Gene editing systems including CRISPR nucleases and their uses

Abstract

1. A gene editing system comprising: (a) a type V CRISPR nuclease polypeptide or a first nucleic acid encoding a type V CRISPR nuclease polypeptide; (b) a reverse transcriptase (RT) polypeptide or a second nucleic acid encoding a RT polypeptide; (c) a guide RNA (gRNA) or a third nucleic acid encoding a gRNA, wherein the gRNA comprises one or more binding sites recognizable by a type V CRISPR nuclease (CRISPR nuclease binding site) and a spacer sequence specific for a target sequence within a genomic site of interest, wherein the target sequence is adjacent to a protospacer adjacent motif (PAM); and (d) a reverse transcription donor RNA (RT donor RNA) or a fourth nucleic acid encoding a RT donor RNA, wherein the RT donor RNA comprises a primer binding site (PBS) and a template sequence.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority under 35 U.S.C. §119(e) to U.S. Provisional Application No. 63 / 195,621, filed June 1, 2021, U.S. Provisional Application No. 63 / 236,047, filed August 23, 2021, U.S. Provisional Application No. 63 / 272,937, filed October 28, 2021, and U.S. Provisional Application No. 63 / 299,695, filed January 14, 2022, the contents of each of which are incorporated by reference herein in their entirety.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. The ASCII copy created on Jun. 1, 2022 is named 116928-0042-0001WO00_SEQ.txt and is 388,313 bytes in size. [Background technology]

[0003] Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) genes, collectively known as the CRISPR-Cas or CRISPR / Cas system, are an adaptive immune system in archaea and bacteria that defends certain species against foreign genetic elements. Summary of the Invention

[0004] The present disclosure is based at least in part on the development of a gene editing system comprising a V-type CRISPR nuclease polypeptide (e.g., Cas12i2 polypeptide), a reverse transcriptase, a guide RNA (gRNA) that mediates cleavage at a target gene site by the CRISPR nuclease polypeptide, and a reverse transcription donor RNA that mediates synthesis of a desired sequence that is integrated into a target genome site. As reported herein, the gene editing system disclosed herein has successfully edited genes at various genome sites with high editing efficiency and accuracy. Without being bound by theory, the gene editing system disclosed herein exhibits at least one of the following advantageous features:

[0005] 1. Many of the editing template RNAs described herein, such as those specific to Cas12i polypeptides, do not require a transactivating CRISPR RNA (tracrRNA) component and are therefore smaller than primed editing guide RNAs (pegRNAs). In addition, many of the CRISPR nuclease-reverse transcriptase fusions described herein, such as Cas12i polypeptide-reverse transcriptase fusions, are smaller than Cas9-reverse transcriptase fusions. Both of these aspects are favorable in terms of delivery and synthesis costs.

[0006] 2. The editing template RNA described herein can be designed to have a primer binding site (PBS) that is longer than the PBS of the pegRNA. This feature can increase the efficiency of editing incorporation into the target nucleic acid.

[0007] 3. The gene editing system described herein, which includes an editing template RNA designed to bind only to the non-PAM strand (i.e., the complementary strand of the strand in which the PAM motif is present, also described herein as the target strand), can incorporate editing over a wider window compared to the primed editing system. In particular, the Cas12i polypeptide-reverse transcriptase system can rewrite the entire recognition sequence of the Cas12i polypeptide and the RNA guide. Thus, these gene editing systems can be more efficient in avoiding retargeting of the target nucleic acid by the CRISPR nuclease-reverse transcriptase fusion and the editing template RNA.

[0008] Accordingly, provided herein are gene editing systems, pharmaceutical compositions or kits comprising such gene editing systems, methods of using the gene editing systems to produce genetically modified cells, and the resulting cells so produced.

[0009] In some embodiments, the disclosure features a gene editing system that includes: (a) a Type V CRISPR nuclease polypeptide or a first nucleic acid encoding a Type V CRISPR nuclease polypeptide; (b) a reverse transcriptase (RT) polypeptide or a second nucleic acid encoding a RT polypeptide; (c) a guide RNA (gRNA) or a third nucleic acid encoding a gRNA, where the gRNA comprises one or more binding sites recognizable by a Type V CRISPR nuclease (CRISPR nuclease binding site) and a spacer sequence specific for a target sequence within a genomic site of interest, where the target sequence is adjacent to a protospacer adjacent motif (PAM); and (d) a reverse transcription donor RNA (RT donor RNA) or a fourth nucleic acid encoding a RT donor RNA, where the RT donor RNA comprises a primer binding site (PBS) and a template sequence.

[0010] In some embodiments, the V-type CRISPR nuclease polypeptide in any of the gene editing systems disclosed herein is a Cas12 polypeptide.In some examples, the Cas12 polypeptide is a Cas12i polypeptide, for example, a Cas12i2 polypeptide.In some cases, the Cas12i polypeptide is a Cas12i2 polypeptide, and the Cas12i2 polypeptide comprises at least 95% identical amino acid sequence to SEQ ID NO:2.

[0011] In some cases, the Cas12i2 polypeptide comprises one or more mutations at positions D581, G624, F626, P868, I926, V1030, E1035, and / or S1046 of SEQ ID NO: 2. For example, the one or more mutations are amino acid substitutions, optionally, the amino acid substitutions are D581R, G624R, F626R, P868T, I926R, V1030G, E1035R, S1046G, or a combination thereof. In one example, the Cas12i2 polypeptide comprises mutations at positions D581, D911, I926, and V1030 (e.g., amino acid substitutions of D581R, D911R, I926R, and V1030G). In another example, the Cas12i2 polypeptide comprises mutations at positions D581, I926, and V1030 (e.g., amino acid substitutions of D581R, I926R, and V1030G). In yet another example, the Cas12i2 polypeptide comprises mutations at positions D581, I926, V1030, and S1046 (e.g., amino acid substitutions of D581R, I926R, V1030G, and S1046G). In yet another example, the Cas12i2 polypeptide comprises mutations at positions D581, G624, F626, I926, V1030, E1035, and S1046 (e.g., amino acid substitutions of D581R, G624R, F626R, I926R, V1030G, E1035R, and S1046G). In another example, the Cas12i2 polypeptide comprises mutations at positions D581, G624, F626, P868, I926, V1030, E1035, and S1046 (e.g., amino acid substitutions of D581R, G624R, F626R, P868T, I926R, V1030G, E1035R, and S1046G). Exemplary Cas12i2 polypeptides for use in any of the gene editing systems disclosed herein may comprise the amino acid sequence of any one of SEQ ID NOs: 3-7. In some examples, exemplary Cas12i2 polypeptides can comprise the amino acid sequence of SEQ ID NO: 4. In other examples, exemplary Cas12i2 polypeptides can comprise the amino acid sequence of SEQ ID NO: 7.

[0012] In other cases, the Cas12i polypeptide has reduced crRNA processing activity, and optionally, the Cas12i polypeptide comprises a mutation at position H485 and / or H486 of SEQ ID NO:2.

[0013] In some embodiments, any of the gene editing systems disclosed herein may include a V-type CRISPR nuclease polypeptide. Alternatively, the gene editing system may include a first nucleic acid encoding a V-type CRISPR nuclease polypeptide. In some cases, the first nucleic acid is located in a first vector (e.g., a viral vector, such as an adeno-associated viral vector or an AAV vector). In other cases, the first nucleic acid is a messenger RNA (mRNA).

[0014] In any of the gene editing systems disclosed herein, the RT polypeptide can be Moloney Murine Leukemia Virus (MMLV)-RT, Mouse Mammary Tumor Virus (MMTV)-RT, Marathon-RT, or RTx-RT (e.g., MMLV RT can include the amino acid sequence of SEQ ID NO: 29). In some cases, the gene editing system includes a RT polypeptide. Alternatively, the system can include a second nucleic acid encoding a RT polypeptide. In some cases, the second nucleic acid is located in a second vector (e.g., a viral vector, such as an adeno-associated viral vector or an AAV vector). In one example, the gene editing system includes a vector (e.g., a viral vector), and the vector includes both a first nucleic acid encoding a V-type CRISPR polypeptide and a second nucleic acid encoding a RT polypeptide. In another example, the second nucleic acid encoding RT is a second mRNA. In one example, the gene editing system includes a single RNA molecule, and the single RNA molecule includes both a first mRNA encoding a V-type CRISPR polypeptide and a second mRNA encoding RT.

