Compositions and methods for improved site-specific modification

Abstract

The present disclosure provides proteins, compositions, methods, and kits for improved gene editing efficiency. In some embodiments, the present disclosure provides fusion proteins comprising a Cas nuclease and a reverse transcriptase, a DNA polymerase, a DNA ligase, or a combination thereof.

Description

TECHNICAL FIELD

[0001] The present disclosure provides proteins, compositions, methods, and kits for improved gene editing efficiency. In some embodiments, the present disclosure provides a fusion protein comprising a Cas nuclease and a reverse transcriptase, a DNA polymerase, a DNA ligase, or a combination thereof. BACKGROUND ART

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

[0003] Non-Patent Document 1 describes the development of prime editing that utilizes a programmable nickase fused to a reverse transcriptase that can insert short sequences at the cleavage site, wherein the programmable nickase generates single-strand breaks. However, prime editing can only insert short sequences of up to 22 base pairs, 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 via cellular equilibration. PRIOR ART DOCUMENTS NON-PATENT DOCUMENTS

[0004] NON-PATENT DOCUMENT 1 Anzalone et al. (Nature 576:149-157 (2019)) SUMMARY OF THE INVENTION MEANS FOR SOLVING THE PROBLEMS

[0005] In some embodiments, the present disclosure provides a fusion protein comprising (i) a Cas nuclease and (ii) a reverse transcriptase, DNA polymerase, DNA ligase, or a combination thereof, wherein the Cas nuclease is capable of generating double-stranded polynucleotide cleavage.

[0006] In some embodiments, the present disclosure provides a fusion protein comprising (i) a Cas nuclease and (ii) a reverse transcriptase, DNA polymerase, DNA ligase, or a combination thereof, wherein the Cas nuclease is capable of generating double-stranded polynucleotide cleavage.

[0007] In some embodiments, the Cas nuclease is Cas9 or Cas12. In some embodiments, Cas9 is type IIB Cas9. In some embodiments, Cas9 comprises a polypeptide sequence having at least 90% identity with SEQ ID NO: 1.

[0008] In some embodiments, the fusion protein comprises a Cas nuclease and a reverse transcriptase. In some embodiments, the reverse transcriptase is MMLV reverse transcriptase or R2 reverse transcriptase. In some embodiments, the reverse transcriptase comprises a polypeptide sequence having at least 90% identity to one of SEQ ID NOs: 2-3.

[0009] In some embodiments, the fusion protein comprises a Cas nuclease and a DNA polymerase. In some embodiments, the DNA polymerase is phi29 DNA polymerase, T4 DNA polymerase, DNA polymerase mu, DNA polymerase delta, or DNA polymerase epsilon, Rev3, DNA polymerase I, or a Klenow fragment of DNA polymerase I. In some embodiments, the DNA polymerase comprises a polypeptide sequence having at least 90% identity to any one of sequence numbers 4-6.

[0010] In some embodiments, the fusion protein comprises a Cas nuclease and a DNA ligase. In some embodiments, the DNA ligase is a T4 DNA ligase. In some embodiments, the DNA ligase comprises a polypeptide sequence having at least 90% identity to SEQ ID NO: 7.

[0011] In some embodiments, the fusion protein further comprises a DNA-binding or RNA-binding domain. In some embodiments, the DNA-binding domain is a zinc finger DNA-binding domain, a transcription factor, or an adeno-associated virus Rep protein. In some embodiments, the RNA-binding domain is an MS2 coat protein (MCP2). In some embodiments, the RNA-binding domain comprises a KH domain. In some embodiments, the RNA-binding domain is heteronuclear ribonucleoprotein K (hnRNPK). In some embodiments, the DNA-binding domain can bind single-stranded DNA (ssDNA). In some embodiments, the DNA-binding domain is a far upstream element-binding protein (FUBP). In some embodiments, the DNA-binding or RNA-binding domain comprises a polypeptide sequence having at least 90% identity to one of sequence numbers 8-11.

[0012] In some embodiments, the fusion protein further includes a polypeptide linker between (i) and (ii).

[0013] In some embodiments, the fusion protein comprises a polypeptide sequence having at least 90% identity with any one of SEQ ID NOs: 18-26.

[0014] In some embodiments, the present disclosure provides compositions comprising (a) a fusion protein provided herein, and (b) a polynucleotide that forms a complex with the fusion protein and comprises (i) a guide sequence and (ii) a template sequence for a reverse transcriptase, DNA polymerase, or DNA ligase.

[0015] In some embodiments, the polynucleotide comprises RNA. In some embodiments, the guide sequence comprises RNA and the template sequence comprises DNA. In some embodiments, the template sequence comprises a debase site, a triethylene glycol (TEG) linker, or both. In some embodiments, the guide sequence is about 15 to about 20 nucleotides long. In some embodiments, the polynucleotide further comprises tracrRNA. In some embodiments, the composition comprises a second nucleotide comprising tracrRNA.

[0016] In some embodiments, the template sequence includes a primer-binding sequence and a target sequence. In some embodiments, the primer-binding sequence and the target sequence include DNA. In some embodiments, the target sequence includes DNA. In some embodiments, the template sequence is about 25 to about 10,000 nucleotides long. In some embodiments, the primer-binding sequence is about 4 to about 30 nucleotides long. In some embodiments, the target sequence is about 5 to about 9,800 nucleotides long.

[0017] In some embodiments, the polynucleotide includes a spacer between the guide sequence and the template sequence. In some embodiments, the spacer is about 10 to about 200 nucleotides long. In some embodiments, the spacer includes a stop sequence for reverse transcriptase or DNA polymerase. In some embodiments, the spacer includes two or more stop sequences. In some embodiments, the stop sequence includes a secondary structure. In some embodiments, the secondary structure is a hairpin loop.

[0018] In some embodiments, the Disclosure provides compositions comprising (a) a fusion protein provided herein, (b) a guide polynucleotide that forms a complex with the fusion protein and includes a guide sequence, and (c) a template polynucleotide that includes a template sequence for a reverse transcriptase, DNA polymerase, or DNA ligase.

[0019] In some embodiments, the guide polynucleotide is RNA. In some embodiments, the template polynucleotide comprises RNA. In some embodiments, the template sequence comprises DNA. In some embodiments, the template sequence comprises an abasic site, a triethylene glycol (TEG) linker, or both. In some embodiments, the guide sequence is about 15 to about 20 nucleotides in length. In some embodiments, the guide polynucleotide further comprises tracrRNA. In some embodiments, the composition further comprises a third polynucleotide comprising tracrRNA.

[0020] In some embodiments, the template sequence is about 25 to about 10000 nucleotides in length. In some embodiments, the template sequence comprises a sequence of interest. In some embodiments, the sequence of interest is about 5 nucleotides to about 9800 nucleotides in length. In some embodiments, the sequence of interest comprises DNA.

[0021] In some embodiments, the template polynucleotide further comprises a primer-binding sequence. In some embodiments, the primer-binding sequence is about 10 to about 20 nucleotides in length. In some embodiments, the primer-binding sequence and the sequence of interest comprise DNA.

[0022] In some embodiments, the template polynucleotide further comprises a stop sequence for reverse transcriptase or DNA polymerase. In some embodiments, the template polynucleotide comprises two or more stop sequences. In some embodiments, the stop sequence comprises a secondary structure. In some embodiments, the secondary structure is a hairpin loop.

[0023] In some embodiments, the template polynucleotide comprises an adeno-associated virus (AAV) vector comprising the sequence of interest.

[0024] In some embodiments, the present disclosure provides a polynucleotide encoding the fusion protein provided herein. In some embodiments, the present disclosure provides a vector comprising the polynucleotide encoding the fusion protein provided herein.

[0025] In some embodiments, the present disclosure provides a cell comprising the fusion protein provided herein. In some embodiments, the present disclosure provides a cell comprising the polynucleotide encoding the fusion protein provided herein, or the vector provided herein.

[0026] In some embodiments, the present disclosure provides a cell comprising the composition provided herein.

[0027] In some embodiments, the present disclosure provides a method for providing a site-specific modification at a target sequence in a target polynucleotide, the method comprising contacting the target polynucleotide with the composition provided herein.

[0028] In some embodiments, the target polynucleotide is DNA. In some embodiments, the guide sequence is capable of hybridizing to the target sequence. In some embodiments, the contacting is performed under conditions sufficient for a Cas nuclease to generate double-stranded polynucleotide cleavage at the target sequence.

[0029] In some embodiments, the template sequence comprises a sequence of interest. In some embodiments, the template sequence comprises a primer-binding sequence capable of hybridizing to the target sequence.

[0030] In some embodiments, the contacting is performed under conditions sufficient for a reverse transcriptase to transcribe the complementary strand of the sequence of interest. In some embodiments, the method further comprises cleaving the template sequence to generate a double-stranded sequence comprising the sequence of interest. In some embodiments, the cleavage is performed by RNase H.

[0031] In some embodiments, the contact is carried out under conditions sufficient to generate a double-stranded sequence containing the target sequence by the DNA polymerase. In some embodiments, the contact is carried out under conditions sufficient to ligate the target sequence to the cleaved target sequence by the DNA ligase.

[0032] In some embodiments, a double-stranded sequence containing the target sequence is inserted into the cleaved target sequence by non-homologous end joining (NHEJ). In some embodiments, a double-stranded sequence containing the target sequence is inserted into the cleaved target sequence by DNA ligase.

[0033] In some embodiments, the method further includes generating a second double-stranded polynucleotide cleavage at a second target sequence in the target polynucleotide. In some embodiments, the sequence of interest substitutes the sequence of the target polynucleotide between the target sequence and the second target sequence.

[0034] In some embodiments, the present disclosure provides a kit comprising a fusion protein provided herein.

[0035] In some embodiments, the kit further comprises a vector for expressing polynucleotides and / or polynucleotides that form a complex with a fusion protein. In some embodiments, the kit further comprises a vector for expressing template polynucleotides and / or template polynucleotides that include a template sequence for a reverse transcriptase, DNA polymerase, or DNA ligase. In some embodiments, the kit further comprises a nucleotide containing tracrRNA. In some embodiments, the kit further comprises RNase H.

[0036] In some embodiments, the Cas9-RT fusion is used in combination with pegRNA and a DNAPK inhibitor to enhance gene editing efficiency. [Brief explanation of the drawing]

[0037] [Figure 1A-1D]Figures 1A–1D illustrate exemplary methods described in embodiments of this specification. Figures 1A and 1B show a fusion protein called a Primed Insertion (PRINS) fused to a Cas9 “NHEJ-promoting domain,” e.g., reverse transcriptase, DNA polymerase, or DNA ligase. In Figure 1A, the “SPRINgRNA” (single-primed insertion guide RNA) contains the target sequence ("ins") and a primer-binding site (PBS). In Figure 1B, the fusion protein further includes a DNA or RNA-binding domain (e.g., MCP2, ZF, TALE, FBP, Pumilio, HUH, or SNAP), and the target sequence with PBS is provided as an isolated polynucleotide. Figure 1C illustrates the mechanism of action of the PRINS complex shown in Figure 1A. The Cas9 nuclease generates double-strand cleavage at the target polynucleotide. Using the template sequence in the Cas9 complex containing PBS and the target sequence, a double-strand insertion sequence containing a replica of the target sequence is generated. The generated double-stranded insertion sequence can then be ligated to the target polynucleotide cleaved by the NHEJ. Figure 1D shows a further embodiment for combining insertions and deletions. The Cas9 nuclease generates a double-stranded break in the target polynucleotide. Using the template sequence in the Cas9 complex containing PBS and the target sequence, a double-stranded insertion sequence containing a replica of the target sequence is generated. The generated double-stranded insertion sequence can then be ligated by the NHEJ to another break generated downstream by a second CRISPR / Cas complex. The sequence between the two CRISPR / Cas complexes is replaced with the target sequence. [Figure 2A-2E]Figures 2A–2E illustrate exemplary methods described in embodiments of this specification. Figure 2A shows a Cas9-RT fusion protein (PRINS) having a guide RNA (gRNA) containing an insertion sequence that induces a double-strand break in the target sequence. PRINS binds to the gRNA for elongation. Figure 2B shows the result of elongation, with the elongated sequence indicated by a dashed line. Figure 2C shows the generation of a double-strand break in the elongated sequence by, for example, RNase H. Figure 2D shows the incorporation of the elongated sequence into the cleaved target sequence by NHEJ. Figure 2E shows the inserted sequence. [Figure 3A-3B] Figures 3A and 3B show a comparison of Cas9 editing (Figure 3A) versus PRINS editing (Figure 3B) at the AAVS1 site in Example 1. The relative editing frequency was determined by RIMA as described in Example 1. Insertions are indicated by ellipses. Figure 3B shows that PRINS facilitates template insertion of the sequence AAGATG and that PRINS promotes insertion via Cas9. All insertions originate from the original sequence AAGATG. [Figure 4] Figure 4 shows an exemplary method described in embodiments of this specification. A Cas nuclease is guided by gRNA to a target sequence to generate a double-strand DNA break. The template sequence includes a primer-binding sequence that hybridizes with the cleaved DNA, functions as a primer, and contains the target sequence. For example, a reverse transcriptase fused to a Cas9 nuclease synthesizes a first cDNA from the primer. A DNA strand complementary to the first cDNA is produced by a polymerase, such as DNA polymerase. The first cDNA and the DNA strand complementary to the first cDNA hybridize to generate a double-strand sequence that can be inserted into the cleaved DNA by a DNA repair pathway, such as NHEJ. [Figures 5A-5D]Figures 5A-5D, relating to Example 2, show a comparison of prime editing using prime editing guide RNA (pegRNA) (as described by Anzalone et al., Nature 576:149-157 (2019)) versus PRINS editing using single-primed insertion guide RNA (springRNA) at the AAVS1 site for inserting the AAGATG sequence. Relative editing frequencies were determined by fragment analysis as described herein. A comparison of Figure 5A (PRINS) with Figure 5B (primed editing) shows that PRINS is more efficient than prime editing. Figures 5C and 5D demonstrate the NHEJ dependency of PRINS. Figures 5C and 5D show a comparison of PRINS (Figure 5C) and prime editing (Figure 5D) insertion frequencies in the presence of DNA-dependent protein kinase inhibitors involved in NHEJ. [Figure 6] Figure 6 shows the effect of inserting the AAGATG sequence at the AAVS1 site using pegRNA and springRNA together with PRINS, in relation to Example 3. The relative editing frequency was determined by fragment analysis as described herein. As shown in Figure 6, pegRNA and springRNA can promote DNA insertion by PRINS via either a pathway similar to prime editing or a pathway similar to PRINS (primed edit insertion). [Figure 7] Figure 7 shows the effects of using PRINS editing or prime editing in the presence or absence of the DNA-dependent kinase (DNA-PK) inhibitor AZD7648 in Example 4. Specific incorporation was determined by NGS Amplicon-Seq as described herein. The bar graph shows the mean of n=2 as a standard deviation. Bars labeled "#1" or "#2" refer to various spring RNAs (in the case of PRINS editing) or various pegRNAs (in the case of prime editing). [Figure 8]Figure 8 relates to Example 5. Figure 8 summarizes the editing efficiency when using either a DNA template sequence containing spring RNA ("DNA tail") or an RNA template sequence containing spring RNA ("RNA tail"), as described herein, with a Cas9+RT ("PE0") fusion, a Cas9+DNA polymerase D ("PE0 PolD") fusion, a Cas9+Phi29 DNA polymerase ("PE0 Phi") fusion, or a Cas9 control. [Figure 9] Figure 9 relates to Example 5. Figure 9 shows editing patterns using the Cas9+RT ("PE0") fusion protein and three different guide RNAs (one containing an RNA tail ("123RNA MS") and two containing DNA tails ("123DNA" and "123DNA PS")) as described herein. The top, middle, and bottom panels of Figure 9 show editing patterns of PE0 using the 123RNA MS tail, the 123DNA tail, or the 123DNA PS tail, respectively. [Figure 10] Figure 10 relates to Example 5. Figure 10 shows editing patterns using the Cas9+DNA polymerase D ("PE0 PolD") fusion protein and three different guide RNAs (one containing an RNA tail ("123RNA MS") and two containing DNA tails ("123DNA" and "123DNA PS")) as described herein. The top, middle, and bottom panels of Figure 10 show editing patterns of PE0 PolD using the 123RNA MS tail, the 123DNA tail, or the 123DNA PS tail, respectively. [Figure 11]Figure 11 relates to Example 5. Figure 11 shows editing patterns using the Cas9+Phi29 DNA polymerase ("PE0 Phi") fusion protein and three different guide RNAs (one containing an RNA tail ("123RNA MS") and two containing DNA tails ("123DNA" and "123DNA PS")) as described herein. The top, middle, and bottom panels of Figure 11 show editing patterns of PE0 Phi using the 123RNA MS tail, the 123DNA tail, or the 123DNA PS tail, respectively. [Figure 12] Figure 12 relates to Example 5. Figure 12 shows editing patterns using Cas9 and three different guide RNAs (one containing an RNA tail ("123RNA MS") and two containing DNA tails ("123DNA" and "123DNA PS")) as described herein. The top, middle, and bottom panels of Figure 12 show editing patterns of Cas9 using the 123RNA MS tail, the 123DNA tail, or the 123DNA PS tail, respectively. [Figure 13] Figure 13 relates to Example 6. Figure 13 shows exemplary guide RNA designs for PRINS editing (indicated as "PRINS#1" and "PRINS#2") and prime editing (indicated as "PE#1" and "PE#2"). As shown in Figure 13, the prime editing guide RNA includes an additional 3' homology region. Figure 13 discloses Sequence IDs 44 and 45 in the order listed. [Figure 14A-14B] Figures 14A and 14B relate to Example 6. Figures 14A and 14B show the effects of using different guide RNAs shown in Figure 13, with or without the DNA-PK inhibitor AZA7648, using PRINS editing or prime editing. Specific incorporation was determined by NGS Amplicon-Seq as described herein. The bar graphs represent the mean of n=2 as a standard deviation. [Figure 15]Figure 15 relates to Example 7. Figure 15 shows an exemplary schematic diagram of the diphtheria toxin selection system described herein. As shown in Figure 15, the intron of HbEGF, the DT receptor, was selected as a PRINS editing or Cas9 editing target. Only a large deletion of the biallelic gene provides DT resistance to the cell. [Figure 16] Figure 16 relates to Example 7. Figure 16 shows microscopic images of cells transfected with Cas9-RT fusion (PRINS edited, "PE0"), Cas9, or Cas9 nickase-RT fusion (PRIMS edited, "PE2"), and three different guide RNAs. The positive control shows cells transfected with HbEGF-targeting Cas9. [Figure 17] Figure 17 relates to Example 8. Figure 17 shows exemplary schematic diagrams of two Cas9+RT fusion proteins containing an MCP domain either between Cas9 and RT ("PRINS_MS2_v1") or downstream of RT ("PRINS_MS2_v2"), as described herein. Three different polynucleotide systems were tested: (1) guide RNA and template polynucleotide for reverse transcriptase fused to the MS2 aptamer as isolated polynucleotides, (2) control, non-target guide RNA, and (3) guide RNA fused to the reverse transcriptase template. [Figure 18] Figure 18 relates to Example 8. Figure 18 shows the editing efficiency of PRINS editing for inserting the desired sequence AAGATG using the Cas9+RT+MCP fusion protein with the three different polynucleotide systems described in Figure 17. [Figure 19] Figure 19 relates to Example 9 and shows exemplary guide RNAs for Cas12 and target EXM1. Figure 19 discloses Sequence ID No. 41. [Figure 20]Figure 20 shows the results of PRINS editing with Cas9-DNA polymerase fusion protein for Example 10. The insertion frequency of spring RNA insertion sequences was analyzed in cells transfected with Cas9, Cas9-RT ("PE0"), or various DNA polymerases: Klenow fragments lacking 3'→5' exonuclease activity ("Cas9-Klenow exo-"), Klenow fragments with 3'→5' exonuclease activity ("Cas9-Klenow exo+"), or Cas9 fused to REV3 polymerase ("Cas9-REV3"). Each circle represents the exact insertion frequency for each independent transfection. The dotted line represents the mean value (i.e., background value) of insertions by Cas9 alone, and the difference from the background for each condition tested was calculated by multiple comparison ANOVA (Brown-Forsythe and Welch adjusted). The mean and standard deviation of 10 to 15 measurements are shown as a whisker plot. ***: p<0.0005; ****: p<0.0001. [Figure 21A-21C] Figures 21A-21C show the results of PRINS editing using a Cas9-DNA polymerase fusion protein with chimeric spring RNA for Example 11. Co-transfection of Cas9-DNA polymerase with a chimeric spring RNA ("DiHP") containing both DNA and RNA insertion sequences and PBS, or a spring RNA ("DiRP") containing only a DNA insertion sequence, increases the overall insertion efficiency, as shown in Figure 27A, and increases the frequency of insertion of the desired sequence, as shown in Figure 27B. In Figures 27A and 27B, each symbol (circle, square, or hexagon) represents the editing observed for each sample. Circles represent spring RNA, squares represent DiHP, and hexagons represent DiRP. The mean and standard deviation are shown in the whisker plot. Figure 27C shows typical editing patterns of Cas9, PE0, and Cas9-DNA polymerase fusion protein with spring RNA, DiHP, and DiRP. In Figure 27C, insertions are represented by shaded rectangles along with their specified arrangement, while deletions are represented by connecting lines. [Figure 22]Figure 22 shows the results of PRINS editing by Cas9-RT using spring RNA with modifications (abasic site or TEG linker) in Example 12. Co-transfection of Cas9-RT with the modified spring RNA increased the frequency of insertions of the desired length, resulting in more precise modifications. [Figures 23A-23B] Figures 23A-23B relate to Example 13. Figure 23A shows the electrogram of the AAVS1 locus after PRINS editing with PE0 (upper panel) and individually expressed Cas9 and RT (lower panel) after amplification with fluorescently labeled PCR primers and degradation by capillary electrophoresis. Asterisks represent the DNA product corresponding to the wild-type sequence, and large molecules with 6 bp insertions correspond to the PRINS-edited sequence. Figure 23B shows the results of PRINS editing of individually expressed Cas9, PE0, Cas9 and RT, and individually expressed Cas9-LigD and RT. Co-expression of Cas9-LigD and RT improved the insertion of the desired sequence compared to co-expression of Cas9 and RT. Circles represent individual editing measurements of >4 biological replications. Mean and standard deviation are shown by crossbar and whisker plots. Statistical differences were calculated by ANOVA (****: p<0.0001). [Figures 24A-24B] Figures 24A and 24B show the results of PRINS editing efficiency with and without mismatches in springRNA PBS for Example 14. Figure 24A shows that PRINS editing using springRNA without nucleic acid base mismatches had a relative insertion frequency of 37.13% for 6 bp insertion sequences. Figure 24B shows that PRINS editing using springRNA with a 2 bp nucleic acid base mismatch at the 3' end of PBS had a relative insertion frequency of 59.59% for 4 nt insertion sequences (the original 6 bp sequence minus the 2 bp mismatch). [Figure 25]Figure 25 relates to Example 15 and shows the results of PRINS editing in cells partially deficient in one of the following DNA repair genes: PRKDC (also known as DNAPK), LIG4, TP53BP1, PARP1, POLQ, LIG3, and ATM. The experiment was performed three times in the presence of DMSO control ("d") or a DNAPK inhibitor ("i"). The left panel shows the experiment using Cas9-RT fusion ("PE0") and spring RNA. The right panel shows the experiment using PE0 and pegRNA. [Figures 26A-26B] Figures 26A-26B relate to Example 16. Sequence ID 46 in Figures 26A-26B shows a spring RNA containing a tracrRNA scaffold for MHCas9, a 6 bp insertion sequence, and PBS. Figure 26A shows the most efficient PRINS editing events by MHCas9-RT (Sequence IDs 47 and 48). Figure 26B shows the 10 most frequent PRINS editing events by MHCas9-RT, indicating that RT mediates not only template insertions but also the elongation of overhang sequences (CCCs) generated by MHCas9, as shown by the three most frequent editing events. [Figures 27A-27B] Figures 27A and 27B show the results of targeted substitution / insertion and deletion using Cas9-RT with pegRNA for Example 17. Figure 27A shows the frequency of A-to-G substitution at the AAVS1 locus using DMSO or a DNAPK inhibitor (DNAPKi). Figure 27B shows the frequency of single-nucleotide deletion at the AAVS1 locus using DMSO or DNAPKi. [Modes for carrying out the invention]

