plugRNA and ssDNA donors. Different lengths of homologous sequences (Figure 17A) were tested against the ssDNA donor or ssDNA with gaps or flaps. Genomic DNA was harvested from transfected cells after 48 hours, amplified, and analyzed by ILLUMINA next-generation sequencing.

[0271] result: It was observed that the absolute and relative precise editing efficiency of KING was further improved by optimizing the length of the homologous sequence of the ssDNA donor. See FIG. 17B. In plugRNA1.5, the highest absolute precise editing efficiency of KING editing was observed when a ssDNA donor containing a 10bp or 50bp homologous sequence was used. In plugRNA1.4, the highest absolute precise editing efficiency of KING editing was observed when a ssDNA donor containing a 50bp homologous sequence was used. FIG. 17B further demonstrated that KING editing using a donor containing a gap or a flap allows for the introduction of precise insertions.

[0272] Example 14. KING editing with dsDNA donors In this example, KING editing was tested with dsDNA donors of various configurations, as illustrated in FIG. 18A.

[0273] method: FugeneHD was used to transfect HEK293T cells with a plasmid inducing the expression of Cas9. After 24 hours, plugRNA and ssDNA donors were assembled using the following conditions: denaturation at 95C for 2 minutes, followed by a constant ramp to 4C at a rate of 0.1°C / sec. RNAiMAX was used to transfect cells with 2pmol of assembled plugRNA and ssDNA / dsDNA donors. Different designs of dsDNA or control ssDNA (Figure 18A) were tested. Genomic DNA was harvested from transfected cells after 48 hours, amplified, and analyzed by ILLUMINA next-generation sequencing.

[0274] result: It was observed that precise insertions were introduced by KING editing with blunt, overhang, or bridge designed dsDNA donors. The absolute and relative precise editing efficiencies were further improved by modifying the phosphorothioate linkages of the donor oligos when using blunt and overhang designed donors (Figure 18B).

[0275] Example 15. KING editing with Cas9(H840A) nickase In this example, KING editing was examined by Cas9(H840A) nickase.

[0276] method: HEK293T cells were transfected with a plasmid that induces the expression of SpCas9 (H840A) nickase using FugeneHD. After 24 hours, the plugRNA and ssDNA donor were assembled using the following conditions: denaturation at 95C for 2 minutes, followed by a constant ramp to 4C at a rate of 0.1°C / sec. Cells were transfected with 2pmol of assembled plugRNA and ssDNA donor using RNAiMAX. Different designs of ssDNA (Figure 19A) were tested. Genomic DNA was harvested from transfected cells after 48 hours, amplified, and analyzed by ILLUMINA next-generation sequencing.

[0277] result: It was observed that precise insertions were introduced by KING editing using SpCas9(H840A) nickase: A relative precise editing efficiency of up to 36% was achieved using SpCas9(H840A) nickase (Figure 19B), which was higher than that achieved with SpCas9 nuclease (<5%).

[0278] Example 16. KING editing upon treatment with DNA-dependent protein kinase (DNA-PK) inhibitors In this example, KING editing was examined with a DNA-PK inhibitor. DNA-PK is part of the non-homologous end joining (NHEJ) repair pathway.

[0279] method: Prior to transfection, HEK293T cells were pretreated with 1 μM of DNAPK inhibitor (AZD7648) for 1 hour. DMSO was used as a mock control. After 1 hour, HEK293T cells were transfected with plasmids inducing the expression of Cas9 and PEn using FugeneHD reagent. After 24 hours, cells were transfected with 2 pmol of synthetic RNA using RNAiMAX. Synthetic plugRNA was annealed to the donor sequence by denaturing at 95° C. for 2 minutes and then slowly cooled to 4° C. Several different plugRNA and donor configurations were tested (see FIG. 20A).

[0280] Cells were harvested 48 hours after RNA transfection, and the AAVS1 locus was amplified by PCR and sequenced using the Illumina platform. Data were analyzed by aligning reads to a reference sequence, and editing frequency was analyzed by RIMA (PMID:30032200).

[0281] The results show that Cas9 and PEn in combination with synthetic oligonucleotides achieved KING insertion, and pretreatment with a DNAPK inhibitor improved precise editing in HEK293T cells.