[0015] In some embodiments, the gene editing system disclosed herein comprises a fusion polypeptide comprising a type V CRISPR nuclease polypeptide and a RT polypeptide, or a nucleic acid (e.g., a vector, e.g., a viral vector) encoding the fusion polypeptide. Alternatively, the gene editing system comprises a type V CRISPR nuclease polypeptide and a RT polypeptide as two separate polypeptides.

[0016] In any of the gene editing systems disclosed herein, the spacer sequence can be 20 to 30 nucleotides in length. In some examples, the spacer sequence is 20 nucleotides in length.

[0017] In some embodiments, the PAM comprises a 5'-TTN-3' motif. In some cases (e.g., in the context of a Cas12i2 polypeptide), the PAM may be located 5' to the target sequence.

[0018] In some embodiments, one or more CRISPR nuclease binding sites are direct repeat sequence(s). In some cases, each of the direct repeat sequences is 23-36 nucleotides in length. In one example, the direct repeat sequence is 23 nucleotides in length. In some examples, the direct repeat sequence is at least 90% identical to any one of SEQ ID NOs: 15-17 and 241-247 (e.g., SEQ ID NO: 17), or a fragment thereof that is at least 23 nucleotides in length. In a specific example, the direct repeat sequence is any one of SEQ ID NOs: 15-17 and 241-247 (e.g., SEQ ID NO: 17), or a fragment thereof that is at least 23 nucleotides in length.

[0019] In some embodiments, the gene editing system disclosed herein comprises a gRNA. Alternatively, the gene editing system comprises a third nucleic acid encoding the gRNA. In some examples, the third nucleic acid is located in a third vector, and the third vector is optionally a viral vector. In some examples, the gene editing system may comprise a vector, for example a viral vector, and the vector comprises a third nucleic acid encoding the gRNA and a first and / or second nucleic acid encoding a V-type CRISPR nuclease polypeptide and / or a RT polypeptide.

[0020] In some embodiments, the PBS in the RT donor RNA of any of the gene editing systems disclosed herein can be 5-100 nucleotides in length. In some examples, the PBS is 10-60 nucleotides in length. In specific examples, the PBS is 10-30 nucleotides in length. In some cases, the PBS binds to a PBS target site adjacent to the complementary region of the target sequence. The PBS target site is upstream of the complementary region of the target sequence. For example, the PBS target site can be 3-10 nucleotides (e.g., 4-10 nucleotides) upstream of the complementary region of the target sequence. Alternatively, the PBS target site can overlap the complementary region of the target sequence. In other cases, the PBS target site is adjacent to or overlaps with the target sequence.

[0021] In some embodiments, the template sequence in the RT donor RNA of any of the gene editing systems disclosed herein can be 5-100 nucleotides in length. For example, the template sequence can be 30-50 nucleotides in length. In some cases, the template sequence can be homologous to a genomic site of interest and includes one or more nucleotide diversity relative to the genomic site of interest. In some examples, at least one nucleotide diversity is located within the target sequence. Alternatively or additionally, at least one nucleotide diversity is located within the PAM.

[0022] In some embodiments, any of the gene editing systems disclosed herein comprises a RT donor RNA. Alternatively, the gene editing system comprises a fourth nucleic acid encoding the RT donor RNA. In some examples, the fourth nucleic acid is located in a fourth vector, and the fourth vector is optionally a fourth viral vector. In some cases, the gene editing system comprises a vector, for example a viral vector, and the vector comprises a nucleic acid encoding the RT donor RNA and one or more additional nucleic acids encoding a guide RNA, a V-type CRISPR nuclease polypeptide, and a RT polypeptide.

[0023] In some embodiments, the gene editing system disclosed herein comprises a single RNA molecule, the single RNA molecule comprises a gRNA and a RT donor RNA. Such a single RNA comprises a CRISPR nuclease binding site, a spacer sequence, a PBS, and a template sequence, which may be arranged in any suitable order. In some examples, the single RNA molecule further comprises a linker between the gRNA and the RT donor RNA. Such a linker may comprise a hairpin structure. In one example, the single RNA molecule comprises, from 5' to 3', a CRISPR nuclease binding site, a spacer sequence, a template sequence, and a PBS. In another example, the single RNA molecule comprises, from 5' to 3', a CRISPR nuclease binding site, a spacer sequence, a linker, a template sequence, and a PBS. In yet another example, the single RNA molecule comprises, from 5' to 3', a template sequence, a PBS, a CRISPR nuclease binding site, and a spacer sequence. In yet another example, the single RNA molecule comprises, from 5' to 3', a template sequence, a PBS, a linker, a CRISPR nuclease binding site, and a spacer sequence.

[0024] In some cases, any of the single RNA molecules disclosed herein may further comprise a 5' end-protected fragment, a 3' end-protected fragment, or both. Each of the 5' end-protected fragment and the 3' end-protected fragment may form a secondary structure, such as a hairpin, a pseudoknot, or a triplex structure. In some examples, the 5' end-protected fragment and / or the 3' end-protected fragment is an exoribonuclease-resistant RNA (xrRNA), a transfer RNA (tRNA), or a truncated tRNA. In specific examples, the 5' end-protected fragment, the 3' end-protected fragment, or both may comprise one or more CRISPR nuclease binding sites. The 5' end-protected fragment, the 3' end-protected fragment, or both may further comprise one or more segments that are not homologous to any human sequence (cannot bind to any human sequence via base pairing).

[0025] In some embodiments, the gene editing system disclosed herein includes any of the gRNAs and any of the RT donor RNAs as two separate RNA molecules. In some examples, the gRNA, the RT donor RNA, or both may further include a 5'-end protected fragment and / or a 3'-end protected fragment. Each of the protected fragments may form a secondary structure, such as a hairpin, a pseudoknot, or a triplex structure. In some examples, the 5'-end protected fragment and / or the 3'-end protected fragment is an exoribonuclease-resistant RNA (xrRNA), a transfer RNA (tRNA), or a truncated tRNA. In other examples, the 5'-end protected fragment and / or the 3'-end protected fragment includes one or more of the CRISPR nuclease binding sites, and optionally includes one or more segments that are not homologous to any human sequence.

[0026] Any of the gene editing systems disclosed herein may include one or more lipid nanoparticles (LNPs), where the one or more LNPs include a V-type CRISPR nuclease polypeptide or encoding nucleic acid, a RT polypeptide or encoding nucleic acid, a guide RNA or encoding nucleic acid, a RT donor RNA or encoding nucleic acid, or any combination thereof. Alternatively, the gene editing system may include (i) one or more lipid nanoparticles (LNPs), where the one or more LNPs together include up to three components selected from a V-type CRISPR nuclease polypeptide or encoding nucleic acid, a RT polypeptide or encoding nucleic acid, a guide RNA or encoding nucleic acid, a RT donor RNA or encoding nucleic acid, and (ii) one or more vectors encoding the remaining components in the gene editing system. In some cases, the one or more vectors can be one or more viral vectors, for example, one or more adeno-associated virus (AAV) vectors.

[0027] In some examples, the gene editing system disclosed herein comprises a V-type CRISPR nuclease polypeptide, a RT polypeptide, a gRNA, and a RT donor RNA. In some cases, the V-type CRISPR nuclease polypeptide and / or the RT polypeptide form a complex (e.g., a ribonucleoprotein (RNP) complex) with the gRNA and / or the RT donor RNA.

[0028] In some embodiments, the present disclosure also provides pharmaceutical compositions comprising any of the gene editing systems disclosed herein and a pharma- ceutically acceptable carrier, and kits comprising components of the gene editing systems.

[0029] In another aspect, the disclosure also features a method for gene editing a cell, the method including contacting a host cell with any of the gene editing systems disclosed herein or a pharmaceutical composition including such a gene editing system to gene edit the host cell. In some examples, the host cell is cultured in vitro. In other examples, the contacting step is performed by administering the gene editing system to a subject including the host cell.

[0030] Also within the scope of the present disclosure are populations of genetically modified cells that can be produced by the gene editing system disclosed herein. In some instances, the genetically modified cells can include cells that are not editable by the gene editing system, for example, can include one or more modifications in the PAM, the target sequence, or both.