[0038] This disclosure relates to improved CRISPR systems and their components, as well as methods for using them. Generally, a CRISPR system, such as a CRISPR / Cas system, includes elements that facilitate the formation of a CRISPR complex, such as a guide polynucleotide and a Cas protein, at a target polynucleotide, such as a site of a target DNA sequence. In natural CRISPR systems (e.g., bacterial immune CRISPR / Cas9 systems), foreign DNA is incorporated into a CRISPR array, which then generates CRISPR-RNA (crRNA). The crRNA contains a protospacer region complementary to the foreign DNA site and hybridizes with transactivated CRISPR-RNA (tracrRNA), which is also encoded by the CRISPR system. The tracrRNA can form secondary structures, such as stem-loops, and bind to the Cas9 protein. The crRNA / tracrRNA hybrid associates with Cas9, and the crRNA / tracrRNA / Cas9 complex recognizes and cleaves the foreign DNA with the protospacer sequence, conferring immunity against the invading virus or plasmid.

[0039] Since its initial discovery, extensive research has focused on the potential applications of the CRISPR system in genetic engineering, including gene editing (see, e.g., Jinek et al., Science 337(6096):816-821(2012); Cong et al., Science 339(6121):819-823(2013); and Mali et al., Science 339(6121):823-826(2013)). The CRISPR / Cas system utilizes the components of the natural CRISPR system described herein and is used in a wide range of organisms and cell lines for site-directed genome modification, such as gene editing. In addition to gene editing, the CRISPR system has several other applications, such as regulatory gene expression, gene circuit construction, and functional genomics (see Sander and Joung, Nat Biotechnol 32:347-355(2014)).

[0040] Unless otherwise defined herein, scientific and technical terms used herein shall have the meanings generally understood by those skilled in the art. Furthermore, unless the context requires otherwise, singular terms shall include plural forms, and plural terms shall include singular forms. As used herein, “a” or “an” (one) may mean one or more. As used herein, and in conjunction with the word “comprising,” the words “a” or “an” may mean one or more. As used herein, “another” or “further” may mean at least two or more.

[0041] A nucleic acid molecule is "hybridizable" or "hybridized" if, under appropriate temperature and solution ionic strength conditions, the single-stranded form of the nucleic acid molecule can anneal to another nucleic acid molecule, such as cDNA, genomic DNA, or RNA. Hybridization and washing conditions are known and exemplified in Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor (1989), particularly in Chapter 11 and Table 11.1. Temperature and ionic strength conditions determine the stringency of hybridization. The stringency of hybridization conditions can be selected to achieve the selective formation or maintenance of a desired hybridization product of two complementary nucleic acid polynucleotides in the presence of other polynucleotides that may potentially cross-react or interfere. Stringent conditions are sequence-dependent; typically, longer complementary sequences hybridize more specifically at higher temperatures than shorter complementary sequences. Typically, stringent hybridization conditions for a particular polynucleotide include defined ionic strength, concentration of chemical denaturant, pH, and thermal melting point (T) at the concentration of the hybridization partner. m) (i.e., the temperature at which 50% of the sequence hybridizes to substantially complementary sequences) is about 5°C to about 10°C lower. Generally, nucleotide sequences hybridize under stringent conditions more readily when they have a higher percentage of G and C bases than nucleotide sequences with a lower percentage of G and C bases. Stringency can usually be increased by raising the temperature, raising the pH, lowering the ionic strength, and / or by increasing the concentration of chemical nucleic acid denaturants (e.g., formamide, dimethylformamide, dimethyl sulfoxide, ethylene glycol, propylene glycol, and ethylene carbonate). Stringent hybridization conditions typically include salt concentrations or ionic intensities less than approximately 1 M, 500 mM, 200 mM, 100 mM, or 50 mM; hybridization temperatures greater than approximately 20°C, 30°C, 40°C, 60°C, or 80°C; and chemical modifier concentrations greater than approximately 10%, 20%, 30%, 40%, or 50%. Since many factors can influence the stringency of hybridization, the combination of parameters may be more important than the absolute value of any single parameter.

[0042] For example, exemplary low-stringency hybridization conditions corresponding to a Tm of 55°C include 5× sodium citrate-buffered saline (SSC), 0.1% SDS, 0.25% milk, and no formamide; or 30% formamide, 5× SSC, and 0.5% SDS. For higher Tm temperatures of approximately 55°C to approximately 65°C... m An example of a moderate stringency hybridization condition corresponding to this is 40% formamide and 5× or 6× SCC. An example of a high stringency hybridization condition corresponding to the highest Tm above 65°C is 50% formamide and 5× or 6× SCC.

[0043] Further exemplary hybridization conditions include a buffer solution at a temperature of approximately 15°C to 40°C with an ionic strength of approximately 20 mM to 200 mM and a pH of approximately 6.5 to 8.5 (for example, a phosphate, Tris, or HEPES buffer solution with approximately 20 mM to 200 mM of buffering component). For example, the buffer may contain salts at concentrations of approximately 10 mM to 1 M, 20 mM to 500 mM, 30 mM to 100 mM, 40 mM to 80 mM, or 50 mM. Exemplary salts include NaCl, KCl, (NH4)2SO4, Na2SO4, and CH3COONH4.

[0044] The term “complementary” is used to describe the relationships between nucleotide bases that can hybridize with one another. For example, with respect to DNA, adenosine is complementary to thymine, and cytosine is complementary to guanine. Accordingly, this disclosure also includes isolated nucleic acid fragments complementary to complete sequences disclosed or used herein, and nucleic acid sequences substantially similar thereto.

[0045] The term "homologous recombination" refers to the insertion of a foreign polynucleotide (e.g., DNA) into another nucleic acid (e.g., DNA) molecule, such as the insertion of a vector into a chromosome. In some cases, the vector targets a chromosomal site specific to homologous recombination. For specific homologous recombination, the vector typically contains a sufficiently long region homologous to the chromosomal sequence to enable complementary binding and integration of the vector into the chromosome. Longer complementary regions and greater degrees of sequence similarity can increase the efficiency of homologous recombination. In some embodiments, the fusion proteins or compositions described herein facilitate homologous recombination by generating cleavage, such as double-strand breaks in the nucleic acid sequence.

[0046] As used herein, the term “operably linked” means that the polynucleotide of interest, for example, a polynucleotide encoding a nuclease, is linked to a regulatory element so as to enable the expression of the polynucleotide. In some embodiments, the regulatory element is a promoter. In some embodiments, the polynucleotide expressing the polypeptide of interest is operably linked to a promoter on an expression vector.

[0047] A “vector” is any means of cloning and / or transferring a nucleic acid into a host cell. A vector may be a replicon to which another DNA segment can be attached, resulting in the replication of the attached segment. A “replicon” is any genetic element (e.g., plasmid, phage, cosmid, chromosome, virus) that functions as an autonomous unit of DNA replication in vivo, i.e., can replicate under its own control. In some embodiments, a vector is an episomal vector that is removed / disappeared from a population of cells after many cell generations by asymmetric distribution, for example. The term “vector” includes both viral and nonviral means for introducing nucleic acids into cells in vitro, ex vivo, or in vivo. Nucleic acids can be manipulated using a number of vectors known in the art to incorporate response elements and promoters into genes. A vector may contain one or more regulatory regions, and / or selection markers useful for selecting, measuring, and monitoring the results of nucleic acid introduction (e.g., which tissues it is introduced into, the duration of expression).

[0048] Possible vectors include, for example, plasmids or modified viruses, e.g., bacteriophages, e.g., lambda derivatives, or plasmids, e.g., PBR322 or pUC plasmid derivatives, or Bluescript vectors. For example, insertion of DNA fragments corresponding to response elements and promoters into a preferred vector can be achieved by ligating the appropriate DNA fragments into a selection vector having complementary sticky ends. Alternatively, the ends of a DNA molecule can be enzymatically modified, or any site can be produced by ligating polynucleotides (linkers) into the DNA ends. Such vectors can be modified to contain a selection marker gene that provides selection of cells incorporating a marker into their cellular genome. Such markers enable the identification and / or selection of host cells that incorporate the marker and express the protein encoded by that marker.

[0049] Viral vectors, particularly retroviral vectors, are used in a wide range of gene delivery applications in cells and living animals. Viral vectors that can be used include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, pox, baculoviruses, vaccinia, herpes simplex, Epstein-Var, adenoviruses, geminiviruses, and kalimovirus vectors. In some embodiments, viral vectors are used to provide the polynucleotides described herein. In some embodiments, viral vectors are used to provide polynucleotides encoding the polypeptides described herein.

[0050] Vectors can be introduced into desired host cells by known methods, including but not limited to transfection, transduction, cell fusion, and lipofection. Vectors may include various regulatory elements, such as promoters. In some embodiments, vector designs can be based on constructs designed by Mali et al., Nat Methods 10:957-63 (2013).

[0051] Polynucleotides and / or vectors provided herein can be propagated using methods known in the art. Once suitable host systems and growth conditions are established, recombinant expression vectors can be propagated and prepared in large quantities. Expression vectors that can be used as described herein include, but are not limited to, the following vectors or derivatives thereof: human or animal viruses, e.g., vaccinia virus or adenovirus; insect viruses, e.g., baculovirus; yeast vectors; bacteriophage vectors (e.g., lambda); and plasmids and cosmid DNA vectors.

[0052] The term "plasmid" refers to an external chromosome element, usually in the form of a circular double-stranded DNA molecule, that often carries genes that are not part of the cell's central metabolism. Such elements may be autonomous replicating sequences, genomic integration sequences, phages, or polynucleotides derived from any resource, and may be linear, circular, or supercoiled single-stranded or double-stranded DNA or RNA, in which numerous polynucleotides are bound or recombined into a unique construct that can introduce promoter fragments and DNA sequences for a selected gene product into the cell, along with appropriate 3' untranslated sequences. In some embodiments, plasmids are used to provide the polynucleotides described herein. In some embodiments, plasmids are used to provide polynucleotides encoding the polypeptides described herein.

[0053] As used herein, the term “transfection” means the introduction of an exogenous nucleic acid molecule, such as a vector, into a cell. A “transfected” cell is one in which an exogenous nucleic acid molecule is present inside the cell, and a “transformed” cell is one in which an exogenous nucleic acid molecule inside the cell induces a phenotypic change in the cell. The transfected nucleic acid molecule can be incorporated into the genomic DNA of a host cell and / or maintained by the cell outside the chromosome for a transient or prolonged period. A host cell or organism expressing an exogenous nucleic acid molecule or fragment is referred herein to as a “recombinant,” “transformed,” or “transgenic” organism. In some embodiments, this disclosure provides a host cell containing an expression vector comprising one of the expression vectors described herein, for example, a nuclease, a fusion protein, or a polynucleotide encoding a variant thereof.

[0054] The term "host cell" refers to a cell into which a recombinant expression vector has been introduced, or it may also refer to the offspring of such a cell. For example, because mutations or environmental influences may cause modifications in later generations, such progeny may not be identical to the parent cell, but they are still included within the scope of the term "host cell."

[0055] The terms “peptide,” “polypeptide,” and “protein” are used interchangeably herein and refer to polymeric forms of amino acids of any length, including coded and non-coding amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides having a modified peptide backbone.

[0056] The starting point of a protein or polypeptide is known as the "N-terminus" (also called the amino-terminus, NH2-terminus, N-terminal region, or amine-terminus), which refers to the free amine (-NH2) group of the first amino acid residue of the protein or polypeptide. The ending point of a protein or polypeptide is known as the "C-terminus" (also called the carboxyl-terminus, carboxyl-terminus, C-terminal region, or COOH-terminus), which refers to the free carboxyl group (-COOH) of the last amino acid residue of the protein or polypeptide.

[0057] As used herein, “amino acid” refers to a compound containing both a carboxyl (-COOH) and an amino (-NH2) group. “Amino acid” refers to both natural and unnatural (i.e., synthetic) amino acids. Examples of 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). Non-natural or synthetic amino acids include side chains different from those of the natural amino acids provided above, and may include, for example, fluorophores, post-translational modifications, metal ion chelators, photocages and photocrosslinking moieties, unique reactive functional groups, and NMR, IR, and X-ray crystal probes. Exemplary non-natural 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). Non-natural amino acids may also include naturally occurring compounds that are not typically incorporated into proteins or polypeptides, such as citrulline (Cit), selenocysteine ​​(Sec), and pyrrolidine (Pyl).

[0058] "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 an amino acid residue by an amino acid different from that wild-type or naturally occurring amino acid. The substituted amino acid may be synthetic or 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-natural or synthetic amino acid. Substitution variants may be described using abbreviations. For example, a substitution variant in which the fifth amino acid residue is substituted can be abbreviated as "X5Y", where "X" is the wild-type or naturally occurring amino acid to be substituted, "5" is the amino acid residue position in the amino acid sequence of the protein or polypeptide, and "Y" is the substitution, or a non-wild-type or non-naturally occurring amino acid.

[0059] An "isolated" polypeptide, protein, peptide, or nucleic acid is a molecule taken from its natural environment. It is also understood that an "isolated" polypeptide, protein, peptide, or nucleic acid may be formulated with excipients, such as diluents or auxiliaries, and may 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.