[0282] Figure 20B shows the absolute desired editing percentages of different combinations of transfected plugRNA:donor. Pretreatment with DNA-PK inhibitor improved the absolute desired editing (light grey) compared to the DMSO control (dark grey).

[0283] Example 17. KING editing in the presence of co-expressed ligase In this example, KING editing was performed in the presence of a ligase or a ligase adaptor protein (also referred to as a "ligase recruiting protein").

[0284] method: HEK293T cells were transfected with plasmid(s) driving the expression of Cas9 and ligase / ligase adaptor using FugeneHD reagent. A panel of three different donor configurations (Figure 21A), each with nine different ligase conditions, was tested: ligases of bacterial origin (T4 DNA ligase, LigD and PBCV1); ligases of human origin (Lig1, Lig3α, Lig4); and human DNA repair proteins (XRCC1, XRCC4, PCNA). Cas9 and ligase were expressed from separate plasmids.

[0285] Cells were harvested 48 hours after RNA transfection, and the AAVS1 locus was amplified by PCR and sequenced using the Illumina platform. Data were analyzed by aligning reads to a reference sequence, and editing frequency was analyzed by RIMA (PMID:30032200).

[0286] Figure 21B shows the absolute desired editing percentages for combinations of co-expressed Cas9 and different ligases. The results show that KING insertion was achieved by Cas9 in the presence of ligase.

[0287] Example 18. Summary of compositions of different donors and their potency in in vitro assays and in cells The table in Figure 22 summarizes the configurations of the plugRNA:donor complex as shown in the Examples herein and the editing efficiency of each configuration, for example, "-" means that no KING editing was observed, and "++++" means that editing was highly efficient.

[0288] Example 19. Dual Hybridization Design In this example, a dual hybridization configuration (Figure 23A) was tested.

[0289] method: HEK293T cells were transfected with a plasmid that induces the expression of Cas9 using FugeneHD. After 24 hours, the cells were transfected with 2 pmol of synthetic RNA using RNAiMAX. The synthetic plugRNA was annealed to the donor sequence by denaturing at 95°C for 2 minutes and then slowly cooled to 4°C.

[0290] Cells were harvested 48 hours after transfection and the AAVS1 locus was amplified by PCR and sequenced using the Illumina platform. Data were analyzed by aligning reads to a reference sequence and editing frequency was analyzed by RIMA (PMID:30032200).

[0291] result: Prime edit insertions were observed. The results in Figure 23B show the frequency of all edits and precise edit (PE) insertions.

Claims

1. 1. A polynucleotide comprising: (i) an RNA guide sequence; (ii) a Cas binding region; and (iii) a DNA template sequence, wherein the DNA template sequence is at a 3' end of the polynucleotide.

2. 2. The polynucleotide of claim 1, wherein the DNA template sequence comprises modified nucleotides, non-B-type DNA structures, DNA polymerase recruiting moieties, DNA ligase recruiting moieties, or combinations thereof.

3. 3. The polynucleotide of claim 2, wherein the modified nucleotide comprises an abasic site, a covalent linker, a heterologous nucleic 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.

4. The polynucleotide of claim 3 , wherein the covalent linker comprises triethylene glycol (TEG).

5. The polynucleotide of claim 3, wherein the DNA damage comprises 8-oxoguanine, thymine-glycol, or a combination thereof.

6. 4. The polynucleotide of claim 3, wherein the DNA photoproduct comprises a cyclobutane pyrimidine dimer (CPD), a pyrimidine (6-4) pyrimidone photoproduct, or a combination thereof.

7. The polynucleotide of claim 3 , wherein the modified deoxyribonucleoside comprises deoxyuridine.

8. 4. The polynucleotide of claim 3, wherein the methylated nucleotide comprises 5-hydroxymethylcytosine, 5-methylcytosine, or a combination thereof.

9. 3. The polynucleotide of claim 2, wherein the non-B-type DNA structure comprises a hairpin, a cruciform, a Z-DNA, an H-DNA (triple helix DNA), a G-quadruplex DNA (quadruplex DNA), a slip DNA, a sticky DNA, or a combination thereof.

10. The polynucleotide of claim 2 , wherein the DNA polymerase recruitment portion comprises a DNA polymerase recruitment protein linked to the DNA template sequence.