[0031] In yet another aspect, the disclosure features a gene-editing RNA molecule comprising: (i) one or more binding sites recognizable by a type V CRISPR nuclease (CRISPR nuclease binding site); (ii) a spacer sequence specific to a target sequence within a gene site, the target sequence being adjacent to a protospacer adjacent motif (PAM); (iii) a primer binding site (PBS); and (iv) a template sequence. In some embodiments, the gene-editing RNA molecule may further comprise one or more linkers, such as those disclosed herein.

[0032] In some examples, the RNA molecule comprises, from 5' to 3', a CRISPR nuclease binding site, a spacer sequence, a template sequence, and a PBS. In other examples, the RNA molecule comprises, from 5' to 3', a CRISPR nuclease binding site, a spacer sequence, a linker, a template sequence, and a PBS. In yet other examples, the RNA molecule comprises, from 5' to 3', a template sequence, a PBS, a CRISPR nuclease binding site, and a spacer sequence. In yet other examples, the RNA molecule comprises, from 5' to 3', a template sequence, a PBS, a linker, a CRISPR nuclease binding site, and a spacer sequence.

[0033] Any of the gene editing RNA molecules disclosed herein may further comprise a 5' end protection fragment, a 3' end protection fragment, or both. Each of the protection fragments may form a secondary structure, such as a hairpin, a pseudoknot, or a triplex structure. In some examples, the 5' end protection fragment and / or the 3' end protection fragment is an exoribonuclease-resistant RNA (xrRNA), a transfer RNA (tRNA), or a truncated tRNA. In other examples, the 5' end protection fragment and / or the 3' end protection fragment comprises one or more of the CRISPR nuclease binding sites, and optionally comprises one or more segments that are not homologous to any human sequence.

[0034] In addition, the present disclosure features a set of gene editing RNA molecules (two separate RNA molecules) including: (i) a guide RNA that includes one or more binding sites (CRISPR nuclease binding sites) recognizable by V-type CRISPR nuclease and a spacer sequence specific to a target sequence within a gene site, the target sequence being adjacent to a protospacer adjacent motif (PAM); and (ii) a reverse transcription donor RNA (RT donor RNA) or a fourth nucleic acid encoding the RT donor RNA, the RT donor RNA including a primer binding site (PBS) and a template sequence. In some examples, the gRNA, the RT donor RNA, or both further include a 5'-end protection fragment and / or a 3'-end protection fragment. Each of the protection fragments can form a secondary structure, such as a hairpin, a pseudoknot, or a triplex structure. In some examples, the 5'-end protected fragment and / or the 3'-end protected fragment are exoribonuclease-resistant RNA (xrRNA), transfer RNA (tRNA), or truncated tRNA. In other examples, the 5'-end protected fragment and / or the 3'-end protected fragment include one or more of the CRISPR nuclease binding sites, and optionally include one or more segments that are not homologous to any human sequence.

[0035] Also provided herein is a DNA molecule or set of DNA molecules encoding the gene-editing RNA molecule or set of gene-editing RNA molecules disclosed herein. In some examples, the DNA molecule or set of DNA molecules is contained within a vector or set of vectors, and optionally the vector or set of vectors is a viral vector.

[0036] Additionally, a fusion polypeptide comprising a CRISPR nuclease and a reverse transcriptase is provided herein. Any of such CRISPR nuclease-RT fusion polypeptides can be used in the gene editing system disclosed herein. In some embodiments, the CRISPR nuclease is a V-type CRISPR nuclease, such as a Cas12i polypeptide. In some examples, the Cas12i polypeptide is a Cas12i2 polypeptide, such as those disclosed herein. In specific examples, the fusion polypeptide can include the amino acid sequence of SEQ ID NOs: 25-26 and 219-223.

[0037] In some embodiments, the Cas12i polypeptide is a Cas12i4 polypeptide. In some instances, the Cas12i4 polypeptide can be fused with a reverse transcriptase, such as MMLV RT. Such a fused Cas12i4-RT fusion polypeptide can include the amino acid sequence of SEQ ID NO:53.

[0038] Any nucleic acid encoding any of the CRISPR nuclease-RT fusion polypeptides, including vectors, for example, expression vectors (e.g., viral vectors), are within the scope of this disclosure.

[0039] The details of one or more embodiments of the invention are set forth in the description below. Other features and advantages of the invention will become apparent from the following drawings and detailed description of several embodiments, and from the appended claims.

[0040] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure, which can be better understood by reference to the drawings in conjunction with the detailed description of specific embodiments presented herein. [Brief description of the drawings]

[0041] [Figure 1A] The present disclosure includes schematic diagrams showing exemplary gene editing systems. Schematic diagram showing a gene editing system including a CRISPR nuclease (e.g., Cas12i polypeptide) fused to a reverse transcriptase polypeptide and an RNA guide fused to a RT donor RNA at the 3' end of the RNA guide. The RT donor RNA includes a reverse transcription template sequence and a PBS. The PBS includes substantial complementarity with the PAM strand (also known as the non-target strand) of the target nucleic acid. [Figure 1B] Included are schematic diagrams showing exemplary gene editing systems disclosed herein. Shown is a Cas9 nickase fused to a reverse transcriptase (left) and a Cas12i nickase fused to a reverse transcriptase (right). Using a RT donor RNA fused to the 3' end of an RNA guide, edits are incorporated into the PAM strand of the target nucleic acid.

[0042] [Diagram 2] Schematic diagram showing an exemplary gene editing system including a CRISPR nuclease (e.g., Cas12i polypeptide) fused to a reverse transcriptase polypeptide and an RNA guide fused to a RT donor RNA at the 5' end of the RNA guide. The RT donor RNA includes a PBS and a reverse transcription template sequence. The PBS includes complementarity with the PAM strand of the target nucleic acid.

[0043] [Diagram 3] Schematic diagram showing a CRISPR nuclease (e.g., Cas12i polypeptide), a reverse transcriptase polypeptide, an RNA guide, and a RT donor RNA. The RT donor RNA contains a reverse transcription template sequence and a PBS. Edits are incorporated into the genome after cleavage by the CRISPR nuclease.

[0044] [Figure 4] Schematic diagram showing a CRISPR nuclease (e.g., Cas12i polypeptide), a reverse transcriptase polypeptide, an RNA guide, and an RNA reverse transcription template sequence. The RT donor RNA comprises a PBS and a reverse transcription template sequence. Edits are incorporated into the genome in the presence of the CRISPR nuclease.

[0045] [Diagram 5] Schematic diagram showing an exemplary gene editing system including a CRISPR nuclease (e.g., Cas12i polypeptide) fused to a reverse transcriptase polypeptide and an RNA guide containing a mismatch with a target nucleic acid and fused to a RT donor RNA at the 3' end of the RNA guide. The RT donor RNA includes a PBS. The PBS includes complementarity with the non-PAM strand (also known as the target strand or TS) of the target nucleic acid.

[0046] [Figure 6A] Included are schematic diagrams showing exemplary gene editing systems disclosed herein. Schematic diagram showing exemplary gene editing systems including a CRISPR nuclease (e.g., Cas12i polypeptide) fused to a reverse transcriptase polypeptide and an RNA guide fused to an RT donor RNA at the 3' end of the RNA guide. The RT donor RNA includes a reverse transcription template sequence and a PBS. When the spacer sequence of the RNA guide and the PBS bind to the target nucleic acid, the reverse transcription template sequence forms a loop of unpaired nucleotides. The PBS includes complementarity with the non-PAM strand of the target nucleic acid. The variant Cas12i2 cleavage sites in the PAM strand and the non-PAM strand are indicated by triangles. Using the RT donor RNA fused to the 3' end of the RNA guide, edits are incorporated into the non-PAM strand of the target nucleic acid.

[0047] [Figure 6B] 1 includes a schematic diagram showing an exemplary gene editing system disclosed herein, showing the arrangement of the edit, the reverse transcription template sequence, and the PBS, where the length of the reverse transcription template sequence and the PBS can be varied.

[0048] [Figure 7] Schematic diagram showing an exemplary gene editing system including a CRISPR nuclease (e.g., Cas12i polypeptide) fused to a reverse transcriptase polypeptide and an RNA guide fused to a RT donor RNA at the 5' end of the RNA guide. The RT donor RNA includes a PBS and a reverse transcription template sequence. The PBS includes complementarity with the non-PAM strand of a target nucleic acid.