[0060] When the term “recombinant” is used in reference to nucleic acid molecules, peptides, polypeptides, or proteins, it means novel combinations of genetic material not known to exist in nature, or those resulting from such combinations. 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.

[0061] The term "domain," when used in relation to polypeptides or proteins, refers to a distinct functional and / or structural unit within a protein. Domains may be responsible for specific functions or interactions that contribute to the overall role of a protein. Domains can exist in a variety of biological contexts. Similar domains can be found in proteins with different functions. Alternatively, domains with low sequence identity (i.e., less than approximately 50%, 40%, 30%, 20%, 10%, 5%, or 1%) may have the same function.

[0062] When used in relation to polypeptides or proteins, the term “motif” generally refers to a set of conserved amino acid residues, typically shorter than 20 amino acids, that may be important to the function of a protein. Certain sequence motifs can mediate common functions in various proteins, such as protein binding or targeting to specific intracellular locations. Examples of motifs include, but are not limited to, nuclear localization signals, microbody targeting motifs, motifs that prevent or promote secretion, and motifs that promote protein recognition and binding. Motif databases and / or motif search tools are publicly known in the art and include, for example, PROSITE (expasy.ch / sprot / prosite.html), Pfam (pfam.wustl.edu), PRINTS (biochem.ucl.ac.uk / bsm / dbbrowser / PRINTS / PRINTS.html), and Minimotif Miner.

[0063] As used herein, “engineered” protein means a protein that contains one or more modifications to achieve a desired property. Exemplary modifications include, but are not limited to, insertions, deletions, substitutions, and / or fusions with another domain or protein. A “fusion protein” (also called a “chimeric protein”) is a protein that typically contains at least two domains encoded by two distinct genes, which are joined together so as to be transcribed and translated as a single unit, thereby producing a single polypeptide having the functional properties of each domain. Examples of engineered proteins in this disclosure include nucleases and fusion proteins, such as Cas nucleases and reverse transcriptases, DNA polymerases, or DNA ligases.

[0064] In some embodiments, the engineered protein is derived from a wild-type protein. As used herein, “wild-type” protein or nucleic acid is a naturally occurring, unmodified protein or nucleic acid. For example, wild-type Cas9 protein can be isolated from the organism Streptococcus pyogenes. Wild-type may be contrasted with “mutant,” which contains one or more modifications in the amino acid and / or nucleotide sequence of the protein or nucleic acid. In some embodiments, the engineered protein may have substantially the same activity as the wild-type protein, e.g., more than 80%, more than 85%, more than 90%, more than 95%, or more than 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.

[0065] As used herein, the terms “sequence similarity” or “% similarity” refer to the degree of identity or correspondence between nucleic acid sequences or amino acid sequences. In the context of polynucleotides, “sequence similarity” refers to nucleic acid sequences in which a change in one or more nucleotide bases results in a substitution of one or more amino acids, but does not affect the functional properties of the protein encoded by the polynucleotide. Alternatively, “sequence similarity” may refer to a modification of a polynucleotide, such as the deletion or insertion of one or more nucleotide bases, which does not substantially affect the functional properties of the resulting transcript. Therefore, it is understood that this disclosure encompasses more than specific exemplary sequences. Methods for forming nucleotide base substitutions are known, as are methods for determining the retention of the biological activity of the encoded polypeptide.

[0066] Furthermore, those skilled in the art will recognize that the similar polynucleotides encompassed by this disclosure are also defined by their ability to hybridize with the sequences exemplified herein under stringent conditions. The similar polynucleotides of this disclosure are identical to the polynucleotides disclosed herein by 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%.

[0067] In the context of polypeptides, "sequence similarity" refers to two or more polypeptides in which more than 40% of amino acids are identical, or more than 60% of 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 their functional similarity as follows: Positively charged side chains: Arg, His, Lys; Load side chain: Asp, Glu; Polar uncharged side chains: Ser, Thr, Asn, Gln; Hydrophobic side chains: Ala, Val, Ile, Leu, Met, Phe, Tyr, Trp; Others: Cys, Gly, Pro.

[0068] In some embodiments, the similar polypeptides of this 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.

[0069] In some embodiments, the similar polypeptides of this disclosure have amino acids that are functionally identical by 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%.

[0070] Sequence similarity can be determined by sequence alignment using methods known in the art, such as BLAST, MUSCLE, Clustal (including ClustalW and ClustalX), and T-Coffee (including heteromorphs, such as M-Coffee, R-Coffee, and Expresso).

[0071] The percentage of identity of a polynucleotide or polypeptide can be determined when the polynucleotide or polypeptide sequences are aligned within a specified comparison window. In some embodiments, sequence identity is determined by aligning only specific portions of two or more sequences. In some embodiments, sequence similarity is determined by aligning only specific domains of two or more sequences. The comparison window may be a segment of at least 10 to over 1000 residues, at least 20 to about 1000 residues, or at least 50 to 500 residues, in 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), modified as described in Karlin and Altschul, Proc Nat Acad Sci USA 90:5873-5877 (1993). Such algorithms are incorporated into the BLAST program, for example, the BLAST+ or NBLAST and XBLAST programs described in Altschul et al., J Mol Biol, 215:403-410 (1990). BLAST protein searching can be performed using a program, for example, the XBLAST program, with a score of 50 and a word length of 3, to obtain amino acid sequences homologous to the protein molecules of this disclosure. If a gap exists between two sequences, Gapped BLAST can be used as described in Altschul et al., Nucleic Acids Res 25(17):3389-3402 (1997). When using the BLAST and Gapped BLAST programs, the default parameters of each program (e.g., XBLAST and NBLAST) can be used.

[0072] In some embodiments, the polypeptide or polynucleotide has sequence identity of 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%, at least 99%, or 100% with a reference polypeptide or polynucleotide (or a fragment of a reference polypeptide or polynucleotide) provided herein. In some embodiments, the polypeptide or polynucleotide has sequence identity of 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% with a reference polypeptide or polynucleotide (or a fragment of a reference polypeptide or nucleic acid molecule) provided herein.

[0073] As used herein, “complex” refers to a group of two or more associated polynucleotides and / or polypeptides. In the context of complex formation, the term “associate” or “associate” refers to molecules that are bound to one another via electrostatic, hydrophobic / hydrophilic, and / or hydrogen bonding interactions, without covalent bonding. Molecules containing different parts covalently bonded to each other are known. In some embodiments, a complex is formed when all components of the complex are present together, i.e., it is a self-assembling complex. In some embodiments, a complex is formed via chemical interactions between different components of the complex, such as hydrogen bonding. In some embodiments, a polynucleotide, e.g., an RNA polynucleotide, complexes with a protein or polypeptide, e.g., an RNA-induced protein, through secondary structure recognition of the polynucleotide by the protein or polypeptide.

[0074] Fusion protein The fusion protein disclosed herein provides improved gene editing efficiency compared to wild-type Cas nucleases.

[0075] In some embodiments, the present disclosure provides a fusion protein comprising (i) a Cas nuclease and (ii) a reverse transcriptase, or a DNA polymerase, or a DNA ligase, wherein the Cas nuclease is capable of generating double-stranded polynucleotide cleavage.

[0076] As described herein, fusion proteins typically comprise at least two domains having different functions. In some embodiments, the fusion protein comprises a Cas nuclease. Generally, the Cas nuclease is part of the CRISPR / Cas system. As described herein, the CRISPR / Cas system can be used for site-directed genome modification. The CRISPR / Cas system may comprise a Cas nuclease and a guide polynucleotide (e.g., guide RNA). In some embodiments, the guide polynucleotide comprises a polypeptide-binding segment that binds and / or activates the Cas nuclease and a guide sequence (e.g., crRNA) that hybridizes to a target sequence. As used herein, “segment” refers to a portion, section, or region of a molecule, e.g., a continuous stretch of nucleotides in a guide polynucleotide molecule. The definition of “segment” is not limited to a specific number of total base pairs unless otherwise specifically defined. In some embodiments, the guide polynucleotide comprises tracrRNA. In some embodiments, the guide polynucleotide does not include tracrRNA, and tracrRNA is provided as an isolated polynucleotide in the CRISPR / Cas system. In some embodiments, tracrRNA activates the Cas nuclease. In some embodiments, activation of the Cas nuclease initiates or increases nuclease activity. In some embodiments, activation of the Cas nuclease includes binding of the nuclease to a target sequence in the target polynucleotide.

[0077] The CRISPR / Cas system can be classified into types I to VI based on the nuclease proteins within the system. For example, Cas9 can be found in the type II system, while Cas12 can be found in the type V system. Each type can be further classified into subtypes. For example, type II may include subtypes II-A, II-B, and II-C, and type V may include subtypes VA and VB. The classification of the CRISPR / Cas system and Cas nucleases is further discussed, 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). Unless otherwise specified, the Cas nucleases described herein may include any type or variant.

[0078] In some embodiments, Cas nucleases can produce double-stranded polynucleotide cleavage, such as double-stranded DNA cleavage. Generally, Cas nucleases can contain one or more nuclease domains, such as RuvC and HNH, which can cleave double-stranded DNA. In some embodiments, Cas nucleases contain a RuvC domain and an HNH domain, each of which cleaves one strand of double-stranded DNA. In some embodiments, Cas nucleases produce blunt ends. In some embodiments, the RuvC and HNH of Cas nucleases cleave each DNA strand at the same position, thereby producing blunt ends. In some embodiments, Cas nucleases produce sticky ends. In some embodiments, the RuvC and HNH of Cas nucleases cleave each DNA strand at different positions (i.e., cut at an "offset"), thereby producing 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,” sticky ends are produced by staggered cuts on double-stranded nucleic acids (e.g., DNA). Sticky or adhesive ends have unpaired nucleotides or protruding single strands with “overhangs,” such as 3' or 5' overhangs.

[0079] In some embodiments, the Cas nuclease is Cas9. Cas9 is found in the type II CRISPR / Cas system described herein. Exemplary Cas9 proteins include, but are not limited to, those derived 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, Cas9 refers to the polypeptide of Sequence ID No. 1.

[0080] In some embodiments, Cas9 is type IIB Cas9. Generally, type IIB Cas9 proteins can produce sticky ends as described herein. Exemplary type IIB Cas9 proteins include, but are not limited to, those derived from Legionella pneumophila, Francisella novicida, Parasutterella excrementihominis, Sutterella wadsworthensis, Wolinella succinogenes, and numerous other bacteria. In some embodiments, type IIB Cas9 is derived from the sequenced intestinal metagenome MH0245_GL0161830.1 (MHCas9). Further type IIB Cas9 proteins are described, for example, in International Publication No. 2019 / 099943.

[0081] In some embodiments, Cas9 comprises SEQ ID NO: 1. In some embodiments, Cas9 comprises a polypeptide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or about 100% sequence identity to SEQ ID NO: 1. In some embodiments, the Disclosure provides a polynucleotide encoding a polypeptide having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or about 100% sequence identity to SEQ ID NO: 1. In some embodiments, Cas9 is encoded by a polynucleotide that is codon-optimized for expression in host cells.

[0082] In some embodiments, the Cas nuclease is Cas12. The Cas12 nuclease is also sometimes known as the "Cpf1" or "C2c1" nuclease and is found in the V-type CRISPR / Cas system described herein. The Cas12 nuclease is typically smaller than the Cas9 nuclease and can produce sticky ends. Exemplary Cas12 proteins include, but are not limited to, those derived from Francisella novicida, Acidaminococcus sp., Lachnospiraceae sp., Prevotella sp., and many other bacteria. Further Cas12 nucleases are described, for example, in U.S. Patent 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).

[0083] In some embodiments, Cas12 is the sequence number 38 Includes. In some embodiments, Cas12 is sequence number 38 It has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or about 100% sequence identity. In some embodiments, the present disclosure relates to the sequence number. 38 The present invention provides polynucleotides encoding polypeptides having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or about 100% sequence identity. In some embodiments, Cas12 is encoded by a polynucleotide that is codon-optimized for expression in host cells.

[0084] In some embodiments, the Cas nuclease is Cas14. Originally discovered in archaea, the Cas14 nuclease is typically a small enzyme that targets single-stranded DNA (ssDNA) and does not require a PAM sequence. Cas14 is found in the DPANN superphylum of archaea and is further described, for example, in Harrington et al., Science 362:839-842 (2018) and U.S. Patent Application Publication No. 2020 / 0087640.

[0085] In some embodiments, Cas14 is the sequence number 39 Includes. In some embodiments, Cas14 is sequence number 39 It has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or about 100% sequence identity. In some embodiments, the present disclosure relates to the sequence number. 39 The present invention provides polynucleotides encoding polypeptides having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or about 100% sequence identity. In some embodiments, Cas14 is encoded by a polynucleotide that is codon-optimized for expression in host cells.

[0086] In some embodiments, the fusion protein comprises a Cas nuclease and a reverse transcriptase, DNA polymerase, DNA ligase, or a combination thereof.

[0087] In some embodiments, the fusion protein includes a reverse transcriptase. Reverse transcriptase (sometimes abbreviated as RT) is an enzyme used to produce DNA (e.g., complementary DNA or cDNA) from an RNA template in a process called reverse transcription. A typical reverse transcription reaction is initiated using an RNA template and primers that bind to the ends of the RNA template. In some embodiments, the reverse transcriptase binds to a primer (e.g., PBS) and synthesizes a cDNA strand (e.g., based on the RNA template) in a process that provides a first cDNA. Figure 4 provides an exemplary and non-limiting overview of the use of Cas nuclease, reverse transcriptase, polymerase, and NHEJ for inserting the sequence of interest. In some embodiments, an RNase, e.g., RNase H, removes the RNA template. In some embodiments, the reverse transcriptase includes RNase activity, e.g., RNase H. In some embodiments, a DNA strand complementary to the first cDNA is then synthesized by DNA polymerase to produce a double-stranded sequence. In some embodiments, the reverse transcriptase includes DNA polymerase activity. In some embodiments, a DNA repair mechanism, such as NHEJ, can be used to insert a double-stranded sequence containing the target sequence into a double-stranded polynucleotide.

[0088] Examples of reverse transcriptases include, but are not limited to, AMV reverse transcriptase, MMLV (M-MuLV) reverse transcriptase, R2 reverse transcriptase, and HIV reverse transcriptase. In some embodiments, the reverse transcriptase is MMLV reverse transcriptase or R2 reverse transcriptase. In some embodiments, the reverse transcriptase is capable of DNA polymerase activity.

[0089] In some embodiments, the Cas nuclease of the fusion protein generates double-stranded polynucleotide cleavage at a target polynucleotide, e.g., a target sequence in a target DNA sequence. In some embodiments, one strand of the cleaved DNA serves as a primer for the reverse transcriptase of the fusion protein. In some embodiments, a template polynucleotide containing a template sequence for the reverse transcriptase is provided, and the reverse transcriptase generates a first cDNA. In some embodiments, the template sequence is RNA, and the RNase removes the template sequence. In some embodiments, the reverse transcriptase includes RNase activity. In some embodiments, the template sequence is removed by an isolation RNase. In some embodiments, the RNase is RNase H. In some embodiments, a DNA strand complementary to the first cDNA is generated by a DNA polymerase, e.g., isolation DNA polymerase, or a reverse transcriptase having DNA polymerase activity. In some embodiments, the first cDNA and the DNA strand complementary to the first cDNA are hybridized to form a double-stranded sequence. In some embodiments, the double-stranded sequence can be inserted into the cleaved target sequence. In some embodiments, the double-stranded sequence is inserted into the cleaved target sequence by a DNA repair pathway. In some embodiments, the DNA repair pathway is non-homologous end joining (NHEJ), microhomology-mediated end joining (MMEJ), homology-oriented repair (HDR), or a combination thereof. In some embodiments, the double-stranded sequence is inserted into the cleaved target sequence by ligation, for example, using a DNA ligase.

[0090] In some embodiments, the reverse transcriptase comprises one of SEQ ID NOs: 2-3. In some embodiments, the reverse transcriptase has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or about 100% sequence identity to one of SEQ ID NOs: 2-3. In some embodiments, the disclosure provides a polynucleotide encoding a polynucleotide having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or about 100% sequence identity to one of SEQ ID NOs: 2-3. In some embodiments, the reverse transcriptase is encoded by a polynucleotide that is codon-optimized for expression in a host cell.

[0091] In some embodiments, the fusion protein includes DNA polymerase, an enzyme that synthesizes DNA by adding nucleotides to an existing single DNA strand. In some embodiments, DNA polymerase generates a double-stranded sequence from a first synthetic strand produced by reverse transcriptase. In some embodiments, DNA polymerase generates double-stranded DNA from a single-stranded DNA template (ssDNA).

[0092] In some embodiments, the Cas nuclease of the fusion protein generates a double-stranded polynucleotide cleavage at a target polynucleotide, for example, at a target sequence in a target DNA sequence. In some embodiments, a template polynucleotide, for example, an ssDNA template, is provided, and the DNA polymerase of the fusion protein generates a double-stranded sequence from the ssDNA template. In some embodiments, the double-stranded sequence can be inserted into the cleaved target sequence. In some embodiments, the double-stranded sequence is inserted into the cleaved target sequence by a DNA repair pathway. In some embodiments, the DNA repair pathway is non-homologous end joining (NHEJ), microhomology-mediated end joining (MMEJ), or homology-oriented repair (HDR). In some embodiments, the double-stranded sequence is inserted into the cleaved target sequence by ligation, for example, using a DNA ligase.

[0093] Examples of DNA polymerases include, but are not limited to, DNA polymerases (Pol) I, II, III, IV, and V; DNA polymerases (Pol) α, β, λ, γ, σ, μ, δ, ε, η, ι, κ, ζ, θ, Rev1, and Rev3; isothermal DNA polymerases, including, for example, Bst, T4, and Φ29 (phi29) DNA polymerase; and thermostable DNA polymerases, including, for example, Taq, Pfu, KOD, Tth, and Pwo DNA polymerase. In some embodiments, the DNA polymerase is part of a DNA repair pathway. In some embodiments, the DNA repair pathway DNA polymerase is Polβ, Polγ, Polσ, or Polμ. In some embodiments, the DNA polymerase is Rev3. DNA repair pathways are further described herein. In some embodiments, the DNA polymerase has high processing capacity, i.e., the DNA polymerase can process a large number of nucleotides in a single binding event. In some embodiments, high-capacity DNA polymerases can bind sequences of over 100 bp, over 200 bp, over 300 bp, over 400 bp, over 500 bp, over 600 bp, over 700 bp, over 800 bp, over 1 kp, over 5 kp, over 10 kp, over 50 kb, or over 100 kb per binding event. In some embodiments, high-capacity DNA polymerases are advantageous for the synthesis of sequences with secondary structures such as long templates and high GC content. In some embodiments, high-capacity DNA polymerases are Polα, Polδ, Polε, or Φ29 DNA polymerases. In some embodiments, the DNA polymerases are phi29 DNA polymerase, T4 DNA polymerase, DNA polymerase μ (mu), DNA polymerase δ (delta), or DNA polymerase ε (epsilon). In some embodiments, the DNA polymerase of the fusion protein comprises a catalytically active fragment or truncation of DNA polymerase. As used herein, the “catalystically active” fragment, truncation, or domain of an enzyme means that the fragment or truncation has substantially the same activity as the full-length or wild-type enzyme (e.g., DNA polymerase).In some embodiments, the catalytically active fragments, truncations, or domains of the enzymes herein have about 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, or more than 200% of the activity of the full-length or wild-type enzyme (e.g., DNA polymerase). In some embodiments, the catalytically active fragments, truncations, or domains of the enzymes herein have one or more improved properties compared to the full-length or wild-type enzyme (e.g., DNA polymerase), such as improved stability and / or processing capacity. In some embodiments, the DNA polymerase is the Klenow fragment of E. coli DNA polymerase I. In some embodiments, the DNA polymerase is a truncation of Rev3, as described in Lee et al., PNAS (2014), doi:10.1073 / pnas.1324001111.