11. 11. The polynucleotide of claim 10, wherein the DNA polymerase recruitment protein comprises proliferating cell nuclear antigen (PCNA), single stranded DNA binding protein (SSBP), tumor necrosis factor alpha induced protein (TNFAIP), polymerase delta interacting protein (PolDIP), X-ray repair cross-complementing protein (XRCC), 5-hydroxymethylcytosine-binding embryonic stem cell specific (HMCES) protein, RAD1, RAD9, HUS1, or a combination thereof.

12. The polynucleotide of claim 2, wherein the DNA ligase recruitment portion comprises a 5' adenylation of the DNA template sequence.

13. 1. A polynucleotide comprising: (i) an RNA guide sequence; (ii) a Cas binding region; and (iii) a DNA template sequence, wherein the DNA template sequence is at a 3' end of the polynucleotide, and wherein the DNA template sequence comprises phosphorothioate linkages.

14. The polynucleotide of any one of claims 1 to 13, wherein the DNA template sequence comprises a primer binding sequence and a sequence of interest.

15. 15. The polynucleotide of claim 14, wherein the Cas binding region comprises RNA, or the Cas binding region comprises a combination of RNA and DNA.

16. 16. The polynucleotide of Claim 15, wherein the Cas binding region comprises a tracrRNA.

17. The polynucleotide of claim 15, wherein the Cas binding region is capable of hybridizing to a tracrRNA.

18. 18. The polynucleotide of Claim 17, wherein the tracrRNA is capable of binding to a Cas nuclease.

19. 19. The polynucleotide of claim 18, wherein the Cas nuclease is Cas9 or Cas12a.

20. The polynucleotide of claim 18, wherein the Cas nuclease is a type II-B Cas.

21. 18. The polynucleotide of Claim 17, wherein the tracrRNA is capable of binding to Cas nickase.

22. 22. The polynucleotide of claim 21, wherein the Cas nickase is a Cas9 nickase, a Cas12a nickase, or a type II-B Cas nickase.

23. 23. The polynucleotide of claim 22, wherein the DNA template sequence is from about 8 to about 10,000 nucleotides in length.

24. 24. The polynucleotide of claim 23, wherein the primer binding sequence is from about 4 to about 300 nucleotides in length.

25. 25. The polynucleotide of claim 24, wherein the sequence of interest is from about 4 to about 100 nucleotides in length.

26. 26. The polynucleotide of claim 25, wherein the RNA guide sequence is about 15 to about 25 nucleotides in length.

27. 27. The polynucleotide of Claim 26, further comprising a spacer located between the Cas binding region and the DNA template sequence.

28. 28. The polynucleotide of claim 27, wherein the spacer comprises a stop sequence for a DNA polymerase.

29. 30. The polynucleotide of claim 28, wherein the spacer comprises two or more termination sequences.

30. 30. The polynucleotide of claim 29, wherein the termination sequence is comprised of a secondary structure.

31. 31. The polynucleotide of claim 30, wherein the secondary structure is comprised of a stem loop.

32. 32. The polynucleotide of claim 31, wherein the spacer is from about 10 to about 200 nucleotides in length.

33. A cell comprising the polynucleotide of claim 32.

34. Cas nuclease or Cas nickase; (i) a guide sequence, (ii) a Cas binding region, and (iii) a DNA template sequence, wherein the DNA template sequence is at a 3' end of the polynucleotide; A composition comprising:

35. Cas nuclease or Cas nickase; (i) a first polynucleotide comprising a guide sequence, (ii) a Cas binding region, and (iii) a first hybridization region, wherein the first hybridization region is at a 3' end of the first polynucleotide; (i) a second polynucleotide comprising a second hybridization region that is complementary to the first hybridization region, and (ii) a DNA template sequence; A composition comprising:

36. Cas nuclease or Cas nickase; (i) a first polynucleotide comprising a guide sequence; and (ii) a first hybridization region, wherein the first hybridization region is at a 3' end of the first polynucleotide; (i) a second hybridization region complementary to the first hybridization region, (ii) a Cas binding region, and (iii) a second polynucleotide comprising a DNA template sequence; A composition comprising:

37. The composition of any one of claims 34 to 36, wherein the DNA template sequence comprises a primer binding sequence and a sequence of interest.