[0049] [Figure 8A] Included are schematic diagrams showing exemplary Cas12i2 RNA guide-RT donor RNA fusions. Schematic diagram of variant Cas12i2 RNA guide fused to RT donor RNA tested in Example 1. The spacer of the RNA guide binds to the non-PAM strand adjacent to 5'-TTT-3'PAM. The RT donor RNA comprises a reverse transcription template sequence and a PBS. When the spacer sequence and the PBS bind to the target nucleic acid, the reverse transcription template sequence forms a loop of unpaired nucleotides. The PBS comprises complementarity with the non-PAM strand of the target nucleic acid. In this schematic diagram, the PBS is 13 nucleotides long and the reverse transcription template sequence is 34 nucleotides long. The PBS is designed such that complementarity with the non-PAM strand begins at the cleavage site (triangle). The sequences are SEQ ID NOs: 65-67 from top to bottom.

[0050] [Figure 8B] Schematic diagram showing exemplary Cas12i2 RNA guide-RT donor RNA fusions. Exemplary RNA guide-RT donor RNA fusions targeting the AAVS1_T7 genomic site tested in Example 1 are shown. Various PBS lengths were tested (13, 30, and 60 nucleotides). RNA guide-RT donor RNA fusions were designed to introduce substitutions (S), insertions (I), deletions (D), or hairpins (H) into the target sequence. The sequences are, from top to bottom, SEQ ID NOs: 74-80 and 87-89.

[0051] [Figure 8C]1 includes a schematic diagram showing exemplary Cas12i2 RNA guide-RT donor RNA fusions. Shown are the encoded edits (substitutions, insertions, and deletions) introduced into the AAVS1_T7 genomic site (top panel), the EMX1_T6 genomic site (middle panel), and the VEGFA_T5 genomic site (bottom panel), as described in Example 1. The sequences, from top to bottom, are SEQ ID NOs: 248-259.

[0052] [Figure 9A] FIG. 1 includes a diagram showing gene editing efficiency resulting from an exemplary gene editing system disclosed herein. The percentage of analyzed NGS reads with indels and encoded edits induced by variant Cas12i2 of SEQ ID NO: 4, C-terminal and N-terminal Cas12i2-MMLV RT fusions of SEQ ID NO: 25 and SEQ ID NO: 26, and RNA guide targeting AAVS1_T6 genomic site is shown.

[0053] [Figure 9B] Figure 1 includes a diagram showing gene editing efficiency resulting from an exemplary gene editing system disclosed herein. The percentage of analyzed NGS reads with indels and encoded edits induced by N-terminal and C-terminal Cas12i2-MMLV RT fusions of SEQ ID NO:26 and SEQ ID NO:25, and RNA guide-RT donor RNA fusions targeting the AAVS1_T6 genomic site are shown. The RNA guide-RT donor RNA fusions have PBS lengths of 13, 30, or 60 nucleotides and are designed to introduce substitutions (S), insertions (I), deletions (D), or hairpins (H) into the AAVS1_T6 genomic site.

[0054] [Figure 9C]FIG. 1 includes a diagram showing gene editing efficiency resulting from an exemplary gene editing system disclosed herein. The percentage of analyzed NGS reads with indels and encoded edits induced by variant Cas12i2 of SEQ ID NO: 4, C-terminal and N-terminal Cas12i2-MMLV RT fusions of SEQ ID NO: 25 and SEQ ID NO: 26, and RNA guide targeting AAVS1_T7 genomic site is shown.

[0055] [Figure 9D] Figure 1 includes a diagram showing gene editing efficiency resulting from an exemplary gene editing system disclosed herein. The percentage of analyzed NGS reads with indels and encoded edits induced by N-terminal and C-terminal Cas12i2-MMLV RT fusions of SEQ ID NO: 26 and SEQ ID NO: 25, and RNA guide-RT donor RNA fusions targeting the AAVS1_T7 genomic site are shown. The RNA guide-RT donor RNA fusions have PBS lengths of 13, 30, or 60 nucleotides and are designed to introduce substitutions (S), insertions (I), deletions (D), or hairpins (H) into the AAVS1_T7 genomic site.

[0056] [Figure 9E] FIG. 1 includes a diagram showing gene editing efficiency resulting from an exemplary gene editing system disclosed herein. The percentage of analyzed NGS reads with indels and encoded edits induced by variant Cas12i2 of SEQ ID NO: 4, C-terminal and N-terminal Cas12i2-MMLV RT fusions of SEQ ID NO: 25 and SEQ ID NO: 26, and RNA guide targeting EMX1_T6 genomic site are shown. [Figure 9F]Figures showing gene editing efficiency resulting from exemplary gene editing systems disclosed herein are included. The percentage of analyzed NGS reads with indels and encoded edits induced by N-terminal and C-terminal Cas12i2-MMLV RT fusions of SEQ ID NO:26 and SEQ ID NO:25, and RNA guide-RT donor RNA fusions targeting EMX1_T6 genomic site are shown. The RNA guide-RT donor RNA fusions have PBS lengths of 13, 30, or 60 nucleotides and were designed to introduce substitutions (S), insertions (I), deletions (D), or hairpins (H) into the EMX1_T6 genomic site.

[0057] [Figure 9G] FIG. 1 includes a diagram showing gene editing efficiency resulting from an exemplary gene editing system disclosed herein. It shows the percentage of analyzed NGS reads with indels and encoded edits induced by variant Cas12i2 of SEQ ID NO: 4, C-terminal and N-terminal Cas12i2-MMLV RT fusions of SEQ ID NO: 25 and SEQ ID NO: 26, and RNA guide targeting VEGFA_T2 genomic site.

[0058] [Figure 9H] Figure 1 includes a diagram showing gene editing efficiency resulting from an exemplary gene editing system disclosed herein. The percentage of analyzed NGS reads with indels and encoded edits induced by N-terminal and C-terminal Cas12i2-MMLV RT fusions of SEQ ID NO: 26 and SEQ ID NO: 25, and RNA guide-RT donor RNA fusions targeting VEGFA_T2 genomic sites are shown. The RNA guide-RT donor RNA fusions have PBS lengths of 13, 30, or 60 nucleotides and are designed to introduce substitutions (S), insertions (I), deletions (D), or hairpins (H) into VEGFA_T2 genomic sites.

[0059] [Figure 9I]FIG. 1 includes a diagram showing gene editing efficiency resulting from an exemplary gene editing system disclosed herein. It shows the percentage of analyzed NGS reads with indels and encoded edits induced by variant Cas12i2 of SEQ ID NO: 4, C-terminal and N-terminal Cas12i2-MMLV RT fusions of SEQ ID NO: 25 and SEQ ID NO: 26, and RNA guide targeting VEGFA_T5 genomic site.

[0060] [Figure 9J] Figures showing gene editing efficiency resulting from exemplary gene editing systems disclosed herein are included. The percentage of analyzed NGS reads with indels and encoded edits induced by N-terminal and C-terminal Cas12i2-MMLV RT fusions of SEQ ID NO:26 and SEQ ID NO:25 and RNA guide-RT donor RNA fusions targeting VEGFA_T5 genomic sites are shown. The RNA guide-RT donor RNA fusions have PBS lengths of 13, 30, or 60 nucleotides and are designed to introduce substitutions (S), insertions (I), deletions (D), or hairpins (H) into VEGFA_T5 genomic sites.

[0061] [Figure 10] FIG. 1 is a schematic diagram showing a Cas12i polypeptide (e.g., Cas12i2 nickase) fused to a reverse transcriptase enzyme. Using a RT donor RNA fused to the 5' or 3' end of the RNA guide, the encoded edit is incorporated into the PAM strand of the target nucleic acid. The ends of the RNA guide-RT donor RNA can be protected to prevent exonuclease or endonuclease activity. The PBS length can vary from about 3 to 100 nucleotides and can include substantial complementarity with the PAM strand. A structured RNA, e.g., a hairpin, can be introduced between the spacer and the reverse transcription template sequence.

[0062] [Figure 11]Schematic diagram showing an RNA guide-RT donor RNA further fused to a second direct repeat (DR)-spacer sequence. The additional DR-spacer inhibits exonuclease activity.

[0063] [Figure 12A] Included are schematic diagrams showing exemplary designs of editing template RNA (gene editing RNA). Schematic diagram showing editing template RNA (5'-nuclease binding sequence-DNA binding sequence-reverse transcription template-PBS-3') further comprising 3'-end protection. 3'-end protection can be chemical end protection (top of figure) or hairpin (bottom of figure). Hairpin can be nuclease binding sequence, e.g., direct repeat sequence.