[0094] In some embodiments, the DNA polymerase comprises one of SEQ ID NOs: 4-6. In some embodiments, the DNA polymerase has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or about 100% sequence identity to one of SEQ ID NOs: 4-6. In some embodiments, the disclosure provides a polynucleotide encoding a polypeptide having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or about 100% sequence identity to one of SEQ ID NOs: 4-6. In some embodiments, the DNA polymerase is encoded by a polynucleotide that is codon-optimized for expression in a host cell.

[0095] In some embodiments, the fusion protein includes a DNA ligase. A DNA ligase is an enzyme that facilitates the binding of DNA strands by catalyzing the formation of phosphodiester bonds. DNA ligases can repair single-strand or double-strand breaks in DNA. In some embodiments, the DNA ligase ligates single-strand DNA. In some embodiments, the DNA ligase ligates the blunt ends of double-strand DNA. In some embodiments, the DNA ligase ligates the sticky ends of double-strand DNA. In some embodiments, the DNA ligase facilitates the recombination of a double-stranded insertion sequence into a double-stranded polynucleotide. In some embodiments, if two double-stranded polynucleotide cleavage occurs at a target polynucleotide (e.g., at a first target site and a second target site), the DNA ligase can facilitate the recombination of the double-stranded polynucleotide, thereby removing the sequence between the first and second target sites.

[0096] In some embodiments, the Cas nuclease of the fusion protein generates double-stranded polynucleotide cleavage at a target polynucleotide, for example, a target sequence in a target DNA sequence. In some embodiments, a template polynucleotide, for example, a DNA template, is provided, and the DNA ligase of the fusion protein ligates the template polynucleotide to the cleaved target sequence. In some embodiments, the DNA template is a double-stranded polynucleotide containing blunt ends. In some embodiments, the DNA template is a double-stranded polynucleotide containing sticky ends. In some embodiments, the DNA template is a single-stranded polynucleotide.

[0097] Examples of DNA ligases include, but are not limited to, E. coli DNA ligase, Taq DNA ligase, T4 DNA ligase, T7 DNA ligase, DNA ligases I, III, and IV, and Ampligase DNA ligase. In some embodiments, the DNA ligase is T4 ligase.

[0098] In some embodiments, the DNA ligase comprises SEQ ID NO: 7. In some embodiments, the DNA ligase has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or about 100% sequence identity to SEQ ID NO: 7. In some embodiments, the disclosure provides a polynucleotide encoding a polypeptide having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or about 100% sequence identity to SEQ ID NO: 7. In some embodiments, the DNA ligase is encoded by a polynucleotide that is codon-optimized for expression in host cells.

[0099] In some embodiments, the fusion protein further comprises a DNA-binding or RNA-binding domain. In some embodiments, the DNA-binding or RNA-binding domain of the fusion protein brings the fusion protein and the template polynucleotide into close proximity. In some embodiments, the DNA-binding or RNA-binding domain facilitates the binding of the template polynucleotide to the fusion protein. In some embodiments, the DNA-binding or RNA-binding domain improves the efficiency of reverse transcriptase, DNA polymerase, or DNA ligase reactions by bringing the template polynucleotide and the fusion protein into close proximity. In some embodiments, the DNA-binding or RNA-binding domain increases the efficiency of incorporating the double-stranded sequence resulting from the reverse transcriptase or DNA polymerase reaction into the cleaved target sequence.

[0100] In some embodiments, the fusion protein further comprises a DNA-binding domain. Therefore, in some embodiments, the fusion protein comprises a Cas nuclease, a reverse transcriptase, and a DNA-binding domain. In some embodiments, the fusion protein comprises a Cas nuclease, a DNA polymerase, and a DNA-binding domain. In some embodiments, the fusion protein comprises a Cas nuclease, a DNA ligase, and a DNA-binding domain. The DNA-binding domain can be found as part of transcription factors in viruses, bacteria, and eukaryotes (such as mammals). In some embodiments, the DNA-binding domain binds to single-stranded DNA. In some embodiments, the DNA-binding domain binds to double-stranded DNA. In some embodiments, the DNA-binding domain binds to both single-stranded and double-stranded DNA. Examples of DNA-binding domains that bind double-stranded DNA include, but are not limited to, helix-turn-helix (HTH), zinc finger (ZF), transcriptional activation-like effector (TALE), nuclear small RNA activating protein (SNAP), leucine zipper, winged helix, helix-loop-helix, HMG-box, Wor3, and OB-fold. Examples of DNA-binding domains that bind single-stranded DNA include, but are not limited to, T4 gene 32 protein (T4g32), HUH enzymes such as viral Rep proteins, and far upstream element-binding protein 1 (FUBP). Further DNA-binding domains are provided, for example, in Alberts B et al. Molecular Biology of the Cell. 4th edition. New York: Garland Science; 2002. DNA-Binding Motifs in Gene Regulatory Proteins; Yesudhas et al., Genes (Basel) 8(8):192 (2017); and Vidangos et al., Biopolymers 99(12):1082-1096 (2013).In some embodiments, the DNA-binding domain is a zinc finger DNA-binding domain, a transcription factor, or an adeno-associated virus Rep protein. In some embodiments, the DNA-binding domain is a far upstream element-binding protein (FUBP).

[0101] In some embodiments, the fusion protein further comprises an RNA-binding domain. Therefore, in some embodiments, the fusion protein comprises a Cas nuclease, a reverse transcriptase, and an RNA-binding domain. In some embodiments, the fusion protein comprises a Cas nuclease, a DNA polymerase, and an RNA-binding domain. In some embodiments, the fusion protein comprises a Cas nuclease, a DNA ligase, and an RNA-binding domain. The RNA-binding domain can be found as part of an RNA processing protein and is involved, for example, in RNA biosynthesis, maturation, transport, cellular localization, and stability. In some embodiments, the RNA-binding domain comprises an RNA recognition motif. In some embodiments, the RNA-binding domain comprises a double-stranded RNA-binding motif. In some embodiments, the RNA-binding domain comprises a zinc finger. In some embodiments, the RNA-binding domain comprises a KH domain, such as heteronuclear ribonucleoprotein K (hnRNPK). Examples of RNA-binding domains include, but are not limited to, NOVA1, ADAR, CPSF, TAP / NXF1:p15, ZBP1, Elav, Sxl, tra-2, FOG-1, MOG-1, MOG-4, MOG-5, RNP-4, GLD-1, GLD-3, DAZ-1, PGL1, OMA-1, OMA2, MEC-8, UNC-75, EXC-7, Pumilio, Nanos, FMRP, CPEB, Staufen1, FXR1, and MCP2. Further examples of RNA-binding domains are provided, for example, in Lunde et al., Nat Rev Mol Cell Biol 8(6):479-490(2007) and Glisovic et al., FEBS Lett 582(14):1977-1986(2008). In some embodiments, the RNA-binding domain is the MS2 coat protein (MCP2). In some embodiments, the RNA-binding domain includes a KH domain. In some embodiments, the RNA-binding domain is hnRNPK.

[0102] In some embodiments, the DNA-binding or RNA-binding domain comprises one of sequence numbers 8-11. In some embodiments, the DNA-binding or RNA-binding domain comprises a polypeptide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or about 100% sequence identity with one of sequence numbers 8-11. In some embodiments, the disclosure provides polynucleotides encoding polypeptides having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or about 100% sequence identity with one of sequence numbers 8-11.

[0103] In some embodiments, the fusion proteins provided herein have sequence identity of at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or about 100% with respect to any one of SEQ ID NOs.

[0104] In some embodiments, the fusion protein further comprises a nuclear localization signal (NLS). As used herein, “nuclear localization signal” or “nuclear localization sequence” (NLS) refers to a polypeptide that “tags” a protein for transport into the cell nucleus by nuclear transport; i.e., a protein having an NLS is transported into the cell nucleus. Typically, an NLS comprises a positively charged Lys or Arg residue exposed on the protein surface. Exemplary nuclear localization sequences include, but are not limited to, NLS derived from SV40 large T-antigen, nucleoplasm, EGL-13, c-Myc, and TUS-proteins. In some embodiments, the NLS comprises the sequence PKKKRKV (SEQ ID NO: 14). In some embodiments, the NLS comprises the sequence AVKRPAATKKAGQAKKKKLD (SEQ ID NO: 29). In some embodiments, the NLS comprises the sequence PAAKRVKLD (SEQ ID NO: 30). In some embodiments, the NLS comprises the sequence MSRRRKANPTKLSENAKKLAKEVEN (SEQ ID NO: 31). In some embodiments, the NLS includes the sequence KLKIKRPVK (SEQ ID NO: 32). Other nuclear localization sequences include, but are not limited to, the acidic M9 domain of hnRNP A1, the sequence KIPIK (SEQ ID NO: 33) in the yeast transcription repressor Matα2, and PY-NLS.

[0105] In some embodiments, the fusion protein further comprises a linker ligating the Cas nuclease domain, as well as a reverse transcriptase, DNA polymerase, or DNA ligase. In some embodiments, the linker is long and / or flexible enough to position the Cas nuclease without steric hindrance from the reverse transcriptase, DNA polymerase, or DNA ligase. In some embodiments, the linker is long and / or flexible enough to allow the reverse transcriptase, DNA polymerase, or DNA ligase to carry out their respective reactions without steric hindrance from the Cas nuclease. In some embodiments, the linker is about 3 to about 100 amino acids long. In some embodiments, the linker is about 5 to about 80 amino acids long. In some embodiments, the linker is about 10 to about 60 amino acids long. In some embodiments, the linker is about 20 to about 50 amino acids long. In some embodiments, the linker is about 25 to about 40 amino acids long. Exemplary linker sequences are described herein, for example, SEQ ID NOs. 15-16.

[0106] Polynucleotides In some embodiments, the present disclosure provides compositions comprising: (a) a fusion protein provided herein; and (b) a polynucleotide that forms a complex with the fusion protein and comprises (i) a guide sequence and (ii) a template sequence for a reverse transcriptase or DNA lipopolymerase.

[0107] In some embodiments, the polynucleotide of the composition is RNA. In some embodiments, the polynucleotide includes a guide polynucleotide component. As described herein, the CRISPR / Cas system includes a guide polynucleotide, for example, guide RNA. In some embodiments, the guide polynucleotide is RNA. RNA guide polynucleotides may be referred to herein as “guide RNA,” “gRNA,” or “DNA-targeting RNA.”

[0108] In some embodiments, the guide polynucleotide includes a guide sequence. In some embodiments, the guide polynucleotide includes a guide sequence and a polypeptide-binding segment. In some embodiments, the guide sequence can hybridize with a target sequence in the target polynucleotide. In some embodiments, the polypeptide-binding segment of the guide polynucleotide binds to a Cas nuclease. In some embodiments, the polypeptide-binding segment binds to a Cas nuclease in a fusion protein provided herein. In some embodiments, the polypeptide-binding segment binds to and / or activates the Cas nuclease.

[0109] In some embodiments, the polynucleotide of the composition includes a guide sequence that can hybridize with a target sequence in the target polynucleotide. In some embodiments, the polynucleotide of the composition includes a polypeptide-binding segment that can bind to the Cas nuclease of the fusion protein, thereby forming a complex with the fusion protein. In some embodiments, the polynucleotide further includes tracrRNA. In some embodiments, the composition further includes a second polynucleotide containing tracrRNA. In some embodiments, the tracrRNA activates the Cas nuclease. In some embodiments, the activation of the Cas nuclease initiates or increases its nuclease activity. In some embodiments, the activation of the Cas nuclease includes binding of the nuclease to the target sequence. In some embodiments, the Cas nuclease generates a double-stranded polynucleotide at the target sequence in the target polynucleotide.

[0110] In some embodiments, the guide sequence is about 10 to about 40 nucleotides long. In some embodiments, the guide sequence is about 12 to about 30 nucleotides long. In some embodiments, the guide sequence is about 15 to about 20 nucleotides long. 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 long. In some embodiments, the guide sequence is long enough to hybridize to the target sequence.

[0111] In some embodiments, the polynucleotide of the composition includes a template sequence. In some embodiments, the template sequence includes a primer-binding sequence and a target sequence. In some embodiments, the template sequence includes a homology region with the target sequence. In some embodiments, the homology region is the primer-binding sequence. In some embodiments, the template sequence includes mismatched nucleotides with respect to the target sequence following the primer-binding sequence. In some embodiments, the template sequence includes 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mismatched nucleotides with respect to the target sequence following the primer-binding sequence. As used herein, “mismatched nucleotides” refers to nucleotides that do not form base pairs. In some embodiments, template sequences containing mismatched nucleotides have a higher insertion frequency than template sequences that do not contain mismatched nucleotides. In some embodiments, the template sequence includes one or more homology addition regions with respect to the target sequence. In some embodiments, the template sequence includes two homology regions. In some embodiments, the template sequence includes at least two homology regions. In some embodiments, the template sequence includes a first homology region, the target sequence, and a second homology region in the 5' to 3' direction. In some embodiments, an additional homology region facilitates the insertion of the target sequence into the target sequence. In some embodiments, the template sequence is single-stranded. In some embodiments, the template sequence is double-stranded. In some embodiments, the template sequence comprises DNA. In some embodiments, the target sequence comprises DNA. In some embodiments, the target sequence and primer-binding sequence comprises DNA. In some embodiments, the template sequence comprises RNA. In some embodiments, the template sequence comprises xeno nucleic acid (XNA). As used herein, XNA refers to a nucleic acid whose polymer chain contains a non-natural backbone. For example, instead of the ribose sugar in the DNA or RNA backbone, XNA may contain hexose, threose, glycol, cyclohexenyl, deoxyribose, etc. XNA is further described, for example, in Schmidt, M. (2010), Bioessays 32(4):322-331.In some embodiments, the template sequence includes an aptamer. In some embodiments, the template sequence includes modifications that prevent elongation of the target sequence by reverse transcriptase and / or DNA polymerase. In some embodiments, the modifications include an abbase site (also known as an aprine / apyrimidine site or AP site), a triethylene glycol (TEG) linker, or both. In some embodiments, the modifications prevent excessive elongation of the target sequence, thereby improving the insertion accuracy of the target sequence.

[0112] In embodiments where the fusion protein comprises a Cas nuclease and a reverse transcriptase, the polynucleotide comprises a template sequence for the reverse transcriptase. In some embodiments, the Cas nuclease of the fusion protein generates a double-stranded polynucleotide cleavage at a target polynucleotide, e.g., a target sequence in a target DNA sequence, and one strand of the cleaved DNA hybridizes to a primer-binding sequence on the template sequence, which then functions as a primer for the reverse transcriptase to reverse transcribe the template sequence. In some embodiments, the sequence of interest is reverse transcribed by the reverse transcriptase to produce a first cDNA. In some embodiments, a DNA strand complementary to the first cDNA is produced by a DNA polymerase, thereby generating a double-stranded sequence containing the sequence of interest. In some embodiments, the double-stranded sequence containing the sequence of interest is inserted into the cleaved target sequence, for example, by a ligation or DNA repair pathway as described herein. In some embodiments, the double-stranded sequence containing the target sequence further includes a recognition site for an endonuclease, transposase, or recombinase, which incorporates the double-stranded sequence into a target polynucleotide. In some embodiments, homology regions on the template sequence described herein facilitate the insertion of the double-stranded sequence containing the target sequence into a cleaved target sequence.

[0113] In embodiments where the fusion protein comprises a Cas nuclease and a DNA polymerase, the polynucleotide comprises a template for the DNA polymerase. In some embodiments, the Cas nuclease of the fusion protein generates a double-stranded polynucleotide cleavage at the target polynucleotide, e.g., the target sequence in the target DNA sequence, and one strand of the cleaved DNA hybridizes to a primer-binding sequence on the template sequence, which functions as a primer for the DNA polymerase. In some embodiments, the DNA polymerase synthesizes a DNA strand complementary to the target sequence, thereby generating a double-stranded sequence containing the target sequence. In some embodiments, the double-stranded sequence containing the target sequence is inserted into the cleaved target sequence, for example, by a ligation or DNA repair pathway described herein. In some embodiments, the double-stranded sequence containing the target sequence further comprises a recognition site for an endonuclease, transposase, or recombinase, and the endonuclease, transposase, or recombinase incorporates the double-stranded sequence into the target polynucleotide. In some embodiments, homology regions on the template sequence described herein facilitate the insertion of a double-stranded sequence containing the sequence of interest into a cleaved target sequence.

[0114] In some embodiments, the template sequence is about 10 to about 25,000 nucleotides long. In some embodiments, the template sequence is about 15 to about 20,000 nucleotides long. In some embodiments, the template sequence is about 20 to about 15,000 nucleotides long. In some embodiments, the template sequence is about 25 to about 10,000 nucleotides long. In some embodiments, the template sequence is approximately 10, 15, 20, 25, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 2500, 5000, 7500, 10000, 15000, 20000, or 25000 nucleotides long. In some embodiments, the template sequence is more than about 10 nucleotides long, more than about 15 nucleotides long, more than about 20 nucleotides long, more than about 25 nucleotides long, more than about 30 nucleotides long, more than about 35 nucleotides long, more than about 40 nucleotides long, more than about 45 nucleotides long, or more than about 50 nucleotides long.

[0115] In some embodiments, the primer-binding sequence is about 3 to about 50 nucleotides long. In some embodiments, the primer-binding sequence is about 4 to about 30 nucleotides long. In some embodiments, the primer-binding sequence is about 5 to about 40 nucleotides long. In some embodiments, the primer-binding sequence is about 7 to about 30 nucleotides long. In some embodiments, the primer-binding sequence is about 10 to about 20 nucleotides long. In some embodiments, the primer-binding sequence is about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 12, about 15, about 17, about 20, about 22, about 25, about 27, about 30, about 32, about 35, about 38, or about 40 nucleotides long. In some embodiments, the primer-binding sequence is long enough to hybridize with the region of the cleaved target DNA sequence.