38. 38. The composition of claim 37, wherein the DNA template sequence comprises modified nucleotides, non-B-type DNA structures, DNA polymerase recruiting moieties, DNA ligase recruiting moieties, or combinations thereof.

39. 39. The composition of claim 38, wherein the modified nucleotide comprises an abasic site, a covalent linker, a heterologous nucleic 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.

40. 40. The composition of claim 39, wherein the modified nucleotide comprises a phosphorothioate linkage.

41. 40. The composition of claim 39, wherein the covalent linker comprises triethylene glycol (TEG).

42. 40. The composition of claim 39, wherein the DNA damage comprises 8-oxoguanine, thymine-glycol, or a combination thereof.

43. 40. The composition of claim 39, wherein the DNA photoproduct comprises a cyclobutane pyrimidine dimer (CPD), a pyrimidine (6-4) pyrimidone photoproduct, or a combination thereof.

44. 40. The composition of claim 39, wherein the deoxyribonucleoside comprises deoxyuridine.

45. 40. The composition of claim 39, wherein the methylated nucleotide comprises 5-hydroxymethylcytosine, 5-methylcytosine, or a combination thereof.

46. 39. The composition of claim 38, wherein the non-B-type DNA structure comprises a hairpin, a cruciform, a Z-DNA, an H-DNA (triple helix DNA), a G-quadruplex DNA (quadruplex DNA), a slip DNA, a sticky DNA, or a combination thereof.

47. 40. The composition of claim 38, wherein the DNA polymerase recruitment moiety is comprised of a DNA polymerase recruitment protein linked to the DNA template sequence.

48. 48. The composition of claim 47, wherein the DNA polymerase recruitment protein comprises proliferating cell nuclear antigen (PCNA), single stranded DNA binding protein (SSBP), tumor necrosis factor alpha induced protein (TNFAIP), polymerase delta interacting protein (PolDIP), X-ray repair cross-complementing protein (XRCC), 5-hydroxymethylcytosine-binding embryonic stem cell specific (HMCES) protein, RAD1, RAD9, HUS1, or a combination thereof.

49. 39. The composition of claim 38, wherein the DNA ligase recruitment portion consists of 5' adenylation of the DNA template sequence.

50. 50. The composition of claim 49, wherein the guide sequence comprises RNA, or wherein the guide sequence comprises a combination of RNA and DNA.

51. 51. The composition of claim 50, wherein the Cas binding region comprises RNA, or the Cas binding region comprises a combination of RNA and DNA.

52. 52. The composition of claim 51 , wherein the Cas binding region comprises a tracrRNA.

53. 52. The composition of claim 51 , wherein the Cas binding region is capable of hybridizing to a tracrRNA, and the composition further comprises a tracrRNA.

54. 54. The composition of claim 53, wherein the tracrRNA is capable of binding to the Cas nuclease or Cas nickase.

55. 55. The composition of claim 54, wherein the composition comprises a Cas nuclease.

56. 56. The composition of claim 55, wherein the Cas nuclease is Cas9 or Cas12a.

57. 56. The composition of claim 55, wherein the composition comprises a Cas nuclease, and the Cas nuclease is a type II-B Cas.

58. 55. The composition of claim 54, wherein the composition comprises a Cas nickase.

59. 59. The composition of claim 58, wherein the Cas nickase is a Cas9 nickase, a Cas12a nickase, or a type II-B Cas nickase.

60. 60. The composition of claim 59, wherein the DNA template sequence is from about 8 to about 500 nucleotides in length.

61. 61. The composition of claim 60, wherein the primer binding sequence is from about 4 to about 30 nucleotides in length.

62. 62. The composition of claim 61, wherein the sequence of interest is from about 4 to about 100 nucleotides in length.

63. 63. The composition of claim 62, wherein the guide sequence is about 15 to about 25 nucleotides in length.

64. 64. The composition of claim 63, wherein the first hybridization region and the second hybridization region are RNA.

65. 64. The composition of claim 63, wherein the first hybridization region and the second hybridization region are single-stranded DNA.

66. 64. The composition of claim 63, wherein the first hybridization region is RNA and the second hybridization region is single-stranded DNA, or the first hybridization region is single-stranded DNA and the second hybridization region is RNA.