[0064] [Figure 12B] Included are schematic diagrams showing exemplary designs of editing template RNA (gene editing RNA). Schematic diagram showing editing template RNA with 5' end protection and editing template RNA without 5' end protection (5'-reverse transcription template-PBS nuclease binding sequence-DNA binding sequence-3'). 5' end protection can be a hairpin (e.g., a nuclease binding sequence, e.g., a direct repeat sequence), as shown at the bottom of the figure.

[0065] [Figure 13A] Figure 1 includes diagrams showing gene editing efficiency resulting from an exemplary gene editing system disclosed herein. Activity of Cas12i2 (SEQ ID NO: 4) and Cas12i2-RT (SEQ ID NO: 25) and RNA guide of SEQ ID NO: 112 or editing template RNA of SEQ ID NO: 123-137 at AAVS1_T7 genomic site (SEQ ID NO: 30) is shown. % NGS reads analyzed as having indels are shown in white bars for Cas12i2 and in gray bars for Cas12i2-RT. % NGS reads analyzed as having coded edits are shown in checkered bars for Cas12i2 and in black bars for Cas12i2-RT.

[0066] [Figure 13B] Figures showing gene editing efficiency resulting from an exemplary gene editing system disclosed herein are included. Activity of Cas12i2 (SEQ ID NO: 4) and Cas12i2-RT (SEQ ID NO: 25) and RNA guide of SEQ ID NO: 114 or editing template RNA of SEQ ID NO: 138-152 at EMX1_T6 genomic site (SEQ ID NO: 34) is shown. % NGS reads analyzed as having indels are shown in white bars for Cas12i2 and in grey bars for Cas12i2-RT. % reads analyzed as having encoded edits are shown in checkered bars for Cas12i2 and in black bars for Cas12i2-RT.

[0067] [Figure 13C] Figure 1 includes diagrams showing gene editing efficiency resulting from an exemplary gene editing system disclosed herein. Activity of Cas12i2 (SEQ ID NO: 4) and Cas12i2-RT (SEQ ID NO: 25) and RNA guide of SEQ ID NO: 116 or editing template RNA of SEQ ID NO: 153-167 on VEGFA_T2 (SEQ ID NO: 36) is shown. % NGS reads analyzed as having indels are shown in white bars for Cas12i2 and in gray bars for Cas12i2-RT. % NGS reads analyzed as having coded edits are shown in checkered bars for Cas12i2 and in black bars for Cas12i2-RT.

[0068] [Figure 13D]Figure 1 includes diagrams showing gene editing efficiency resulting from an exemplary gene editing system disclosed herein. Activity of Cas12i2 (SEQ ID NO: 4) and Cas12i2-RT (SEQ ID NO: 25) and RNA guide of SEQ ID NO: 118 or editing template RNA of SEQ ID NO: 168-182 at VEGFA_T5 genomic site (SEQ ID NO: 38) is shown. % NGS reads analyzed as having indels are shown in white bars for Cas12i2 and in gray bars for Cas12i2-RT. % NGS reads analyzed as having coded edits are shown in checkered bars for Cas12i2 and in black bars for Cas12i2-RT.

[0069] [Figure 14A] 1 includes a schematic diagram showing the steps of the assay used to identify Cas12i2 and the cleavage pattern of an RNA guide or editing template RNA. Oligo constructs including target sequences and barcodes are shown.

[0070] [Figure 14B] Included is a schematic diagram showing the steps of the assay used to identify cleavage patterns of Cas12i2 and the RNA guide or editing template RNA, and treatment of the cleavage products to blunt 5' and 3' overhangs, or to end-repair and fill in 5' overhangs.

[0071] [Figure 14C] 1 includes a schematic diagram showing the steps of the assay used to identify cleavage patterns of Cas12i2 and the RNA guide or editing template RNA. Amplification of cleavage products is shown.

[0072] [Figure 15A] Figure 1 includes diagrams showing gene editing using an exemplary gene editing system disclosed herein. Figure 1 is a schematic diagram showing in vitro cleavage sites (triangles) induced on the PAM and non-PAM strands of the AAVS1_T2 genomic site by Cas12i2 of SEQ ID NO:2.

[0073] [Fig. 15B-E] The figures include diagrams showing gene editing using an exemplary gene editing system disclosed herein. Figure 15B is a histogram of read lengths obtained from the amplification of 5' cleavage products after filling. Figure 15C is a histogram of read lengths obtained from the amplification of 3' cleavage products after filling. Figure 15D is a histogram of read lengths obtained from the amplification of 5' cleavage products after smoothing. Figure 15E is a histogram of read lengths obtained from the amplification of 3' cleavage products after smoothing. The read length histograms are mapped to target sequences as shown on the x-axis of Figures 15B-E.

[0074] [Figure 16A] Shown are in vitro cleavage sites (triangles) induced on the PAM or non-PAM strand of the EMX1_T6 genomic site by Cas12i2 of SEQ ID NO: 2 or variant Cas12i2 of SEQ ID NO: 4. The scale bar (right) represents the cleavage frequency as measured by the number of sequencing reads.

[0075] [Figure 16B] Shown are in vitro cleavage sites (triangles) induced on the PAM or non-PAM strand of the VEGFA_T5 genomic site by Cas12i2 of SEQ ID NO: 2 or variant Cas12i2 of SEQ ID NO: 4. The scale bar (right) represents the cleavage frequency as measured by the number of sequencing reads.

[0076] [Figure 17A] Figures showing gene editing results at exemplary genome sizes are included. Activity with editing template RNAs introducing 4 nucleotide insertions into the AAVS1_T7 genomic site (SEQ ID NO: 30), the EMX1_T6 genomic site (SEQ ID NO: 34), or the VEGFA_T5 genomic site (SEQ ID NO: 38) is shown. The editing template RNAs contained a 34 nucleotide reverse transcription template sequence and a 3, 8, 13, 30, or 60 nucleotide PBS. The ratio of encoded editing to total editing is shown on the y-axis. Sequences, from top to bottom, are SEQ ID NOs: 90-92.

[0077] [Figure 17B] Figures showing gene editing results at exemplary genome sizes are included. Activity by editing template RNA in the introduction of a 4 nucleotide insertion into the AAVS1_T7 genomic site (SEQ ID NO: 30), the EMX1_T6 genomic site (SEQ ID NO: 34), or the VEGFA_T5 genomic site (SEQ ID NO: 38) is shown. The editing template RNA included a 13 nucleotide PBS, and a 14, 24, 34, 44, or 54 nucleotide reverse transcription template sequence. The ratio of encoded editing to total editing is shown on the y-axis. The sequences are SEQ ID NOs: 90-92 from top to bottom.

[0078] [Figure 18] 1 shows the encoded edits integrated within the AAVS1_T7 genomic site (sequence number 32) and the EMX1_T6 genomic site (sequence number 34) in U2OS cells.

[0079] [Figure 19A] The present invention includes schematic diagrams showing gene editing procedures using exemplary gene editing systems disclosed herein. Schematic diagram showing Cas9 prime editor, which includes Cas9 fused to reverse transcriptase and pegRNA. A primer on target DNA is generated after cleavage of PAM strand by Cas9. Hybridization of primer with pegRNA initiates reverse transcription.

[0080] [Figure 19B] The present disclosure includes schematic diagrams showing gene editing procedures using exemplary gene editing systems. Schematic diagram showing V-type CRISPR nuclease fused to reverse transcriptase and editing template RNA. A primer on target DNA is generated after cleavage of non-PAM strand by V-type CRISPR nuclease. Hybridization of primer with editing template RNA initiates reverse transcription.

[0081] [Figure 20A]1 includes a diagram showing editing at various genomic sites with the indicated Cas12i2-RT fusion polypeptides. 2 is a plot showing the % of NGS reads containing indel edits (white bars) or encoded edits (grey bars) introduced by variant Cas12i2-RT fusions of SEQ ID NOs: 219-223 at the AAVS1 genomic site.

[0082] [Figure 20B] 1 includes a diagram showing editing at various genomic sites with the indicated Cas12i2-RT fusion polypeptides. 2 is a plot showing the % of NGS reads containing indel edits (white bars) or encoded edits (grey bars) introduced by variant Cas12i2-RT fusions of SEQ ID NOs: 219-223 at the EMX1 genomic site.