[0116] In some embodiments, the target sequence is approximately 1 to approximately 20,000 nucleotides long. In some embodiments, the target sequence is approximately 2 to approximately 17,000 nucleotides long. In some embodiments, the target sequence is approximately 3 to approximately 15,000 nucleotides long. In some embodiments, the target sequence is approximately 4 to approximately 12,000 nucleotides long. In some embodiments, the target sequence is approximately 5 to approximately 10,000 nucleotides long. In some embodiments, the target sequence is approximately 10 to approximately 9,000 nucleotides long. In some embodiments, the target sequence is approximately 50 to approximately 8,000 nucleotides long. In some embodiments, the target sequence is approximately 100 to approximately 7,000 nucleotides long. In some embodiments, the target sequence is approximately 200 to approximately 6,000 nucleotides long. In some embodiments, the target sequence is approximately 500 to approximately 5,000 nucleotides long. In some embodiments, the target sequences are approximately 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, and 9 The lengths are 50, approximately 1000, approximately 1250, approximately 1500, approximately 1750, approximately 2000, approximately 2500, approximately 3000, approximately 3500, approximately 4000, approximately 4500, approximately 5000, approximately 5500, approximately 6000, approximately 6500, approximately 7000, approximately 7500, approximately 8000, approximately 8500, approximately 9000, approximately 10000, approximately 12500, approximately 15000, approximately 17500, or approximately 25000 nucleotides. In some embodiments, the target sequences are more than approximately 5 nucleotides long, more than approximately 10 nucleotides long, more than approximately 15 nucleotides long, more than approximately 20 nucleotides long, more than approximately 25 nucleotides long, more than approximately 30 nucleotides long, more than approximately 35 nucleotides long, more than approximately 40 nucleotides long, more than approximately 45 nucleotides long, or more than approximately 50 nucleotides long.

[0117] In some embodiments, the polynucleotide of the composition further includes a spacer between the guide sequence and the template sequence. In some embodiments, the spacer includes a stop sequence for the reverse transcriptase or DNA polymerase so that the reverse transcriptase or DNA polymerase is stopped after the complementary strand of the sequence of interest has been transcribed or synthesized. In some embodiments, the spacer includes two or more stop sequences. In some embodiments, the spacer includes one, two, three, four, five, or more than five stop sequences. In some embodiments, multiple stop sequences result in redundancy in stopping the reverse transcriptase or DNA polymerase. In some embodiments, the stop sequences inhibit the activation of the reverse transcriptase and / or DNA polymerase. In some embodiments, the stop sequences facilitate the dissociation of the reverse transcriptase and / or DNA polymerase from the template sequence.

[0118] In some embodiments, the stop sequence includes a secondary structure. In some embodiments, the secondary structure is an inhibitor of reverse transcriptase and / or DNA polymerase activity. In some embodiments, the secondary structure facilitates the dissociation of reverse transcriptase and / or DNA polymerase from the 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.

[0119] In some embodiments, the spacer is about 5 to about 500 nucleotides long. In some embodiments, the spacer is about 10 to about 400 nucleotides long. In some embodiments, the spacer is about 10 to about 300 nucleotides long. In some embodiments, the spacer is about 10 to about 200 nucleotides long. In some embodiments, the spacer is about 20 to about 150 nucleotides long. In some embodiments, the spacer is about 30 to about 100 nucleotides long. In some embodiments, the spacer is about 50 to about 100 nucleotides long. 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 long.

[0120] In some embodiments, the present disclosure provides compositions comprising (a) a fusion protein provided herein, (b) a guide polynucleotide that forms a complex with the fusion protein and includes a guide sequence, and (c) a template polynucleotide that includes a template sequence for a reverse transcriptase or DNA polymerase.

[0121] Guide polynucleotides are described herein. In some embodiments, the guide polynucleotide of the composition comprises a guide sequence that can hybridize with a target sequence. In some embodiments, the guide polynucleotide of the composition comprises a polypeptide-binding segment that can bind to the Cas nuclease of the fusion protein, thereby forming a complex with the fusion protein. In some embodiments, the guide polynucleotide further comprises tracrRNA. In some embodiments, the composition further comprises a third polynucleotide comprising tracrRNA. In some embodiments, tracrRNA activates the Cas nuclease. In some embodiments, activation of the Cas nuclease initiates or increases its nuclease activity. In some embodiments, activation of the Cas nuclease comprises binding of the nuclease to a target sequence.

[0122] In some embodiments, the guide sequence is about 10 to about 40 nucleotides long. In some embodiments, the guide sequence is about 12 to about 30 nucleotides long. In some embodiments, the guide sequence is about 15 to about 20 nucleotides long. 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 long. In some embodiments, the guide sequence is long enough to hybridize to the target sequence.

[0123] The components of the template polynucleotide, such as the template sequence of a reverse transcriptase or DNA polymerase, the primer-binding sequence, the stop sequence, the sequence of interest, and / or additional homology regions, are described herein. In some embodiments, the template sequence is about 10 to about 25,000 nucleotides long. In some embodiments, the template sequence is about 15 to about 20,000 nucleotides long. In some embodiments, the template sequence is about 20 to about 15,000 nucleotides long. In some embodiments, the template sequence is about 25 to about 10,000 nucleotides long. In some embodiments, the template sequence is approximately 10, 15, 20, 25, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 2500, 5000, 7500, 10000, 15000, 20000, or 25000 nucleotides long. In some embodiments, the template sequence is more than about 10 nucleotides long, more than about 15 nucleotides long, more than about 20 nucleotides long, more than about 25 nucleotides long, more than about 30 nucleotides long, more than about 35 nucleotides long, more than about 40 nucleotides long, more than about 45 nucleotides long, or more than about 50 nucleotides long.

[0124] In some embodiments, the template sequence includes the sequence of interest. In some embodiments, the sequence of interest is about 1 to about 20,000 nucleotides long. In some embodiments, the sequence of interest is about 2 to about 17,000 nucleotides long. In some embodiments, the sequence of interest is about 3 to about 15,000 nucleotides long. In some embodiments, the sequence of interest is about 4 to about 12,000 nucleotides long. In some embodiments, the sequence of interest is about 5 to about 10,000 nucleotides long. In some embodiments, the sequence of interest is about 10 to about 9,000 nucleotides long. In some embodiments, the sequence of interest is about 50 to about 8,000 nucleotides long. In some embodiments, the sequence of interest is about 100 to about 7,000 nucleotides long. In some embodiments, the sequence of interest is about 200 to about 6,000 nucleotides long. In some embodiments, the sequence of interest is about 500 to about 5,000 nucleotides long. In some embodiments, the target sequences are approximately 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, and 9 The lengths are 50, approximately 1000, approximately 1250, approximately 1500, approximately 1750, approximately 2000, approximately 2500, approximately 3000, approximately 3500, approximately 4000, approximately 4500, approximately 5000, approximately 5500, approximately 6000, approximately 6500, approximately 7000, approximately 7500, approximately 8000, approximately 8500, approximately 9000, approximately 10000, approximately 12500, approximately 15000, approximately 17500, or approximately 25000 nucleotides. In some embodiments, the target sequences are more than approximately 5 nucleotides long, more than approximately 10 nucleotides long, more than approximately 15 nucleotides long, more than approximately 20 nucleotides long, more than approximately 25 nucleotides long, more than approximately 30 nucleotides long, more than approximately 35 nucleotides long, more than approximately 40 nucleotides long, more than approximately 45 nucleotides long, or more than approximately 50 nucleotides long.

[0125] In some embodiments, the template polynucleotide further comprises the primer-binding sequence described herein. In some embodiments, the primer-binding sequence is about 3 to about 50 nucleotides long. In some embodiments, the primer-binding sequence is about 4 to about 30 nucleotides long. In some embodiments, the primer-binding sequence is about 5 to about 40 nucleotides long. In some embodiments, the primer-binding sequence is about 7 to about 30 nucleotides long. In some embodiments, the primer-binding sequence is about 10 to about 20 nucleotides long. In some embodiments, the primer-binding sequence is about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 12, about 15, about 17, about 20, about 22, about 25, about 27, about 30, about 32, about 35, about 38, or about 40 nucleotides long. In some embodiments, the guide sequence is long enough to hybridize the target sequence cleaved by the Cas nuclease of the fusion protein.

[0126] In some embodiments, the template polynucleotide includes a stop sequence for the reverse transcriptase or DNA polymerase described herein. In some embodiments, the template polynucleotide includes two or more stop sequences. In some embodiments, the spacer includes one, two, three, four, five, or more than five stop sequences. In some embodiments, the stop sequence includes a secondary structure. In some embodiments, the secondary structure is an inhibitor of reverse transcriptase and / or DNA polymerase activity. In some embodiments, the secondary structure facilitates the dissociation of reverse transcriptase and / or DNA polymerase from the 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.

[0127] In embodiments in which the fusion protein further comprises a DNA-binding or RNA-binding domain, the template polynucleotide further comprises a sequence that can bind to the DNA-binding or RNA-binding domain. Regarding FUBP, non-restrictive examples of DNA sequences that bind to DNA-binding domains such as zinc finger DNA-binding domains, transcription factors, and adeno-associated virus Rep proteins include, for example, Bulyk et al., Proc Natl Acad Sci USA 98(13):7158-7163(2001); Fornes et al., Nucleic Acids Res 2019;doi:10.1093 / nar / gkz1001; Gearing et al., PLOS One 14(9):e0215495(2019); Wonderling et al., J Virol 71(3):2528-2534(1997); Benjamin et al., Proc Natl Acad Sci USA 105(47):18296-18301(2008), and Hudson et al., Nat Rev Mol Cell Biol This is described in 15(11):749-760(2014). Non-restrictive examples of RNA sequences for binding to RNA-binding domains such as MCP2 are described, for example, in Castello et al., Mol Cell 63:696-710(2016); Rube et al., Nat Comm 7:11025(2016); Peabody et al., EMBO J 12(2):595-600(1993), and Hudson et al., Nat Rev Mol Cell Biol 15(11):749-760(2014).

[0128] In some embodiments, the template polynucleotide comprises an adeno-associated virus (AAV) vector containing the sequence of interest. AAV is a non-enveloped virus that can be manipulated to deliver the sequence of interest to target cells. See, for example, Naso et al., BioDrugs 31(4):317-334 (2017). In some embodiments, the AAV vector is single-stranded DNA. In some embodiments, the AAV vector comprises an inverted end repeat (ITR), a promoter, the sequence of interest, and a terminator. In some embodiments, the AAV vector comprises an ITR and the sequence of interest. In some embodiments, the AAV vector does not contain viral genes. In some embodiments, the template polynucleotide comprises an AAV vector, and the fusion protein comprises a Cas nuclease and a DNA polymerase. In some embodiments, the AAV vector is about 200, about 300, about 400, about 500, about 600, about 700, about 800, about 900, about 1000, about 2000, about 3000, about 4000, or about 5000 nucleotides long. In some embodiments, the target sequence in the AAV vector is approximately 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1200, 1500, 1700, 2000, 2200, 2500, 2700, 3000, 3200, 3500, 3700, 4000, 4200, 4500, or 4700 nucleotides long.

[0129] In some embodiments, the Disclosure provides polynucleotides encoding fusion proteins provided herein. In some embodiments, the polynucleotides encode polypeptides having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or about 100% sequence identity to any one of SEQ ID NOs. 18-26.

[0130] In some embodiments, the polynucleotides herein, for example, a polynucleotide comprising a polynucleotide encoding a fusion protein, a guide sequence and a template sequence, a guide polynucleotide, and / or a template polynucleotide, are codon-optimized for expression in eukaryotic cells. In some embodiments, the polynucleotides herein are codon-optimized for expression in bacterial cells. In some embodiments, the polynucleotides herein are codon-optimized for expression in mammalian cells. In some embodiments, the polynucleotides herein are codon-optimized for expression in human cells. As used herein, “codon optimization” refers to adjusting codons to match the expression of recombinant or heterologous protein to the host tRNA abundance in order to increase the yield and efficiency of recombinant or heterologous protein expression. Codon optimization methods are known in the art and may be carried out using software programs such as, for example, Integrated DNA Technologies’ Codon Optimization tool, Entelechon’s Codon Usage Table analysis tool, GENEMAKER’s Blue Heron software, Aptagen’s Gene Forge software, and other software such as DNA Builder, OPTIMIZER, and OptimumGene algorithms.

[0131] In some embodiments, the Disclosure provides vectors comprising polynucleotides encoding fusion proteins provided herein. In some embodiments, the Disclosure provides vectors comprising polynucleotides comprising a polynucleotide encoding a fusion protein, a guide sequence and a template sequence, a guide polynucleotide, a template polynucleotide, or a combination thereof. In some embodiments, the polynucleotide encoding the fusion protein and the polynucleotide comprising the guide sequence and the template sequence are located on a single vector. In some embodiments, the polynucleotide encoding the fusion protein and the polynucleotide comprising the guide sequence and the template sequence are located on one or more vectors. In some embodiments, the polynucleotide encoding the fusion protein, the guide polynucleotide, and the template oligonucleotide are located on a single vector. In some embodiments, the polynucleotide encoding the fusion protein, the guide polynucleotide, and the template oligonucleotide are located on one or more vectors.

[0132] Various types of vectors, such as viral and nonviral vectors, are provided herein. 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.

[0133] In some embodiments, the vector is a viral vector. In some embodiments, the viral vector is a retrovirus, adeno-associated virus, pox, baculovirus, vaccinia, herpes simplex virus, Epstein-Barr virus, adenovirus, geminivirus, or kalimovirus vector. In some embodiments, the viral vector is an adenovirus, lentivirus, or adeno-associated virus vector. Viral transduction (administration may be local, targeted, or systemic) using adenovirus, adeno-associated virus (AAV), and lentiviral vectors 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.

[0134] In some embodiments, the vector further comprises a regulatory element operably linked to a polynucleotide encoding a fusion protein, the polynucleotide comprising a guide sequence and a template sequence, a guide polynucleotide, and / or a template polynucleotide. In some embodiments, the regulatory element is a bacterial promoter. In some embodiments, the regulatory element is a viral promoter. In some embodiments, the regulatory element is a mammalian promoter. In some embodiments, the regulatory element is a terminator. Regulatory elements are further described herein.

[0135] In some embodiments, a polynucleotide comprising a fusion protein, a guide sequence, and a template sequence, a guide polynucleotide, and / or a template polynucleotide are introduced into cells via delivery particles. Delivery particles can be used to deliver exogenous biomaterials, such as the polynucleotides and proteins described herein. In some embodiments, the delivery particles are solid, semi-solid, emulsion, or colloid. In some embodiments, the delivery particles are lipid particles, liposomes, micelles, vesicles, or exosomes. In some embodiments, the delivery particles are nanoparticles. The delivery particles are further described, for example, in U.S. Patent Publication No. 2011 / 0293703, No. 2012 / 0251560, No. 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.

[0136] In some embodiments, a polynucleotide comprising a fusion protein, a guide sequence, and a template sequence, the guide polynucleotide, and / or the template polynucleotide are introduced into the cell via a vesicle. In some embodiments, the vesicle comprises an exosome or a liposome. Vesicles engineered to deliver exogenous biomaterials to target cells are described, for example, in Alvarez-Erviti et al., Nat Biotechnol 29:341 (2011), El-Andaloussi et al., Nat Protocols 7:2112-2116 (2012), Wahlgren et al., Nucleic Acid Res 40(17):e130 (2012), Morrissey et al., Nat Biotechnol 23(8):1002-1007 (2005), Zimmerman et al., Nat Letters 441:111-114 (2006), and Li et al., Gene Therapy 19:775-780 (2012).

[0137] cell In some embodiments, the Disclosure provides cells comprising a fusion protein provided herein. In some embodiments, the Disclosure provides cells comprising a polynucleotide encoding a fusion protein provided herein. In some embodiments, the Disclosure provides cells comprising a polynucleotide comprising a polynucleotide encoding a fusion protein, a guide sequence and a template sequence, a guide polynucleotide, a template polynucleotide, or a combination thereof. In some embodiments, the Disclosure provides cells comprising a vector provided herein, for example, comprising a polynucleotide encoding a fusion protein, a polynucleotide comprising a guide sequence and a template sequence, a guide polynucleotide, a template polynucleotide, or a combination thereof.

[0138] In some embodiments, the cells are bacterial cells. In some embodiments, the bacterial cells are laboratory strains. 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, and S. coelicolor. In some embodiments, the bacterial cells consist of bacteria used in the preparation of food and / or beverages.Examples of non-exclusive 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. bcheri, L. casei, L. curvatus, L. fermentum, L. hilgardii, L. jensenii, L. kimchii, L. lactis, L. paracasei Examples include, but are not limited to, Leuconostoc (including L. paracasei, L. plantarum, and L. saucei), Microbacterium, Pediococcus, Propionibacterium, Weissella, and Zymomonas.

[0139] In some embodiments, the cells are eukaryotic cells. In some embodiments, the eukaryotic cells are mammalian cells. In some embodiments, the eukaryotic cells are animal cells. In some embodiments, the eukaryotic cells are mammalian cells. In some embodiments, the eukaryotic cells are animal or human cells, cell lines, or cell strains. Examples of animal or mammalian cells, cell lines, or cell lines include, but are not limited to, mouse myeloma (NSO), Chinese hamster oocyte (CHO), HT1080, H9, HepG2, MCF7, MDBK Jurkat, NIH3T3, PC12, BHK (baby hamster kidney), EBX, EB14, EB24, EB26, EB66, or Ebvl3, VERO, SP2 / 0, YB2 / 0, Y0, C127, L cells, COS (e.g., COS1 and COS7), QC1-3, HEK-293, VERO, PER.C6, HeLA, EB1, EB2, EB3, tumor lysic cells, or hybridoma cells. In some embodiments, the eukaryotic cell is a CHO cell. In some embodiments, the cells are CHO-K1 cells, CHO-K1 SV cells, DG44 CHO cells, DUXB11 CHO cells, CHOS, CHO GS knockout cells, CHO FUT8 GS knockout cells, CHOZN, or CHO-derived cells. CHO GS knockout cells (e.g., GSKO cells) may be, for example, CHO-K1 SV GS knockout cells.

[0140] In some embodiments, eukaryotic cells are human stem cells. Stem cells may be, for example, pluripotent stem cells, such as 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, cells are any differentiated form of the cells described herein. In some embodiments, eukaryotic cells are cells derived from any primary cells in culture.