67. 67. The composition of claim 66, wherein the first hybridization region is from about 4 to about 5000 nucleotides in length.

68. 67. The composition of claim 66, wherein the second hybridization region is from about 4 to about 5000 nucleotides in length.

69. 64. The composition of claim 63, wherein the polynucleotide further comprises a spacer located 5' to the DNA template sequence.

70. 69. The composition of claim 68, wherein the second polynucleotide further comprises a spacer located 5' to the DNA template sequence.

71. 71. The composition of claim 70, wherein the spacer comprises a stop sequence for a DNA polymerase.

72. 72. The composition of claim 71, wherein the spacer comprises two or more termination sequences.

73. 73. The composition of claim 72, wherein the termination sequence is comprised of a secondary structure.

74. 74. The composition of claim 73, wherein the secondary structure is composed of a stem loop.

75. 75. The composition of claim 74, wherein the spacer is from about 10 to about 200 nucleotides in length.

76. 76. The composition of claim 75, wherein the Cas nuclease or the Cas nickase is fused to a DNA polymerase recruitment protein.

77. 77. The composition of claim 76, wherein the DNA polymerase recruitment protein comprises proliferating cell nuclear antigen (PCNA), single stranded DNA binding protein (SSBP), tumor necrosis factor alpha induced protein (TNFAIP), polymerase delta interacting protein (PolDIP), X-ray repair cross-complementing protein (XRCC), 5-hydroxymethylcytosine binding, embryonic stem cell specific (HMCES) protein, RAD1, RAD9, HUS1, or a combination thereof.

78. Cas nuclease or Cas nickase; (i) a guide sequence, (ii) a Cas binding region, (iii) a first hybridization region, and (iv) a primer binding sequence, wherein the primer binding sequence is at a 3' end of the first polynucleotide; (i) a second hybridization region complementary to the first hybridization region, and (ii) a second polynucleotide comprising a sequence of interest (SOI); A composition comprising:

79. Cas nuclease or Cas nickase; (i) a first polynucleotide comprising a guide sequence, (ii) a Cas binding region, and (iii) a first hybridization region, wherein the first hybridization region is at a 3' end of the first polynucleotide; a second polynucleotide comprising: (i) a second hybridization region complementary to the first hybridization region; (ii) a third hybridization region; and (iii) a primer binding sequence, wherein the primer binding sequence is at a 3' end of the second polynucleotide; (i) a fourth hybridization region complementary to the third hybridization region, and (ii) a third polynucleotide comprising a sequence of interest (SOI); A composition comprising:

80. 79. The composition of claim 78, wherein the first polynucleotide and / or the second polynucleotide comprises RNA, DNA, modified nucleotides, or a combination thereof.

81. 81. The composition of claim 80, wherein the first polynucleotide and / or the second polynucleotide further comprises a homologous sequence.

82. 82. The composition of claim 81 , wherein the first polynucleotide further comprises (v) the homologous sequence, and the second polynucleotide comprises the second hybridization region, the SOI, and a further hybridization region that hybridizes to the first polynucleotide, and the second hybridization region and the further hybridization region flank the SOI.

83. 80. The composition of claim 79, wherein any of the first polynucleotide, the second polynucleotide, and / or the third polynucleotide comprises RNA, DNA, modified nucleotides, or a combination thereof.

84. 84. The composition of claim 83, wherein any of the first polynucleotide, the second polynucleotide, and / or the third polynucleotide further comprises a homologous sequence.

85. 84. The composition of claim 83, wherein the modified nucleotide comprises an abasic site, a covalent linker, a heterologous nucleic 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.

86. 86. The composition of claim 85, further comprising an SOI-complementary oligonucleotide comprising a sequence complementary to the SOI.

87. 87. The composition of claim 86, wherein the SOI complementary oligonucleotide further comprises a homologous sequence.

88. 88. The composition of claim 87, wherein the Cas nuclease or the Cas nickase is fused to a DNA polymerase, a DNA polymerase recruitment moiety, a DNA ligase, a DNA ligase recruitment moiety, a DNA binding protein, a DNA repair protein, or a combination thereof.