[0083] [Figure 20C] 1 includes a diagram showing editing at various genomic sites with the indicated Cas12i2-RT fusion polypeptides. 2 is a plot showing the % of NGS reads containing indel edits (white bars) or encoded edits (grey bars) introduced by variant Cas12i2-RT fusions of SEQ ID NOs: 219-223 at the VEGFA genomic site.

[0084] [Figure 21] 1 is a plot showing the % of NGS reads containing indel or encoded edits introduced by variant Cas12i2 (SEQ ID NO: 4) or variant Cas12i2-RT fusion (SEQ ID NO: 219) and an RNA guide or editing template RNA. The RNA guide and editing template RNA were either unmodified or contained terminal phosphorothioate backbone linkages and / or 2'O-methyl nucleotides.

[0085] [Figure 22] 13 is a plot showing the % of NGS reads containing indel edits (white bars) or coded edits (gray bars) introduced by variant Cas12i4-RT fusions at AAVS1 genomic sites.

[0086] [Figure 23] 13A-13C are plots showing the % of NGS reads containing indel edits (white bars) or encoded edits (gray bars) introduced by variant Cas12i2 or variant Cas12i2-RT fusions, RNA guides, and RT donor RNAs at AAVS1, EMX1, or VEGFA genomic sites. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0087] The present disclosure relates to a gene editing system comprising a V-type nuclease or a nucleic acid encoding the same, an RNA guide or a nucleic acid encoding the same, a reverse transcriptase or a nucleic acid encoding the same, and an RT donor RNA or a nucleic acid encoding the same. Also provided herein are pharmaceutical compositions and kits comprising any of the gene editing systems disclosed herein, methods for gene editing a cell using any of the gene editing systems disclosed herein, genetically engineered cells thus produced, and gene editing RNA molecules or sets of RNA molecules contained within the gene editing system, and DNA molecule(s) for producing such gene editing RNA molecules or sets of RNA molecules.

[0088] definition The present disclosure will be described with respect to particular embodiments and with reference to certain figures but the disclosure is not limited thereto but only by the claims. The terms used hereinafter are generally to be understood in their ordinary sense unless otherwise indicated.

[0089] As used herein, the term "activity" refers to biological activity. In some embodiments, activity refers to effector activity. In some embodiments, activity includes enzymatic activity, e.g., the catalytic ability of an effector. For example, activity can include nuclease activity. In another example, activity refers to the ability of an enzyme to generate DNA from RNA, or to introduce an edit into a target sequence.

[0090] As used herein, the term "adjacent to" refers to a nucleotide or amino acid sequence closely adjacent to another nucleotide or amino acid sequence. In some embodiments, a nucleotide sequence is adjacent to (i.e., directly adjacent to) another nucleotide sequence if there are no nucleotides separating the two sequences. In some embodiments, a nucleotide sequence is adjacent to another nucleotide sequence if a small number of nucleotides (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides) separate the two sequences. In some embodiments, a first sequence is adjacent to a second sequence if the two sequences are separated by about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 nucleotides. In some embodiments, a first sequence is adjacent to a second sequence if the two sequences are separated by at most 2 nucleotides, at most 5 nucleotides, at most 8 nucleotides, at most 10 nucleotides, at most 12 nucleotides, or at most 15 nucleotides. In some embodiments, a first sequence is adjacent to a second sequence if the two sequences are separated by 2-5 nucleotides, 4-6 nucleotides, 4-8 nucleotides, 4-10 nucleotides, 6-8 nucleotides, 6-10 nucleotides, 6-12 nucleotides, 8-10 nucleotides, 8-12 nucleotides, 10-12 nucleotides, 10-15 nucleotides, or 12-15 nucleotides.

[0091] As used herein, the term "CRISPR nuclease" refers to an RNA-guided effector capable of binding to nucleic acids and introducing single-stranded or double-stranded breaks. In some embodiments, the CRISPR nuclease is a type II CRISPR nuclease or a type V CRISPR nuclease. In some embodiments, the CRISPR nuclease is an effector described in Makarova et al. "Classification and Nomenclature of CRISPR-Cas Systems: Where from Here?" CRISPR J. 1(5):325-36 (2018).

[0092] As used herein, the terms "type II" and "type II nuclease" refer to a nuclease that includes a RuvC domain and an HNH domain. A type II nuclease can be a type II-A nuclease, a type II-B nuclease, or a type II-C nuclease. In some embodiments, a type II nuclease requires tracrRNA. In some embodiments, a type II nuclease is a Cas9 polypeptide. The Cas9 polypeptide may cleave a double-stranded DNA target or may be a nickase.

[0093] As used herein, the terms "V-type" and "V-type nuclease" refer to an RNA-guided CRISPR nuclease having a RuvC domain. In some embodiments, a V-type nuclease does not require a tracrRNA. In some embodiments, a V-type nuclease requires a tracrRNA. In some embodiments, a V-type nuclease is a Cas12 polypeptide, such as a Cas12a (Cpf1), Cas12b (C2c1), Cas12c, Cas12d, Cas12e, Cas12f, Cas12h, Cas12i, or Cas12j (CasPhi) polypeptide.

[0094] As used herein, the term "Cas12i polypeptide" (also referred to herein as Cas12i) refers to a polypeptide that binds to a target sequence on a target nucleic acid specified by an RNA guide and has at least some amino acid sequence homology with a wild-type Cas12i polypeptide. In some embodiments, the Cas12i polypeptide comprises at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with any one of SEQ ID NOs: 1-5 and 11-18 of U.S. Pat. No. 10,808,245, which is incorporated by reference for the subject matter and purposes referred to herein. In some embodiments, the Cas12i polypeptide comprises at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to any one of SEQ ID NOs: 8, 2, 11, and 9 of the present application. In some embodiments, the Cas12i polypeptide of the present disclosure is a Cas12i2 polypeptide described in WO / 2021 / 202800, the relevant disclosure of which is incorporated by reference for and with respect to the subject matter referenced herein. In some embodiments, the Cas12i polypeptide cleaves the target nucleic acid (e.g., as a nick or double-stranded break).

[0095] The "percent identity" (also known as sequence identity) of two nucleic acid or two amino acid sequences is determined using the algorithm of Karlin and Altschul Proc. Natl. Acad. Sci. USA 87:2264-68, 1990, modified as in Karlin and Altschul Proc. Natl. Acad. Sci. USA 90:5873-77, 1993. Such an algorithm is incorporated into the NBLAST and XBLAST programs (version 2.0) of Altschul, et al. J. Mol. Biol. 215:403-10, 1990. BLAST nucleotide searches can be performed with the NBLAST program, score=100, wordlength=12 to obtain nucleotide sequences homologous to the nucleic acid molecules of the invention. BLAST protein searches can be performed with the XBLAST program, score=50, wordlength=3 to obtain amino acid sequences homologous to the protein molecules of the invention. When gaps exist between the two sequences, Gapped BLAST can be used as described in Altschul et al., Nucleic Acids Res. 25(17):3389-3402, 1997. When utilizing BLAST, Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used.

[0096] As used herein, the term "complex" refers to a grouping of two or more molecules. In some embodiments, a complex includes a polypeptide and a nucleic acid molecule that interact with each other (e.g., bound, contacted, attached). In some embodiments, the term "complex" is used to refer to the association of a CRISPR nuclease (e.g., a V-type nuclease, e.g., a Cas12i polypeptide) and a reverse transcriptase polypeptide. For example, a complex of a CRISPR nuclease (e.g., a Cas12i2 polypeptide disclosed herein) and a reverse transcriptase polypeptide can be a heterodimer of the two polypeptides, e.g., via a dimerization domain (e.g., a leucine zipper), an antibody, a nanobody, or an aptamer. In some embodiments, the term "complex" is used to refer to the association of an RNA guide and a RT donor RNA. In some embodiments, the term "complex" is used to refer to the association of a CRISPR nuclease (e.g., a V-type nuclease, e.g., a Cas12i polypeptide), a reverse transcriptase polypeptide, an RNA guide, and a RT donor RNA. In some embodiments, the term "complex" is used to refer to the association of a reverse transcriptase polypeptide and a RT donor RNA.