[0141] In some embodiments, eukaryotic cells are hepatocytes, e.g., human hepatocytes, animal hepatocytes, or non-parenchymal cells. For example, eukaryotic cells may be human hepatocytes for adherent metabolism studies, human hepatocytes for adherent induction studies, adherent human hepatocytes, human hepatocytes for suspension studies (e.g., 10-donor and 20-donor pooled hepatocytes), human hepatic Kupffer cells, human hepatic stellate cells, canine hepatocytes (e.g., single and pooled beagle hepatocytes), mouse hepatocytes (e.g., CD-1 and C57BI / 6 hepatocytes), rat hepatocytes (e.g., Sprague-Dawley, Wistar Han, and Wistar hepatocytes), monkey hepatocytes (e.g., cynomolgus or rhesus monkey hepatocytes), feline hepatocytes (e.g., domestic shorthair hepatocytes), and rabbit hepatocytes (e.g., New Zealand white hepatocytes).

[0142] In some embodiments, eukaryotic cells are plant cells. For example, plant cells may be from crop plants, such as cassava, maize, sorghum, wheat, or rice. Plant cells may be from algae, trees, or vegetables. Plant cells may be from monocots or dicots, or from crops or cereal plants, production plants, fruits, or vegetables. For example, plant cells can be those of trees, such as citrus trees, such as orange, grapefruit, or lemon trees; peach or nectarine trees; apple or pear trees; nut trees, such as almond, walnut, or pistachio trees; plants of the Solanum genus, such as potato, tomato, eggplant, pepper, and paprika; plants of the Brassica, Lactuca, and Spinacia genera; plants of the Capsicum genus; and plants such as cotton, tobacco, asparagus, carrots, cabbage, broccoli, cauliflower, lettuce, spinach, strawberries, blueberries, raspberries, blackberries, grapes, coffee, and cocoa.

[0143] Site-specific modification methods In some embodiments, the disclosure provides a method for providing site-directed modification in a target sequence in a target polynucleotide, comprising contacting the target polynucleotide with a composition provided herein. In some embodiments, the composition comprises (a) a fusion protein described herein and (b) a polynucleotide described herein comprising a guide sequence and a template sequence. In some embodiments, the composition comprises (a) a fusion protein described herein, (b) a guide polynucleotide described herein and (c) a template oligonucleotide described herein. In some embodiments, the target polynucleotide is double-stranded. In some embodiments, the target polynucleotide is DNA.

[0144] Exemplary methods are illustrated in Figures 1 and 2. Figures 1A and 1B show Cas9 fused to an "NHEJ-promoting domain," e.g., reverse transcriptase, DNA polymerase, or DNA ligase. In Figure 1A, the "SPRINgRNA" (single-priming insertion guide RNA) contains the target sequence ("ins") and a primer-binding site (PBS). In Figure 1B, the fusion protein further includes a DNA or RNA-binding domain (e.g., MCP2, ZF, TALE, FBP, Pumilio, HUH, or SNAP), and the target sequence with PBS is provided as an isolated polynucleotide. Figure 1C shows the mechanism of action of the PRINS complex shown in Figure 1A. The Cas9 nuclease generates double-strand cleavage at the target polynucleotide. The target sequence is replicated using the template sequence in the Cas9 complex containing PBS and the target sequence. The generated double-strand sequence can then be ligated to the target polynucleotide cleaved by NHEJ.

[0145] In some embodiments, the fusion protein comprises a Cas nuclease and a reverse transcriptase. In some embodiments, the template sequence comprises RNA. In some embodiments, a polynucleotide guide sequence or guide polynucleotide in the composition can hybridize to the target sequence. In some embodiments, the fusion protein is guided to the target sequence by hybridizing the guide sequence and the target sequence. In some embodiments, the contact step of the method is carried out under conditions sufficient to allow the Cas nuclease to produce a double-stranded polynucleotide cleavage at the target sequence. In some embodiments, one strand of the cleaved target sequence is a primer for the reverse transcriptase. In some embodiments, the polynucleotide template sequence or template polynucleotide in the composition comprises a primer-binding site that can bind to the primer. In some embodiments, the template sequence comprises the sequence of interest. In some embodiments, the contact step of the method is carried out under conditions sufficient to allow the reverse transcriptase to recognize the primer-binding sequence hybridized to the target sequence and reverse transcribe the complementary strand of the sequence of interest to produce a first cDNA. In some embodiments, a DNA polymerase synthesizes a DNA strand complementary to the first cDNA. In some embodiments, the template sequence is removed from the first cDNA by an RNase so that DNA polymerase can synthesize a DNA strand complementary to the first cDNA, thereby generating a double-stranded sequence containing the target sequence. In some embodiments, where the reverse transcriptase is capable of RNase activity, the template sequence is removed by the reverse transcriptase. In some embodiments, the method further includes providing an RNase to remove the template sequence. In some embodiments, the RNase is RNase H. RNase H can specifically hydrolyze the RNA hybridized to DNA.

[0146] In some embodiments, after the removal of the template sequence from the first cDNA by an RNase, e.g., RNase H, e.g., digestion or cleavage, a DNA polymerase generates a DNA strand complementary to the first cDNA, thereby generating a double-stranded sequence containing the target sequence. In some embodiments where reverse transcriptase is capable of DNA polymerase activity, the DNA strand complementary to the first cDNA is generated by the reverse transcriptase. In some embodiments where the method is performed in cells, the DNA strand complementary to the first cDNA is generated by a native DNA polymerase in the cell. In some embodiments where the method is performed in vitro, the method further includes providing a DNA polymerase to generate a DNA strand complementary to the first cDNA. In some embodiments, the first cDNA and the DNA strand complementary to the first cDNA hybridize to form a double-stranded sequence containing the target sequence. In some embodiments, the double-stranded sequence containing the target sequence can be inserted into the cleaved target sequence. In some embodiments, a double-stranded sequence containing the target sequence is inserted into a cleaved target sequence by a DNA repair pathway, such as non-homologous end joining (NHEJ). In some embodiments, a double-stranded sequence containing the target sequence is inserted into a cleaved target sequence by a DNA ligase. In some embodiments, the double-stranded sequence containing the target sequence further includes a recognition site for an endonuclease, transposase, or recombinase, which incorporates the double-stranded sequence into a target polynucleotide. In some embodiments, homology regions on the template sequence described herein facilitate the insertion of the double-stranded sequence containing the target sequence into a cleaved target sequence.

[0147] In some embodiments, the fusion protein comprises a Cas nuclease and a DNA polymerase. In some embodiments, the template sequence comprises DNA. In some embodiments, the template sequence comprises single-stranded DNA (ssDNA). In some embodiments, a polynucleotide guide sequence or guide polynucleotide in the composition can hybridize to the target sequence. In some embodiments, the fusion protein is guided to the target sequence by hybridizing the guide sequence and the target sequence. In some embodiments, the contact step of the method is carried out under conditions sufficient to allow the Cas nuclease to produce a double-stranded polynucleotide cleavage at the target sequence. In some embodiments, one strand of the cleaved target sequence is a primer for the DNA polymerase. In some embodiments, the polynucleotide template sequence or template polynucleotide in the composition comprises a primer-binding site that can bind to the primer. In some embodiments, the template sequence comprises the sequence of interest. In some embodiments, the contact step of the method is carried out under conditions sufficient to allow the DNA polymerase to recognize the primer-binding sequence hybridized to the target sequence and produce a double-stranded sequence containing the sequence of interest. In some embodiments, a double-stranded sequence containing the target sequence can be inserted into a cleaved target sequence. In some embodiments, the double-stranded sequence containing the target sequence is inserted into the cleaved target sequence by a DNA repair pathway, such as non-homologous end joining (NHEJ). In some embodiments, the double-stranded sequence containing the target sequence is inserted into the cleaved target sequence by a DNA ligase. In some embodiments, the double-stranded sequence containing the target sequence further includes a recognition site for an endonuclease, transposase, or recombinase, which incorporates the double-stranded sequence into the target polynucleotide. In some embodiments, homology regions on the template sequence described herein facilitate the insertion of the double-stranded sequence containing the target sequence into the cleaved target sequence.

[0148] In some embodiments, the method further includes generating a second double-strand polynucleotide cleavage at a second target sequence in the target polynucleotide. In some embodiments, the second target sequence is upstream of the target sequence. In some embodiments, the second target sequence is downstream of the target sequence. In some embodiments, the second double-strand polynucleotide cleavage is generated by a second Cas nuclease. In some embodiments, for example, one end of a double-strand sequence containing the target sequence generated by reverse transcriptase and / or DNA polymerase is bound to the cleaved target sequence, and the other end of the double-strand sequence is bound to the cleaved second target sequence, thereby substituting the target polynucleotide sequence between the target sequence and the second target sequence. Such embodiments are illustrated in Figure 1D. The Cas9 nuclease generates a double-strand break in the target polynucleotide. The target sequence is replicated using a template sequence in a Cas9 complex containing PBS and the target sequence. The generated double-stranded sequence can then be ligated by the NHEJ to another cleavage generated downstream by a second CRISPR / Cas complex. The sequence on the target polynucleotide between the two CRISPR / Cas complexes is replaced with the sequence of interest.

[0149] In some embodiments, a double-stranded sequence containing the target sequence is inserted into the cleaved target sequence by a DNA repair pathway. In embodiments where the method is performed in cells, the double-stranded sequence is inserted into the target sequence by cell-specific DNA repair pathway components. Examples of DNA repair pathways include non-homologous end joining (NHEJ), microhomology-mediated end joining (MMEJ), and homology-oriented repair (HDR). NHEJ does not require a homologous template. Generally, NHEJ has higher repair efficiency than HDR but lower fidelity, although errors are reduced if the double-strand break has a compatible sticky end or overhang. MMEJ 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 direct repair, and HDR repair is typically more fidelity than NHEJ and MMEJ but less efficient. In some embodiments, the method is performed under conditions sufficient for non-homologous end joining (NHEJ).

[0150] In some embodiments, a double-stranded sequence containing the target sequence, generated by, for example, reverse transcriptase and / or DNA polymerase, is inserted into the target sequence that has been cleaved by ligation. In some embodiments, ligation is carried out by a ligase, for example, a DNA ligase. In some embodiments, the method further includes providing a ligase. Ligases are further described herein. In some embodiments, the ligase is a T4 DNA ligase.

[0151] In some embodiments, the double-stranded sequence containing the target sequence, generated by, for example, reverse transcriptase and / or DNA polymerase, further includes a recognition site for an endonuclease, transposase, or recombinase. In some embodiments, the endonuclease, transposase, or recombinase incorporates the double-stranded sequence into the target polynucleotide. The mechanisms of sequence incorporation by endonucleases, transposases, and recombinases are known to those skilled in the art and are further described, for example, 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).

[0152] In some embodiments, the fusion protein comprises a Cas nuclease and a DNA ligase, and the composition comprises a double-stranded template polynucleotide, the double-stranded template polynucleotide comprising a target sequence. In some embodiments, a guide sequence of the polynucleotide in the composition or a guide polynucleotide can be hybridized to the target sequence. In some embodiments, the fusion protein is guided to the target sequence by hybridizing the guide sequence and the target sequence. In some embodiments, the contact step of the method is carried out under conditions sufficient to generate a double-stranded polynucleotide cleavage at the target sequence by the Cas nuclease. In some embodiments, the double-stranded template polynucleotide can be inserted into the cleaved target sequence by ligation. In some embodiments, the template sequence and the cleaved target sequence comprise complementary sticky ends, and the DNA ligase can ligate the sticky ends. In some embodiments, the template sequence and the cleaved target sequence comprise blunt ends, and the DNA ligase can ligate the blunt ends. In some embodiments, the contact step of the method is carried out under conditions sufficient to allow the DNA ligase to ligate a template sequence containing the sequence of interest to the cleaved target sequence, thereby incorporating the template sequence into the cleaved target sequence. Ligases are further described herein. In some embodiments, the ligase is a T4 DNA ligase. In some embodiments, the fusion protein comprises a Cas nuclease and a DNA ligase, the template sequence comprises the sequence of interest and a primer-binding sequence, and the method further comprises contacting the target polynucleotide with a reverse transcriptase. In some embodiments, the reverse transcriptase reverse transcribes the complementary strand of the sequence of interest, thereby forming a double-stranded sequence containing the sequence of interest as described herein. In some embodiments, the DNA ligase of the fusion protein ligates the double-stranded sequence to the cleaved target sequence.

[0153] In some embodiments, the composition comprises a polynucleotide including a guide sequence and a template sequence, the template sequence is located in close proximity to the cleavage site and the fusion protein. In some embodiments, the fusion protein further comprises a DNA-binding domain or RNA-binding domain for binding the template polynucleotide, thereby bringing the template sequence in close proximity to the cleavage site and the fusion protein. In some embodiments, proximity of the template sequence to the fusion protein enhances the activity of reverse transcriptase, DNA polymerase, or DNA ligase. In some embodiments, proximity of the template sequence to the cleavage site enhances the integration of the double-stranded sequence resulting from the reverse transcriptase or DNA polymerase reaction into the cleaved target sequence.

[0154] In some embodiments, the method improves the efficiency of incorporating a double-stranded sequence into a cleaved target sequence by providing the double-stranded sequence in close proximity to the cleaved target sequence. In some embodiments, the method improves the efficiency of incorporating a double-stranded sequence into a cleaved target sequence by reducing religation of the cleaved target sequence. In some embodiments, the method has improved efficiency compared to methods that utilize a Cas nuclease without fusion reverse transcriptase, DNA polymerase, or DNA ligase to generate a double-strand cleavage. In some embodiments, the method has at least 2 times, at least 5 times, at least 10 times, at least 20 times, at least 30 times, at least 40 times, at least 50 times, at least 60 times, at least 70 times, at least 80 times, at least 90 times, at least 100 times, at least 150 times, or at least 200 times or more efficiency compared to methods that utilize a Cas nuclease without fusion reverse transcriptase, DNA polymerase, or DNA ligase to generate a double-strand cleavage. In some embodiments, the method has improved efficiency compared to methods that do not bring the target sequence into close proximity to the cleaved target sequence. In some embodiments, the method has at least 2 times, at least 5 times, at least 10 times, at least 20 times, at least 30 times, at least 40 times, at least 50 times, at least 60 times, at least 70 times, at least 80 times, at least 90 times, at least 100 times, at least 150 times, or at least 200 times or more efficiency compared to methods that do not bring the target sequence into close proximity to the cleaved target sequence.

[0155] In some embodiments, the method can insert a long sequence into a target sequence. For example, the method can insert a sequence of about 10,000 nucleotides into a target sequence, provided that the reverse transcriptase or DNA polymerase has the ability to generate a sequence of that length. Examples of high-capacity reverse transcriptases and DNA polymerases are provided herein. In some embodiments, the sequence to be inserted is longer than about 5 nucleotides, longer than about 10 nucleotides, longer than about 15 nucleotides, longer than about 20 nucleotides, longer than about 25 nucleotides, longer than about 30 nucleotides, longer than about 35 nucleotides, longer than about 40 nucleotides, longer than about 45 nucleotides, or longer than about 50 nucleotides. In some embodiments, the sequence to be inserted is longer than about 1 to about 20,000 nucleotides. In some embodiments, the sequence to be inserted is longer than about 2 to about 17,000 nucleotides. In some embodiments, the sequence to be inserted is longer than about 3 to about 15,000 nucleotides. In some embodiments, the sequence to be inserted is longer than about 4 to about 12,000 nucleotides. In some embodiments, the target sequence is approximately 5 to 10,000 nucleotides long. In some embodiments, the target sequence is approximately 10 to 9,000 nucleotides long. In some embodiments, the target sequence is approximately 50 to 8,000 nucleotides long. In some embodiments, the target sequence is approximately 100 to 7,000 nucleotides long. In some embodiments, the target sequence is approximately 200 to 6,000 nucleotides long. In some embodiments, the target sequence is approximately 500 to 5,000 nucleotides long.

[0156] In some embodiments, the method is carried out in vitro. In some embodiments, the method is carried out in cells. Examples of cells are provided herein.

[0157] kit In some embodiments, the Disclosure provides a kit comprising a fusion protein provided 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, for example, a vector described herein.

[0158] In some embodiments, the kit further comprises a polynucleotide that forms a complex with the fusion protein. In some embodiments, the polynucleotide comprises tracrRNA. In some embodiments, the polynucleotide that forms a complex with the fusion protein is provided on a vector, for example, the vector described herein.

[0159] In some embodiments, the kit further comprises a template polynucleotide containing a template sequence for a reverse transcriptase or DNA polymerase. In some embodiments, the template polynucleotide is provided on a vector, for example, the vector described herein.

[0160] In some embodiments, the kit further comprises a polynucleotide containing tracrRNA. In some embodiments, the tracrRNA conjugates and / or activates the Cas nuclease of the fusion protein. In some embodiments, the polynucleotide containing tracrRNA is provided on a vector, for example, the vector described herein.

[0161] In some embodiments, the kit further comprises a DNA polymerase. In some embodiments, the kit further comprises phi29 DNA polymerase, DNA polymerase mu, DNA polymerase delta, or DNA polymerase epsilon. In some embodiments, the kit further comprises a DNA ligase. In some embodiments, the kit further comprises T4 DNA ligase. In some embodiments, the kit further comprises an RNase. In some embodiments, the kit further comprises an RNase H.

[0162] In some embodiments, the kit further comprises a reaction buffer and / or storage buffer for the fusion protein, DNA polymerase, DNA ligase, and / or RNase. In some embodiments, the kit further comprises reagents for performing DNA cleavage, reverse transcriptase, DNA polymerase, DNA ligase, and / or RNase reactions. In some embodiments, the reagents include ATP, dNTPs, MgCl2, Oligo(dT), and / or RNase inhibitors. In some embodiments, the kit comprises one or more controls, for example, a control target polynucleotide for the fusion protein. For example, the control target polynucleotide can be designed to be specifically cleaved with a certain efficiency by the Cas nuclease of the fusion protein, thereby allowing calibration of the Cas nuclease activity.

[0163] In some embodiments, the kit includes one or more containers. In some embodiments, the kit further includes consumables, such as tubes, vials, or plates designed to contain samples and / or reagents during one or more steps of the method; pipettes or pipette tips for transferring liquid samples and reagents; covers and seals for tubes, vials, plates, and / or other consumables used in the method; racks for holding the consumables; labels for identifying samples; and / or instructions for using the kit to provide site-specific modification in a target sequence of a target polynucleotide, as described herein.