89. 90. The composition of claim 88, wherein the DNA polymerase recruitment moiety comprises proliferating cell nuclear antigen (PCNA), single stranded DNA binding protein (SSBP), tumor necrosis factor alpha induced protein (TNFAIP), polymerase delta interacting protein (PolDIP), X-ray repair cross-complementing protein (XRCC), 5-hydroxymethylcytosine binding, embryonic stem cell specific (HMCES) protein, RAD1, RAD9, HUS1, or a combination thereof.

90. 90. The composition of claim 88, wherein the DNA ligase comprises T4 DNA ligase, PCBV-1 DNA ligase, LigD, a hitoriase protein, or a combination thereof.

91. 90. The composition of claim 88, wherein the DNA ligase recruitment portion consists of 5' adenylation of the DNA template sequence.

92. 92. The composition of claim 91, wherein the DNA binding protein comprises replication protein A (RPA) or a subunit thereof, a single-stranded DNA binding protein (SSBP), or a combination thereof.

93. 89. The composition of claim 88, wherein the DNA repair protein comprises tyrosyl-DNA phosphodiesterase 1 (TDP1), aprataxin, topoisomerase I, or a combination thereof.

94. 94. The composition of claim 93, wherein the Cas binding region comprises a tracrRNA.

95. 94. The composition of claim 93, wherein the Cas binding region is capable of hybridizing to a tracrRNA, and the composition further comprises a tracrRNA.

96. The composition of claim 95, wherein the tracrRNA is capable of binding to the Cas nuclease or Cas nickase.

97. 97. The composition of claim 96, wherein the composition comprises a Cas nuclease.

98. 98. The composition of claim 97, wherein the Cas nuclease is Cas9 or Cas12a.

99. 98. The composition of claim 97, wherein the Cas nuclease is type II-B Cas.

100. 97. The composition of claim 96, wherein the composition comprises a Cas nickase.

101. The composition of claim 100, wherein the Cas nickase is a Cas9 nickase, a Cas12a nickase, or a type II-B Cas nickase.

102. The composition of claim 101, wherein the sequence of interest is from about 4 to about 100 nucleotides in length.

103. 103. The composition of claim 102, wherein the primer binding sequence is from about 4 to about 30 nucleotides in length.

104. The composition of claim 103, wherein the guide sequence is about 15 to about 25 nucleotides in length.

105. 105. The composition of claim 104, wherein the first hybridization region and / or the second hybridization region are each from about 4 to about 5000 nucleotides in length.

106. A cell comprising the composition of claim 105.

107. 107. The cell of claim 106, further comprising an exogenous DNA polymerase, an exogenous DNA ligase, or both.

108. A fusion protein comprising (i) a Cas nuclease or a Cas nickase, and (ii) a DNA polymerase recruitment protein, a DNA ligase, a DNA ligase recruitment moiety, a DNA binding protein, a DNA repair protein, or a combination thereof.

109. The fusion protein of claim 108, wherein the fusion protein comprises a Cas nuclease.

110. The fusion protein of claim 109, wherein the Cas nuclease is Cas9 or Cas12a.

111. The fusion protein of claim 109, wherein the Cas nuclease is a type II-B Cas.

112. The fusion protein of claim 108, wherein the fusion protein comprises a Cas nickase.

113. The fusion protein of claim 112, wherein the Cas nickase is a Cas9 nickase, a Cas12a nickase, or a type II-B Cas nickase.

114. 114. The fusion protein of any one of claims 108-113, wherein the DNA polymerase recruitment protein comprises proliferating cell nuclear antigen (PCNA), single stranded DNA binding protein (SSBP), tumor necrosis factor alpha induced protein (TNFAIP), polymerase delta interacting protein (PolDIP), X-ray repair cross-complementing protein (XRCC), 5-hydroxymethylcytosine-binding embryonic stem cell specific (HMCES) protein, RAD1, RAD9, HUS1, or a combination thereof.

115. 114. The fusion protein of any one of claims 108 to 113, wherein the DNA ligase comprises T4 DNA ligase, PCBV-1 DNA ligase, LigD, a hitoriase protein, or a combination thereof.

116. A fusion protein according to any one of claims 108 to 113, wherein the DNA ligase recruitment moiety consists of 5' adenylation of the DNA template sequence.