[0097] As used herein, the term "binding site recognizable by nuclease" or "nuclease binding sequence" refers to a sequence that can bind to CRISPR nuclease. In some embodiments, the nuclease binding sequence is an RNA sequence. In some embodiments, the nuclease binding sequence is a direct repeat sequence. In some embodiments, the nuclease binding sequence is capable of binding to type II CRISPR nuclease or type V CRISPR nuclease (e.g., binding site recognizable by type II CRISPR nuclease or binding site recognizable by type V CRISPR nuclease).

[0098] As used herein, the term "deletion" refers to the loss of one or more nucleotides in a nucleic acid sequence relative to a reference sequence. No particular process is suggested for how to create a sequence containing a deletion. For example, a sequence containing a deletion can be directly synthesized from individual nucleotides. In other embodiments, the deletion is performed by providing a reference sequence and then modifying it. The nucleic acid sequence can be in the genome of an organism. The nucleic acid sequence can be in a cell. The nucleic acid sequence can be a DNA sequence. The deletion can be a frameshift mutation or a non-frameshift mutation. The deletion described herein refers to an insertion of up to several kilobases.

[0099] As used herein, the term "editing" refers to one or more modifications being introduced into a nucleotide sequence in a target nucleic acid, for example, into a nucleotide sequence at a genomic site of interest. Editing can occur within a target sequence as defined herein. Alternatively, editing can occur outside the target sequence (e.g., adjacent to the target sequence). Editing can be one or more substitutions, one or more insertions, one or more deletions, or a combination thereof.

[0100] As used herein, the terms "fusion" and "fused" refer to the linkage of at least two nucleotide or protein molecules. For example, "fusion" and "fused" can refer to the linkage of at least two polypeptide domains that are encoded by separate genes in nature (e.g., a V-type nuclease and a reverse transcriptase polypeptide). The fusion can be an N-terminal fusion, a C-terminal fusion, or an intramolecular fusion. In some embodiments, the domains are transcribed and translated to produce a single polypeptide. Also, as used herein, the terms "fusion" and "fused" are used to refer to the linkage of two nucleic acid molecules, such as two RNA molecules (e.g., an RNA guide and an RT donor RNA). The fusion can be a 5' fusion, a 3' fusion, or an intramolecular fusion.

[0101] As used herein, the term "insertion" refers to an increase of one or more nucleotides in a nucleic acid sequence relative to a reference sequence. No particular process is suggested for how to create a sequence containing an insertion. For example, a sequence containing an insertion can be directly synthesized from individual nucleotides. In other embodiments, the insertion is performed by providing a reference sequence and then modifying it. The nucleic acid sequence can be in the genome of an organism. The nucleic acid sequence can be in a cell. The nucleic acid sequence can be a DNA sequence. The insertion can be a frameshift mutation or a non-frameshift mutation. An insertion as described herein refers to an insertion of up to several kilobases.

[0102] As used herein, the term "protospacer adjacent motif" or "PAM sequence" refers to a DNA sequence adjacent to a target sequence. In some embodiments, the PAM sequence is required for enzymatic activity. In a double-stranded DNA molecule, the strand containing the PAM motif is referred to as the "PAM strand" and the complementary strand is referred to as the "non-PAM strand." The RNA guide binds to a site in the non-PAM strand that is complementary to the target sequence disclosed herein, and the PAM sequence described herein is present within the PAM strand.

[0103] As used herein, the term "PAM strand" refers to the strand of a (double-stranded) target nucleic acid that contains a PAM motif. In some embodiments, the PAM strand is the coding (e.g., sense) strand. In other embodiments, the PAM strand is non-coding (e.g., antisense strand). The term "non-PAM strand" refers to the complementary strand of the PAM strand. Because the gRNA binds to the non-PAM strand through base pairing, the non-PAM strand is also known as the target strand, and the PAM strand is also known as the non-target strand.

[0104] As used herein, the term "target sequence" refers to a DNA fragment adjacent to a PAM motif (on the PAM strand). The complementary region of the target sequence is on the non-PAM strand. The target sequence may be directly adjacent to the PAM motif. Alternatively, the target sequence and the PAM may be separated by a small sequence segment (e.g., up to 5 nucleotides, e.g., up to 4, 3, 2, or 1 nucleotides). The target sequence may be located at the 3' end of the PAM motif or the 5' end of the PAM motif, depending on the CRISPR nuclease that recognizes the PAM motif, as known in the art. For example, the target sequence is located at the 3' end of the PAM motif for a Cas12i polypeptide (e.g., a Cas12i2 polypeptide such as those disclosed herein).

[0105] As used herein, the term "RNA guide" or "RNA guide sequence" refers to an RNA molecule or modified RNA molecule that facilitates targeting of a CRISPR nuclease described herein to a genomic site of interest. For example, an RNA guide can be a molecule that is complementary to a target sequence in the PAM strand and recognizes (e.g., binds to) a site in the non-PAM strand that is designed to be complementary to, for example, a specific nucleic acid sequence. The RNA guide includes a spacer and a nuclease binding sequence (e.g., a direct repeat (DR) sequence). The terms CRISPR RNA (crRNA), pre-crRNA, and mature crRNA are also used herein to refer to an RNA guide. The 5' or 3' end of the RNA guide can be fused to a RT donor RNA disclosed herein. In some cases, the RNA guide can be a modified RNA molecule that includes one or more deoxyribonucleotides, for example, in a DNA binding sequence contained within the RNA guide, where the DNA binding sequence binds to a complementary sequence of a target sequence. In some examples, the DNA binding sequence can contain a DNA sequence or a DNA / RNA hybrid sequence.

[0106] As used herein, the terms "spacer" and "spacer sequence" (also known as DNA binding sequence) are a portion (DNA sequence) in an RNA guide that is the RNA equivalent of a target sequence. A spacer contains a sequence that is capable of binding to the non-PAM strand via base pairing at a site complementary to the target sequence (in the PAM strand). Such spacers are also known to be specific to the target sequence. In some cases, a spacer can be at least 75% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99%) identical to the target sequence, excluding the RNA-DNA sequence difference. In some cases, a spacer can be 100% identical to the target sequence, excluding the RNA-DNA sequence difference.

[0107] As used herein, the term "complementary" refers to a first polynucleotide having a level of complementarity with a second polynucleotide such that the first polynucleotide (e.g., a spacer sequence of an RNA guide) and the second polynucleotide (e.g., a complementary sequence of a target sequence) can form a double-stranded complex through base pairing to enable an effector polypeptide (e.g., a Cas12i2 polypeptide, a Cas12i2-reverse transcriptase fusion polypeptide, or a variant thereof) that forms a complex with the first polynucleotide to act on (e.g., cleave) the second polynucleotide. In some embodiments, the first polynucleotide can be substantially complementary to the second polynucleotide, i.e., has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% complementarity with the second polynucleotide. In some embodiments, the first polynucleotide is fully complementary to the second polynucleotide, i.e., has 100% complementarity with the second polynucleotide.

[0108] As used herein, the terms "reverse transcriptase" and "RT" typically refer to a multifunctional enzyme that has three enzymatic activities, including an RNA- and DNA-dependent DNA polymerization activity, and an RNase H activity that catalyzes the cleavage of RNA in RNA-DNA hybrids. Reverse transcriptase can generate DNA from an RNA template.

[0109] As used herein, the terms "reverse transcription donor RNA" and "RT donor RNA" refer to an RNA molecule that includes a reverse transcription template sequence (template sequence) and a primer binding site (PBS). The RT donor RNA can be fused to an RNA guide at either the 5' or 3' end of the RNA guide.

[0110] As used herein, the term "PBS target site" refers to a region to which PBS binds. The PBS target site may be adjacent to (e.g., upstream of) a region of the non-PAM strand that is complementary to the target sequence. For example, the PBS target site can be 3-10 nucleotides (e.g., 3 nucleotides or 4 nucleotides) upstream of the region that is complementary to the target sequence. In some cases, the PBS target site may be directly adjacent to the region of the non-PAM strand that is complementary to the target sequence. In other examples, the PBS target site may overlap with the region of the non-PAM strand that is complementary to the target sequence. Alternatively, the PBS target site may be adjacent to, upstream of, or overlap with the target sequence on the PAM strand.