[0164] Embodiments of the present invention are further described in the following sections: [Section 1] A fusion protein comprising (i) a Cas nuclease and (ii) a reverse transcriptase, DNA polymerase, DNA ligase, or a combination thereof, wherein the Cas nuclease is capable of generating double-stranded polynucleotide cleavage. [Section 2] The fusion protein according to item 1 above, wherein the Cas nuclease is Cas9, Cas12, or Cas14. [Section 3] The fusion protein according to item 2, wherein the Cas nuclease comprises a polypeptide sequence having at least 90% identity with any one of Sequence ID No. 1, 29, or 30. [Section 4] The fusion protein described in item 2 above, wherein the Cas9 is type IIB Cas9. [Section 5] The fusion protein according to item 1 above, wherein the fusion protein comprises a Cas nuclease and a reverse transcriptase. [Section 6] The fusion protein according to item 5 above, wherein the reverse transcriptase is MMLV reverse transcriptase or R2 reverse transcriptase. [Section 7] The fusion protein according to item 5 or 6, wherein the reverse transcriptase comprises a polypeptide sequence having at least 90% identity with any one of sequence numbers 2 to 3. [Section 8] The fusion protein according to item 1 above, wherein the fusion protein comprises a Cas nuclease and a DNA polymerase. [Section 9] The fusion protein according to item 7 above, wherein the DNA polymerase is phi29 DNA polymerase, T4 DNA polymerase, DNA polymerase mu, DNA polymerase delta, or DNA polymerase epsilon. [Section 10] The fusion protein according to item 7 or 8, wherein the DNA polymerase comprises a polypeptide sequence having at least 90% identity with any one of sequence numbers 4 to 6. [Section 11] The fusion protein according to item 1 above, wherein the fusion protein comprises a Cas nuclease and a DNA ligase. [Section 12] The fusion protein according to item 11 above, wherein the DNA ligase is T4 DNA ligase. [Section 13] The fusion protein according to item 11 or 12, wherein the DNA ligase comprises a polypeptide sequence having at least 90% identity with SEQ ID NO: 7. [Section 14] A fusion protein according to any one of items 1 to 13 above, further comprising a DNA-binding or RNA-binding domain. [Section 15] The fusion protein according to item 14 above, wherein the DNA-binding domain is a zinc finger DNA-binding domain, a transcription factor, or an adeno-associated virus Rep protein. [Section 16] The fusion protein according to item 14 above, wherein the RNA-binding domain is MS2 coat protein (MCP2). [Section 17] The fusion protein described in item 14 above, wherein the RNA-binding domain includes a KH domain. [Section 18] The fusion protein described in item 17 above, wherein the RNA-binding domain is heteronuclear ribonucleoprotein K (hnRNPK). [Section 19] The fusion protein described in item 14 above, wherein the DNA-binding domain is capable of binding single-stranded DNA (ssDNA). [Section 20] The fusion protein described in item 19 above, wherein the DNA-binding domain is a far upstream element-binding protein (FUBP). [Section 21] The fusion protein according to any one of items 14 to 20, wherein the DNA-binding or RNA-binding domain comprises a polypeptide sequence having at least 90% identity with any one of sequence numbers 8 to 11. [Section 22] The fusion protein according to any one of the above items 1 to 21, further comprising a polypeptide linker between (i) and (ii). [Section 23] The fusion protein according to item 1 above, comprising a polypeptide sequence having at least 90% identity with any one of sequence numbers 18 to 26. [Section 24] a) A fusion protein described in any one of items 1 to 23 above, b) A polynucleotide that forms a complex with the fusion protein and comprises (i) a guide sequence and (ii) a template sequence for the reverse transcriptase, the DNA polymerase, or the DNA ligase, A composition containing the following: [Section 25] The composition according to item 24, wherein the polynucleotide comprises RNA. [Section 26] The composition according to item 24, wherein the guide sequence comprises RNA and the template sequence comprises DNA. [Section 27] The composition according to item 24, wherein the template arrangement comprises a debase site, a triethylene glycol (TEG) linker, or both. [Section 28] The composition according to any one of the above items 24 to 27, wherein the guide sequence is about 15 to about 20 nucleotides in length. [Section 29] The composition according to any one of items 24 to 28, wherein the polynucleotide further comprises tracrRNA. [Section 30] The composition according to any one of items 24 to 28, wherein the composition comprises a second polynucleotide containing tracrRNA. [Section 31] The composition according to any one of items 24 to 30, wherein the template sequence includes a primer-binding sequence and a target sequence. [Section 32] The composition according to item 31, wherein the primer-binding sequence and the target sequence include DNA. [Section 33] The composition according to item 31 above, wherein the target sequence includes DNA. [Section 34] The composition according to any one of the above items 24 to 33, wherein the template sequence has a length of about 25 to about 10,000 nucleotides. [Section 35] The composition according to any one of the above items 24 to 34, wherein the primer-binding sequence is about 4 to about 30 nucleotides in length. [Section 36] The composition according to any one of items 24 to 35 above, wherein the target sequence is approximately 5 nucleotides long to approximately 9000 nucleotides long. [Section 37] The composition according to any one of items 24 to 36, wherein the polynucleotide includes a spacer between the guide sequence and the template sequence. [Section 38] The composition according to item 37, wherein the spacer has a length of about 10 to about 200 nucleotides. [Section 39] The composition according to item 37 or 38, wherein the spacer comprises a stop sequence for the reverse transcriptase or DNA polymerase. [Section 40] The composition according to item 39, wherein the spacer comprises two or more stop arrays. [Section 41] The composition according to item 39 or 40, wherein the termination sequence includes a secondary structure. [Section 42] The composition according to item 41, wherein the secondary structure is a hairpin loop. [Section 43] a) A fusion protein described in any one of items 1 to 23 above, b) A guide polynucleotide that forms a complex with the fusion protein and includes a guide sequence, c) A composition comprising a template polynucleotide containing a template sequence for the reverse transcriptase, the DNA polymerase, or the DNA ligase. [Section 44] The composition according to item 43, wherein the guide polynucleotide is RNA. [Section 45] The composition according to item 43, wherein the template polynucleotide comprises RNA. [Section 46] The composition according to item 43, wherein the template sequence includes DNA. [Section 47] The composition according to item 43, wherein the template arrangement comprises a debase site, a triethylene glycol (TEG) linker, or both. [Section 48] The composition according to any one of the above items 43 to 47, wherein the guide sequence is about 15 to about 20 nucleotides in length. [Section 49] The composition according to any one of the above items 43 to 48, wherein the guide polynucleotide further comprises tracrRNA. [Section 50] The composition according to any one of the above claims 43 to 48, wherein the composition further comprises a third polynucleotide containing tracrRNA. [Section 51] The composition according to any one of the above claims 43 to 50, wherein the template sequence has a length of about 25 to about 10,000 nucleotides. [Section 52] The composition according to any one of the above items 43 to 51, wherein the template sequence includes the sequence to be targeted. [Section 53] The composition according to item 52, wherein the target sequence is approximately 5 nucleotides to approximately 9800 nucleotides in length. [Section 54] The composition according to item 52 or 53, wherein the target sequence includes DNA. [Section 55] The composition according to any one of claims 43 to 54, wherein the template polynucleotide further comprises a primer-binding sequence. [Section 56] The composition according to item 55, wherein the primer-binding sequence is about 4 to about 30 nucleotides in length. [Section 57] The composition according to item 55 or 56, wherein the primer-binding sequence and the target sequence include DNA. [Section 58] The composition according to any one of claims 43 to 57, wherein the template polynucleotide further comprises a stop sequence for the reverse transcriptase or DNA polymerase. [Section 59] The composition according to item 58, wherein the template polynucleotide comprises two or more stop sequences. [Section 60] The composition according to item 58 or 59, wherein the termination sequence includes a secondary structure. [Section 61] The composition according to item 60, wherein the secondary structure is a hairpin loop. [Section 62] The composition according to any one of items 43 to 61, wherein the template polynucleotide comprises an adeno-associated virus (AAV) vector containing the sequence of interest. [Section 63] A polynucleotide encoding the fusion protein described in any one of the above items 1 to 23. [Section 64] A vector comprising the polynucleotide encoding the fusion protein described in any one of items 1 to 23 above. [Section 65] A cell containing the fusion protein described in any one of the above items 1 to 23. [Section 66] A cell containing the polynucleotide encoding the fusion protein described in any one of items 1 to 23 above, or the vector described in item 64 above. [Section 67] Cells containing the composition described in any one of items 24 to 62 above. [Section 68] A method for providing site-specific modification in a target sequence within a target polynucleotide, comprising contacting the target polynucleotide with a composition described in any one of items 24 to 62 above. [Section 69] The method according to item 68, wherein the target polynucleotide is DNA. [Section 70] The method according to item 68 or 69, wherein the guide sequence can hybridize to the target sequence. [Section 71] The method according to any one of items 68 to 70, wherein the contact is carried out under conditions sufficient to cause the Cas nuclease to generate double-stranded polynucleotide cleavage in the target sequence. [Section 72] The method according to any one of items 68 to 71, wherein the template sequence includes the sequence to be targeted. [Section 73] The method according to any one of claims 68 to 72, wherein the template sequence includes a primer-binding sequence that can hybridize to the target sequence. [Section 74] The method according to any one of items 68 to 73, wherein the contact is carried out under conditions sufficient to allow the reverse transcriptase to transcribe the complementary strand of the target sequence. [Section 75] The method according to item 74, further comprising cleaving the template sequence to generate a double-stranded sequence containing the sequence of interest. [Section 76] The method according to item 75, wherein the cleavage is performed by RNase H. [Section 77] The method according to any one of items 68 to 72, wherein the contact is carried out under conditions sufficient to cause the DNA polymerase to generate a double-stranded sequence containing the target sequence. [Section 78] The method according to any one of items 68 to 72, wherein the contact is made under conditions sufficient to cause the DNA ligase to ligate the sequence of interest to the cleaved target sequence. [Section 79] The method according to any one of the above claims 71 to 78, wherein the double-stranded sequence containing the target sequence is inserted into the cleaved target sequence by non-homologous end joining (NHEJ). [Section 80] The method according to any one of items 71 to 78, wherein the double-stranded sequence containing the target sequence is inserted into the cleaved target sequence by a DNA ligase. [Section 81] The method according to any one of claims 68 to 77, further comprising generating a second double-stranded polynucleotide cleavage at a second target sequence in the target polynucleotide. [Section 82] The method according to item 81, wherein the target sequence replaces the sequence of the target polynucleotide between the target sequence and the second target sequence. [Section 83] A kit containing the fusion protein described in any one of the above items 1 to 23. [Section 84] The kit according to item 83, further comprising a polynucleotide that forms a complex with the fusion protein and / or a vector for expressing the polynucleotide. [Section 85] The kit according to item 83, further comprising a template polynucleotide containing a template sequence for the reverse transcriptase, the DNA polymerase, or the DNA ligase, and / or a vector for expressing the template polynucleotide. [Section 86] The kit according to item 83 or 84 above, further comprising a polynucleotide containing tracrRNA. [Section 87] A kit as described in any one of items 83 to 86 above, further comprising RNase H. All references cited herein, such as patents, patent applications, articles, textbooks, etc., and references cited therein, are incorporated herein by reference in their entirety, unless they have already been cited. [Examples]

[0165] Example 1. In this example, Cas9 and Cas9 fused to reverse transcriptase ("PRINS") were introduced into cells along with the corresponding guide RNA.

[0166] HEK293 cells were placed in 1 mL of complete growth medium (DMEM + 10% fetal bovine serum) in a 12-well plate, 2 × 10⁶ cells per well. 5Cells were seeded at the cell density the day before transfection. The CRISPR complex components were prepared by combining 0.55 μg of plasmid expressing wild-type Cas9 or PRINS with 0.55 μg of gRNA targeting the AAVS1 locus, in a total volume of 52 μL. The guide RNA sequences for PRINS are described in SEQ ID NOs. 27-28, targeting the AAVS1 site and inserting the AAGATG sequence. 3.3 μL of FUGENE® HD reagent was added to this mixture. The solution was carefully mixed by pipetting (approximately 15 times) or by simple vortexing, and then incubated at room temperature for 5-10 minutes. 50 μL of the complex was added to each well containing cells, and the wells were shaken.

[0167] Three days after transfection, genomic DNA was extracted, and the edited sequences were amplified using Amplicon-Seq. Rational InDel Meta-Analysis (RIMA) was performed on the Amplicon-Seq data to analyze Cas9-induced changes, as described in Taheri-Ghahfarokhi et al., Nucleic Acids Res 46(16):8417-8434 (2018).

[0168] The results are shown in Figures 3A and 3B. As shown in Figure 3A, most cells transfected with Cas9 had variable-length deletions. In Figure 3B, cells transfected with PRINS had a higher number of insertion events (indicated by ellipses) and higher editing efficiency compared to Cas9.

[0169] Example 2. In this example, the Cas9 nickase fused to RT ("PE") and the Cas9 fused to RT (PRINS), together with the corresponding prime editing guide RNA (pegRNA) for PE and the single primed editing insertion guide RNA (springRNA) for PRINS, were both introduced into cells targeting the AAVS1 site described in Example 1. PE and pegRNA are described in Anzalone et al., Nature 576:149-157 (2019). In summary, the pegRNA comprises a guide sequence complementary to the target sequence, and a template sequence comprising an insertion sequence (AAGATG) flanked by two regions homologous to the target sequence, one of which functions as a primer binding sequence. The springRNA comprises a guide sequence complementary to the target sequence, a template sequence comprising an insertion sequence (AAGATG), and a primer-binding sequence.

[0170] Figures 5A and 5B show the insertion frequencies of PRINS / springRNA and PE / pegRNA, respectively. Relative editing frequencies were determined by fragment analysis (see Yang et al., Nucleic Acids Research 43(9):e59 (2015)). PRINS had an insertion rate of 42.4%, which is more efficient than PE, which only had 14.3% insertion.

[0171] To demonstrate the dependence of PRINS on NHEJ, the same experiment was repeated using 2.5 μM of an inhibitor for the specific DNA-dependent protein kinase (DNAPK) known to be involved in NHEJ. The results in Figures 5C and 5D show the insertion frequencies of PRINS / springRNA and PE / pegRNA, respectively. No effect of DNAPK inhibition was observed for PE (Figure 5D), while PRINS showed reduced insertion frequency in the presence of the DNAPK inhibitor (Figure 5C).

[0172] Example 3. In this example, the Cas9 nickase fused to RT ("PE") and the Cas9 fused to RT (PRINS) were both tested with pegRNA targeting the AAVS1 site, as described in Example 2.

[0173] The insertion frequency was analyzed by fragment analysis as described in Example 2. The results in Figure 6 show that pegRNA can promote insertion by PRINS. As described in Anzalone et al., Nature 576:149-157 (2019), PRINS may be able to utilize pegRNA in a similar manner to PE.

[0174] Example 4. Determination of the mechanism of action of PRINS editing versus prime editing. In this example, the mechanism of action of Cas9 fused to RT for PRINS editing was evaluated and compared with the mechanism of Cas9 nickase fused to RT for prime editing. To determine whether PRINS editing and prime editing utilize non-homologous end joining (NHEJ) for DNA repair, inhibitors of DNA-dependent protein kinases (DNA-PKs), known enzymes in the NHEJ pathway, were introduced.

[0175] HEK-T cells were treated with the DNA-PK inhibitor AZD7648 for 4 hours, and then transfected with components for PRINS editing and prime editing as described above for Example 2. The percentage of specific 6bp incorporations (AAGATG) into the AAVS1 locus was evaluated using NGS Amplicon-Seq.

[0176] The results are shown in Figure 7. The bar graph shows the standard deviation of the mean for n=2. Bars labeled "#1" or "#2" represent various spring RNAs (in the case of PRINS editing) or various pegRNAs (in the case of prime editing). The data showed that PRINS-mediated integration was significantly reduced by DNA-PK inhibition, while prime editing was relatively unaffected.

[0177] Example 5. Evaluation of DNA and RNA template sequences and DNA polymerase fusions. In this example, spring RNA was prepared using either a DNA template sequence ("DNA tail") or an RNA template sequence ("RNA tail"). Fusions of Cas9+RT ("PE0"), Cas9+DNA polymerase D ("PE0 PolD"), Cas9+Phi29 DNA polymerase ("PE0 Phi"), and Cas9 control were tested. Three guide RNAs (one containing an RNA tail ("123RNA MS") and two containing DNA tails ("123DNA" and "123DNA PS")) were synthesized by Agilent. The sequences are shown in Table 1.

[0178] [Table 1]

[0179] On day 1, the fusion protein was transfected into cells using FUGENE, and on day 2, the guide RNA was transfected using RNAiMAX.

[0180] The results are shown in Figures 8–12. Figure 8 provides an overview of the editing efficiency of various proteins. All fusion proteins achieved higher editing efficiency with DNA tail sequences compared to Cas9. The top, middle, and bottom panels of Figures 9–12 show the editing patterns of the indicated proteins (PE0, PE0 PolD, PE0 Phi, or Cas9) using 123RNA MS tails, 123DNA tails, or 123DNA PS tails, respectively. Surprisingly, guide RNA containing DNA tails achieved similar editing patterns using PE0, as shown in Figure 9. Figures 10 and 11 show that DNA polymerases PolD and Phi29 can copy DNA tails but not RNA tails.

[0181] Example 6. Evaluation of the guide array In this embodiment, various guide sequences were designed and their effects on DNA editing by PRINS editing or prime editing were evaluated. As described in the embodiments herein, PRINS editing utilizes a single PRINS guide RNA (springRNA) to target and modify a specific genomic locus. In addition to the spacer and scaffold sequences found in conventional sgRNAs for Cas9 targeting systems, the springRNA includes a 3' extension containing a primer-binding site (PBS) that hybridizes to the target DNA strand and functions as a primer for reverse transcription. The PBS is followed by a DNA synthesis template containing the desired modification. In comparison, the prime editing guide RNA (pegRNA) includes an additional homology region following the DNA synthesis template, as shown in Figure 13.

[0182] To study the effects of different primer designs on PRINS editing and prime editing, HEK-T cells were co-transfected with PRINS editing and prime editing components in the absence or presence of the DNA-PK inhibitor AZD7648, as described above in Example 2 and Example 4.

[0183] The results are shown in Figures 14A and 14B. The data represent the percentage of specific 6bp incorporation (AAGATG) into the AAVS1 locus using PRINS editing (Figure 14A) and prime editing (Figure 14B). The bar graphs represent the standard deviation of the mean for n=2. Bars labeled "#1" or "#2" indicate different springRNA and pegRNA designs, as shown in Figure 13. The results show that PRINS editing works with both springRNA and pegRNA designs. The combination of PRINS editing with pegRNA and a DNA-PK inhibitor yielded the best specific editing, being twice as good as prime editing when using the same pegRNA. Prime editing produced detectable modifications with pegRNA but not with springRNA.

[0184] Example 7. Evaluation of PRINS editing toxicity In this example, the toxicity of PRINS editing compared to Cas9 editing was evaluated by determining the number of large deletions induced after the generation of double-strand breaks.