117. The fusion protein of any one of claims 108 to 113, wherein the DNA binding protein comprises replication protein A (RPA) or a subunit thereof, single-stranded DNA binding protein (SSBP), or a combination thereof.

118. The fusion protein of any one of claims 108 to 113, wherein the DNA repair protein comprises tyrosyl-DNA phosphodiesterase 1 (TDP1), aprataxin, topoisomerase I, or a combination thereof.

119. A polynucleotide encoding the fusion protein of claim 108.

120. A vector comprising the polynucleotide of claim 119.

121. A cell comprising the fusion protein of claim 118, the polynucleotide of claim 119, the vector of claim 120, or a combination thereof.

122. The cell of claim 121, further comprising the polynucleotide of claim 1.

123. 81. A method of providing a targeted insertion in a target DNA in a cell, comprising introducing into the cell the composition of claim 77, wherein the guide sequence is capable of hybridizing to the target DNA.

124. 124. The method of claim 123, wherein the method does not include introducing a DNA polymerase into the cell.

125. 125. The method of claim 124, wherein the Cas nuclease generates a double-stranded break in the target DNA and an endogenous DNA polymerase of the cell extends the DNA template sequence.

126. 124. The method of claim 123, wherein the method further comprises introducing an exogenous DNA polymerase into the cell.

127. 127. The method of claim 126, wherein the Cas nuclease creates a double-stranded break in the target DNA and the exogenous DNA polymerase extends the DNA template sequence.

128. 128. The method of claim 127, wherein the DNA template sequence comprises a primer binding sequence and a sequence of interest, and the DNA polymerase synthesizes a DNA strand complementary to the sequence of interest to form a double-stranded sequence comprising the sequence of interest.

129. 129. The method of claim 128, wherein the double-stranded sequence is inserted into the cleaved target DNA.

130. 130. The method of claim 129, wherein the double-stranded sequence is inserted into the cleaved target DNA by non-homologous end joining (NHEJ).

131. 131. The method of claim 130, wherein the double-stranded sequence is inserted into the cleaved target DNA by a DNA ligase.

132. 132. The method of claim 131, wherein the DNA ligase is an endogenous DNA ligase of the cell.

133. 132. The method of claim 131, wherein the DNA ligase is an exogenous DNA ligase.

134. 107. A method of providing a targeted insertion in a target DNA in a cell, comprising introducing into the cell the composition of claim 105, wherein the guide sequence is capable of hybridizing to the target DNA.

135. 135. The method of claim 134, wherein the method does not include introducing a DNA polymerase or a DNA ligase into the cell.

136. The method of claim 135, wherein the Cas nuclease creates a double-stranded break in the target DNA and an endogenous DNA ligase of the cell ligates the sequence of interest to the cleaved target DNA.

137. 135. The method of claim 134, wherein the method further comprises introducing an exogenous DNA ligase into the cell.

138. 138. The method of claim 137, wherein the Cas nuclease creates a double-stranded break in the target DNA and the exogenous DNA ligase ligates the sequence of interest to the cleaved target DNA.

139. 139. The method of claim 138, wherein the target sequence is a single-stranded sequence that is converted to a double-stranded sequence by a DNA repair pathway of the cell.

140. 106. A method of providing a targeted insertion in a target DNA, comprising contacting the target DNA with the composition of claim 105, wherein the guide sequence is capable of hybridizing to the target DNA.

141. 141. The method of claim 140, further comprising contacting the target DNA with a DNA ligase.

142. 142. The method of claim 141, wherein the Cas nuclease creates a double-stranded break in the target DNA and the DNA ligase ligates the sequence of interest to the cleaved target DNA.

143. 143. The method of claim 142, further comprising contacting the target DNA with a DNA polymerase, a protein in a DNA repair pathway, or a combination thereof.

144. Cas nuclease or Cas nickase; (i) a guide sequence, (ii) a Cas binding region, (iii) a first hybridization region, and (iv) a primer binding sequence, wherein the primer binding sequence is at a 3' end of the first polynucleotide; (i) a second hybridization region complementary to the first hybridization region, and (ii) a second polynucleotide comprising a sequence of interest (SOI); (i) a third hybridization region that is partially complementary to the second polynucleotide, and (ii) a third polynucleotide that includes a sequence of interest (SOI); A composition comprising:

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