[0185] The amount of large deletions was evaluated using diphtheria toxin (DT) selection systems (e.g., those described in U.S. Provisional Application No. 62 / 833,404 and PCT / EP2020 / 060250, filed April 12, 2020). Figure 15 shows a schematic of the experimental design. In summary, the intron of HbEGF, the DT receptor, was selected as the PRINS or Cas9 editing target. Only large deletions of the biallelic gene provide DT resistance to cells, and therefore, cell viability after DT treatment indicates the amount of large deletion.

[0186] Cells were transfected with Cas9-RT fusion (PRINS edited, "PE0"), Cas9, or Cas9 nickase-RT fusion (prime edited, "PE2"), and three different guide RNAs. The results in Figure 16 show that after transfecting the same number of cells with the same amount of DNA, the PE0 plate showed fewer cells compared to the Cas9 plate, indicating fewer large deletions due to PRINS editing. The number of large deletions due to PRINS editing was comparable to that of prime editing with PE2.

[0187] Example 8. Evaluation of exogenous template polynucleotides In this example, the addition of exogenous template polynucleotides not fused to guide RNA for PRINS editing or prime editing was evaluated.

[0188] A schematic diagram of the experimental design is shown in Figure 17. The MCP domain that binds to the MS2 aptamer was fused to the Cas9-RT protein used for PRINS editing, either between Cas9 and RT ("PRINS_MS2_v1") or downstream of RT ("PRINS_MS2_v2"). The reverse transcription template was fused to the MS2 aptamer instead of the guide RNA. PRINS_MS2, the MS2-RT template, and the target gRNA were co-transfected into HEK-T cells and tested for targeted insertion. The control gRNA and the RT template fused to the gRNA served as negative and positive controls, respectively.

[0189] The results in Figure 18 show that, despite lower editing efficiency compared to PRINS editing using a gRNA-fused RT template, the DNA sequence was successfully replicated and inserted specifically from the MS2-RT template by PRINS editing.

[0190] Example 9. Evaluation of Cas12 fusion for PRINS editing In this example, the Cas12-RT fusion protein was evaluated for its PRINS editing and prime editing capabilities.

[0191] RT was fused to LbCas12 (also known as LbCpf1). Guide RNAs were designed for PRINS editing (springRNA) and prime editing (pegRNA) at the EMX1 and DNMT1 sites. An exemplary guide RNA targeting EMX1 is shown in Figure 19 and includes the following sequences, where single underlines indicate insert sequences and double underlines indicate homologous sequences: [ka]

[0192] The insertion at the EMX1 site using the above guide RNA was determined as shown in Table 2.

[0193] [Table 2]

[0194] The mutation types were determined as shown in Table 3.

[0195] [Table 3]

[0196] The results in Tables 2 and 3 show that the DNA sequence was successfully replicated and inserted specifically by the Cas12-RT fusion protein using PRINS editing. The overall editing efficiency was approximately 0.25%.

[0197] Example 10. PRINS editing using Cas9-DNA polymerase fusion. Cas9 fused to DNA polymerase was evaluated for PRINS editing. DNA polymerase has been reported to exhibit reverse transcriptase activity in vitro and in vivo (see, e.g., Ricchetti et al., EMBO J.12(2):387-396(1993)). As shown below, plasmids expressing Cas9, Cas9-RT fusion ("PE0"), or Cas9 fused to DNA polymerase were transfected into HEK293T cells along with plasmids expressing single-priming edit insertion guide RNA (springRNA) targeting the AAVS1 locus. The Cas9-DNA polymerase fusion contained the following DNA polymerase constructs:

[0198] Cas9-Klenow exo + : Codon-optimized Klenow fragment of E. coli DNA polymerase I;

[0199] Cas9-Klenow exo - : Codon-optimized Klenow fragments of E. coli DNA polymerase I with D355A and E357A mutations that disable the 3'→5' exonuclease activity of DNA polymerase;

[0200] Cas9-REV3: This catalytically active truncation of human REV3 polymerase was found to be more stable and expressive compared to full-length REV3 (referred to as REV TR5: see Lee et al., PNAS (2014), doi:10.1073:pnas.1324001111).

[0201] Cells were harvested 72 hours after transfection. Genomic DNA was extracted, the AAVS1 locus was amplified by PCR, and sequencing was performed using the Illumina sequencing platform.

[0202] The results in Figure 20 show that the three Cas9-DNA polymerase fusion proteins were capable of PRINS editing.

[0203] Example 11. PRINS editing using Cas9-DNA polymerase fusion and chimeric spring RNA. Chimeric spring RNAs were evaluated by PRINS editing using Cas9, PE0, and Cas9-DNA polymerase fusion proteins. HEK293T cells were transfected with plasmids expressing Cas9, PEO, or the three Cas9-DNA polymerase fusion proteins described in Example 10 using FUGENE® HD. After 24 hours, the cells were further transfected with 2 pmol of one of the following synthetic spring RNAs using LIPOFECTAMINE® RNAiMAX:

[0204] springRNA - all RNA nucleotides; the sequence includes a guide RNA sequence; a tracrRNA scaffold for Cas9 binding; and a 6-nucleotide insertion sequence ("AATATG") and primer binding site (PBS) at the 3' of springRNA;

[0205] For chimeric spring RNA and DiHP-spring RNA, all RNA nucleotides except for the 10 nucleotides in PBS, including the same sequence as above, are deoxyribonucleotides;

[0206] For chimeric spring RNA and DiRP-spring RNA, all RNA nucleotides except for the inserted sequence are deoxyribonucleotides, and the sequence is the same as described above.

[0207] Cells were harvested 48 hours after transfection. Genomic DNA was extracted, the AAVS1 locus was amplified by PCR, and sequencing was performed using the Illumina sequencing platform.

[0208] The results in Figures 21A-C show that the Cas9-DNA polymerase fusion protein was able to perform PRINS editing with efficiency comparable to PE0 when using spring RNA containing chimeric DNA.

[0209] Example 12. PRINS editing using Cas9-DNA polymerase fusion and modified springRNA. Various chemically modified spring RNAs were evaluated using PRINS editing. HEK293T cells were transfected with a plasmid expressing Cas9 or PE0 using FUGENE® HD. After 24 hours, the cells were further transfected with 2 pmol of one of the following spring RNAs using LIPOFECTAMINE® RNAiMAX:

[0210] springRNA - all RNA nucleotides; the sequence includes a guide RNA sequence; a tracrRNA scaffold for Cas9 binding; and a 6-nucleotide insertion sequence ("AATATG") and a primer binding site (PBS) at 3' of the springRNA;

[0211] All RNA nucleotides except that the spring RNA-spring RNA has the same sequence as above, with the third nucleotide of the inserted sequence replaced by a dSpacer nucleotide 1'2'-dideoxyribose (abase site);

[0212] All RNA nucleotides in spring RNA with a TEG linker, except that the third nucleotide of the inserted sequence is covalently bonded to triethylene glycol (TEG).

[0213] Cells were harvested 48 hours after transfection. Genomic DNA was extracted, the AAVS1 locus was amplified by PCR, and sequencing was performed using the Illumina sequencing platform.

[0214] The results in Figure 22 show that the chemically modified spring RNA prevented excessive elongation of the insertion and improved the accuracy of mutagenesis.

[0215] Example 13. PRINS editing using Cas9-DNA ligase fusion. Cells were transfected with Cas9 and RT on separate expression plasmids and plasmids containing spring RNA, and evaluated for PRINS editing. As shown in Figure 23A, PRINS editing still occurred with co-expression of Cas9 and RT proteins (asterisks indicate wild-type sequences).

[0216] Next, Cas9 fused to DNA ligase was evaluated for PRINS editing. Cas9 was fused to Mycobacterium tuberculosis LigD, a DNA ligase involved in non-homologous end joining of DNA cleavage ("Cas9-LigD"). Plasmids expressing the Cas9-LigD fusion protein were co-transfected with plasmids expressing RT and spring RNA plasmids, and PRINS editing was evaluated.

[0217] The results in Figure 23B show that simultaneous transfection of the Cas9-LigD fusion protein and RT improved the insertion of the desired sequence compared to simultaneous expression of Cas9 and RT.

[0218] Example 14. PBS mismatch in insert and springRNA To reduce the 5'-3' homology of the spring RNA, a mismatch was introduced at the primer-binding site (PBS) of the spring RNA, resulting in two mismatches between the 3' ends of the target DNA strand annealed to the PBS. Typically, the presence of a 3' mismatch with the template reduces the priming efficiency of the DNA. Surprisingly, as shown in Figures 24A-24B, insertion of a 4bp insertion sequence (the original 6bp sequence minus a 2bp mismatch) was more efficient than insertion of a perfectly complementary 6bp insertion. The 4bp insertion with a 2bp mismatch had a relative insertion efficiency of 59.59% (Figure 24B), while the 6bp insertion without a mismatch had a relative insertion efficiency of 37.13% (Figure 24A).

[0219] Example 15. DNA repair pathway and prime editing efficiency of PRINS The PRINS editing efficiency of PE0 using springRNA and the prime editing efficiency of PE0 using pegRNA were evaluated in cell lines partially lacking the following DNA repair genes: PRKDC (also known as DNAPK), LIG4, TP53BP1, PARP1, POLQ, LIG3, and ATM. Cells were also cultured in or without DNAPK inhibitors.

[0220] The results are shown in Figure 25, and the deletion of these genes or inhibition of the PRKDC protein reduced PRINS efficiency, indicating that PRINS editing is dependent on NHEJ pathway enzymes such as PRKDC and TP53BP1. Figure 25 also shows that PRINS editing with PE0 and pegRNA was inversely correlated with NHEJ enzymes, as inhibition or deletion of PRKDC, LIG4, or TP53BP1 resulted in higher insertion efficiency.

[0221] Example 16. Evaluation of Type II-B Cas9 fusion for PRINS editing. A fusion protein containing the type II-B Cas9 protein and Cas9 (MHCas9) derived from the sequenced intestinal metagenogenome MH0245_GL0161830.1 generates sticky ends ("overhangs") and MMLV reverse transcriptase. Spring RNA was designed to bind to MHCas9 and contain a 6-nucleotide insertion sequence targeting the AAVS1 locus as described in Example 10. HEK293T cells were transfected, genomic DNA was extracted, and the targeted insertion was detected using Amplicon-Seq.

[0222] The results in Figure 26A demonstrate that the MHCas9-RT fusion protein successfully performed PRINS-mediated insertions at the target gene locus. The most efficient insertions had an insertion frequency of 0.072%. Figure 26B shows the 10 most frequent editing events by MHCas9-RT. RT not only mediated the insertion of the insertion sequence but also extended overhang sequences (CCCs) generated by MHCas9, as indicated by the three most frequent editing events.

[0223] Example 17. Targeted insertion and deletion using MHCas9-RT fusion The Cas9-RT fusion protein ("PE0") described in the previous example was evaluated for its ability to perform targeted insertions and deletions using pegRNA. In contrast to prime editing using the Cas9 nickase-RT fusion and pegRNA, PE0 using pegRNA introduces double-strand DNA breaks and is therefore repaired by a double-strand DNA break repair pathway that is not involved in prime editing. PegRNA and prime editing are described in Example 2 and Anzalone et al., Nature 576:149-157 (2019).

[0224] HEK293T cells were transfected with a plasmid expressing MHCas9-RT and pegRNA targeting the AAVS1 site, as described in the previous example. Two different pegRNA constructs were tested: 1) a construct providing a one-nucleotide deletion; and 2) a construct for generating an A-to-G substitution at the PAM-3 site. Following transfection, genomic DNA was extracted and processed by NGS, as described in the previous example.

[0225] The results in Figures 27A (substitution of A to G) and 27B (deletion of one nucleotide) demonstrate that PE0 using pegRNA can induce substitutions / insertions and deletions. In the bar graphs of Figures 27A and 27B, the dark gray areas represent desired mutations, and the light gray areas represent undesirable mutations. The experiment was also performed in the presence of a DNAPK inhibitor (DNAPKi), which increased the percentage of desired mutations compared to undesirable mutations.

[0226] array Various polynucleotide and polypeptide sequences are provided herein.

[0227] Amino acid sequence of Cas9 nuclease (SEQ ID NO: 1) [ka]

[0228] Amino acid sequence of Cas12 nuclease (LbCas12a) (SEQ ID NO:) 38 ) [ka]

[0229] Amino acid sequence of Cas14 nuclease (Cas14a1) (SEQ ID NO:) 39 ) [ka]

[0230] Amino acid sequence of MMLV reverse transcriptase (SEQ ID NO: 2) [ka]

[0231] Amino acid sequence of R2 reverse transcriptase (SEQ ID NO: 3) [ka]

[0232] Phi29 DNA polymerase amino acid sequence (SEQ ID NO: 4) [ka]

[0233] Amino acid sequence of DNA polymerase delta (SEQ ID NO: 5) [ka]

[0234] Amino acid sequence of T4 DNA polymerase (SEQ ID NO: 6) [ka]

[0235] Amino acid sequence of T4 DNA ligase (SEQ ID NO: 7) [ka]

[0236] Amino acid sequence of MEPC2 (SEQ ID NO: 8) [ka]

[0237] Amino acid sequence of the Rep protein (SEQ ID NO: 9) [ka]

[0238] Amino acid sequence of T4 gene 32 protein (SEQ ID NO: 10) [ka]

[0239] FUBP amino acid sequence (SEQ ID NO: 11) [ka]

[0240] Nuclear localization sequences (sequences 12-14) MKRTADGSEFESPKKKRKV (Sequence ID 12) SGGSKRTADGSEFEPKKKRKV (Sequence ID 13) PKKKRKV (Sequence ID 14)

[0241] Linker sequence (sequences 15-16) SGGSSGGSSGSETPGTSESATPESSGGSSGGS (Sequence No. 15) SGGSSGGSSGSETPGTSESATPESSG (Sequence No. 16)

[0242] Amino acid sequence of REP_Y156F(1-197)-Cas9 P2A EGFP (SEQ ID NO: 17) [ka]

[0243] Amino acid sequence of Cas9-MMLV RT (SEQ ID NO: 18) [ka]

[0244] Amino acid sequence of MCP2-RT (SEQ ID NO: 19) [ka]

[0245] Amino acid sequence of Cas9-Phi29 (SEQ ID NO: 20) [ka]

[0246] Amino acid sequence of Cas9-PolD (SEQ ID NO: 21) [ka]

[0247] Amino acid sequence of Cas9-R2 RT (SEQ ID NO: 22) [ka]

[0248] Amino acid sequence of Cas9-T4 DNA ligase (SEQ ID NO: 23) [ka]

[0249] Amino acid sequence of Cas9-MCP2 MMLV RT (SEQ ID NO: 24) [ka]

[0250] Amino acid sequence of Cas9-T4 DNA Pol (SEQ ID NO: 25) [ka]

[0251] Amino acid sequence of T4gp32-FUBP (SEQ ID NO: 26) [ka]

[0252] Polynucleotide sequence of AAVS 123 AAGATG gRNA (SEQ ID NO: 27) [ka]

[0253] AAVS 123 AAGATG 20 Polynucleotide sequence of elongated gRNA (SEQ ID NO: 28) [ka]

Claims

1. A fusion protein comprising (i) a Cas nuclease having at least 90% identity with SEQ ID NO: 1, and (ii) a reverse transcriptase, wherein the Cas nuclease is capable of generating double-strand breaks in double-stranded polynucleotides.

2. The fusion protein according to claim 1, wherein the reverse transcriptase is MMLV reverse transcriptase or R2 reverse transcriptase.

3. The fusion protein according to claim 1 or 2, wherein the reverse transcriptase comprises a polypeptide sequence having at least 90% identity with any one of SEQ ID NOs: 2 to 3.

4. The fusion protein according to any one of claims 1 to 3, further comprising a polypeptide linker between (i) and (ii).

5. The fusion protein according to claim 1, comprising a polypeptide sequence having at least 90% identity with SEQ ID NO:

18.

6. a) A fusion protein according to any one of claims 1 to 5, b) A polynucleotide that forms a complex with the fusion protein and comprises (i) a guide sequence and (ii) a template sequence for the reverse transcriptase, A composition comprising, wherein the template sequence comprises a primer-binding sequence and a target sequence.

7. The composition according to claim 6, wherein the polynucleotide comprises RNA.

8. The composition according to claim 6, wherein the template arrangement comprises a debase site, a triethylene glycol (TEG) linker, or both.

9. The composition according to any one of claims 6 to 8, wherein the guide sequence is 15 to 20 nucleotides long.

10. The composition according to any one of claims 6 to 9, wherein the polynucleotide further comprises tracrRNA.

11. The composition according to any one of claims 6 to 10, wherein the composition comprises a second polynucleotide containing tracrRNA.

12. The composition according to any one of claims 6 to 11, wherein the template sequence has a length of 25 to 10,000 nucleotides.

13. The composition according to any one of claims 6 to 12, wherein the primer-binding sequence is 4 to 30 nucleotides long.

14. The composition according to any one of claims 6 to 13, wherein the target sequence is 5 nucleotides to 9,000 nucleotides in length.

15. The composition according to any one of claims 6 to 14, wherein the polynucleotide includes a spacer between the guide sequence and the template sequence.

16. The composition according to claim 15, wherein the spacer has a length of 10 to 200 nucleotides.

17. The composition according to claim 15 or 16, wherein the spacer comprises a stop sequence for the reverse transcriptase.

18. The composition according to claim 17, wherein the spacer includes two or more stop arrays.

19. The composition according to claim 17 or 18, wherein the stop sequence includes a secondary structure.

20. The composition according to claim 19, wherein the secondary structure is a hairpin loop.

21. An in vitro method for providing site-directed modification in a target sequence within a target polynucleotide, comprising contacting the target polynucleotide with a composition according to any one of claims 6 to 20.

22. The method according to claim 21, wherein the target polynucleotide is DNA.

23. The method according to claim 21 or 22, wherein the guide sequence can hybridize to the target sequence.

24. The method according to any one of claims 21 to 23, wherein the contact is carried out under conditions sufficient to cause the Cas nuclease to generate double-stranded polynucleotide cleavage in the target sequence.

25. The method according to any one of claims 21 to 24, wherein the contact is carried out under conditions sufficient for the reverse transcriptase to transcribe the complementary strand of the target sequence.

26. The method according to claim 25, further comprising cleaving the template sequence to generate a double-stranded sequence containing the sequence of interest.

27. The method according to claim 26, wherein the cleavage is performed by RNase H.

28. The method according to claim 26 or 27, wherein the double-stranded sequence containing the target sequence is inserted into the cleaved target sequence by non-homologous end joining (NHEJ).

29. A kit comprising the fusion protein described in any one of claims 1 to 5.

30. The kit according to claim 29, further comprising a polynucleotide that forms a complex with the fusion protein and / or a vector for expressing the polynucleotide.

31. The kit according to claim 29, further comprising a template polynucleotide containing a template sequence for the reverse transcriptase.

32. The kit according to any one of claims 29 to 31, further comprising RNase H.

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