[0111] As used herein, the term "reverse transcription template sequence" or "template sequence" refers to an RNA molecule or a fragment of an RT donor RNA that serves as a template for DNA synthesis by reverse transcriptase. In some embodiments, the reverse transcription template sequence comprises an edit that is incorporated into the genome site where gene editing is required. In some cases, the edit mediated by the reverse transcription template sequence in the RT donor RNA destroys or removes the PAM sequence, the target sequence, or both.

[0112] As used herein, the term "editing template RNA" or "gene editing RNA" (used interchangeably herein) refers to an RNA molecule or set of RNA molecules including an RNA guide (including a spacer and one or more binding sites recognizable by a CRISPR nuclease, such as those disclosed herein) and an RT donor RNA (including a PBS and a reverse transcription template sequence). The gene editing RNA can mediate cleavage at a target sequence within a genomic site of interest by a CRISPR nuclease and can mediate synthesis of a DNA fragment from the free 3' end of the free DNA strand generated by CRISPR nuclease cleavage based on the template sequence in the gene editing RNA. In some embodiments, the editing template RNA or gene editing RNA is a single RNA molecule, and the single RNA molecule includes an RNA guide bound (e.g., fused) to an RT donor RNA. In some embodiments, the editing template RNA includes, from 5' to 3', one or more binding sites recognizable by a CRISPR nuclease, a spacer sequence, a PBS, and an RT donor RNA. In some embodiments, the editing template RNA or gene editing RNA comprises, from 5' to 3', one or more binding sites recognizable by CRISPR nuclease, a spacer, a template sequence, and a PBS. In some embodiments, the editing template RNA or gene editing RNA comprises, from 5' to 3', a template sequence, a PBS, one or more binding sites recognizable by CRISPR nuclease, and a spacer sequence. In some embodiments, the editing template RNA further comprises a linker. For example, in some embodiments, the editing template RNA comprises a linker between one or more binding sites recognizable by CRISPR nuclease and the PBS, or between the spacer sequence and the RT donor RNA.

[0113] As used herein, the term "substitution" refers to one or more nucleotides being replaced with one or more different nucleotides relative to a reference sequence. No particular process is suggested for how to create a sequence containing substitutions. For example, a sequence containing substitutions can be directly synthesized from individual nucleotides. In other embodiments, substitutions are made by providing a reference sequence and then modifying it. The nucleic acid sequence can be in the genome of an organism. The nucleic acid sequence can be in a cell. The nucleic acid sequence can be a DNA sequence. Substitutions as described herein refer to substitutions of up to several kilobases.

[0114] As used herein, the terms "upstream" and "downstream" refer to relative positions within a single nucleic acid (e.g., DNA) sequence. "Upstream" and "downstream" each refer to the 5' to 3' direction in which RNA transcription occurs. A first sequence is upstream of a second sequence when the 3' end of the first sequence occurs before the 5' end of the second sequence. A first sequence is downstream of a second sequence when the 5' end of the first sequence occurs after the 3' end of the second sequence. In some embodiments, the terms "upstream" and "downstream" are used in reference to the non-PAM strand. For example, in some embodiments, the PBS is complementary to the non-PAM strand sequence upstream of the target sequence. Thus, in some embodiments, the PBS binds to a sequence upstream of the sequence to which the spacer sequence binds, and the spacer sequence binds downstream of the sequence to which the PBS binds.

[0115] I. Gene editing systems Prime editing has been developed to introduce substitutions, small insertions, or small deletions into a target sequence. The prime editing approach relies on Cas9 nickase fused to a reverse transcriptase and a prime editing guide RNA (pegRNA). The pegRNA contains a spacer sequence capable of binding to the non-PAM strand (the strand opposite the PAM sequence) of the target locus, a primer binding site (PBS) capable of binding to the PAM strand (the strand containing the PAM sequence) of the target locus, and a reverse transcription template sequence containing the edit. The spacer sequence of the pegRNA binds to the target sequence on the non-PAM strand, and the nickase Cas9 nicks the PAM strand. This exposes a 3' flap on the PAM strand of the target locus, which can hybridize to the PBS. The reverse transcriptase then copies the reverse transcription template, thereby extending the 3' flap. See, for example, FIG. 19A. Through a DNA repair mechanism, the edit is incorporated into the target locus.

[0116] In some embodiments, a gene editing system is provided herein that is capable of editing a target nucleic acid (e.g., at a genomic site of interest), e.g., introducing an insertion, deletion, substitution, or a combination thereof at a genomic site. Editing can occur on either strand of the target nucleic acid. The gene editing system disclosed herein includes at least one protein component or a nucleotide sequence encoding it, and at least one RNA component or a nucleotide sequence encoding it. The protein component has the activity of cleaving the target nucleic acid at a desired site guided by the RNA component, and synthesizing a new DNA sequence using a portion of the RNA component as a template, starting from the free 3' end of the DNA strand generated due to cleavage. The newly synthesized DNA fragment can then be incorporated into the target nucleic acid, e.g., via a DNA repair mechanism in the host cell, which can result in gene editing of the target nucleic acid.

[0117] The protein components in the gene editing system disclosed herein can include a CRISPR nuclease (e.g., a V-type nuclease, e.g., a variant Cas12i polypeptide) and a reverse transcriptase (RT) polypeptide. In some instances, the CRISPR nuclease and the RT polypeptide are two separate polypeptides. In other instances, the CRISPR nuclease and the RT polypeptide are part of a fusion polypeptide.

[0118] The RNA components in the gene editing system disclosed herein may include a guide RNA (gRNA) (also described herein as an RNA guide or CRISPR RNA (crRNA)) that mediates CRISPR nuclease cleavage at a specific site in the target nucleic acid as designed, and a reverse transcription donor RNA (RT donor RNA) that mediates reverse transcription by a RT polypeptide and provides a template sequence for reverse transcription. In some instances, the gRNA and the RT donor RNA are two separate RNA molecules. In other instances, the gRNA and the RT donor RNA are parts of a single RNA molecule.

[0119] As shown herein, without being bound by theory, the gene editing system described herein provides several advantages over the art. For example, template RNA editing has not been demonstrated with V-type CRISPR nuclease, such as Cas12i CRISPR nuclease. V-type nuclease has the advantage of being smaller than Cas9 nuclease. For example, Cas12i2 is 1,054 amino acids long, S.pyogenes Cas9 (SpCas9) is 1,368 amino acids long, S.thermophilus Cas9 (StCas9) is 1,128 amino acids long, FnCpf1 is 1,300 amino acids long, AsCpf1 is 1,307 amino acids long, and LbCpf1 is 1,246 amino acids long. In addition, many V-type nucleases do not require transactivating CRISPR RNA (tracrRNA), and therefore use smaller RNA guides than Cas9 RNA guides. For example, see Table 4 below. Smaller Cas12i polypeptide and RNA guide size is beneficial for delivery. In addition, templated RNA editing has not been demonstrated with any CRISPR nuclease that uses a single edited template RNA that binds to a single strand, e.g., the target strand (non-PAM strand), of the target locus. As shown herein, the gene editing system that comprises Cas12i polypeptide also demonstrates reduced off-target activity compared to the gene editing system that comprises SpCas9 polypeptide. See PCT / US2021 / 025257, which is incorporated by reference in its entirety.

[0120] A. CRISPR nuclease Any of the gene editing systems disclosed herein may include a CRISPR nuclease. In some embodiments, the CRISPR nuclease is capable of binding and / or binds to a nuclease binding sequence described elsewhere herein. In some embodiments, the CRISPR nuclease cleaves DNA at the target sequence. In some embodiments, the CRISPR nuclease is recruited to the target sequence via a DNA binding sequence described elsewhere herein that specifically recognizes and / or binds to the target sequence. In some embodiments, the CRISPR nuclease cleaves one or both strands of DNA at the target sequence. In some embodiments, two or more CRISPR nucleases are recruited to the target sequence, and one or more CRISPR nucleases cleave one or both strands of DNA at or near the target sequence. In such embodiments, the CRISPR nuclease may have or be capable of nuclease activity. In some embodiments, the CRISPR nuclease may have reduced or limited nuclease activity. In some embodiments, the CRISPR nuclease-reverse transcriptase fusion polypeptide described elsewhere herein is capable of binding and binds to at least one nuclease binding sequence in the editing template RNA described elsewhere herein. In some embodiments, the CRISPR nuclease-reverse transcriptase fusion is capable of binding and binds to the target sequence via at least one DNA binding sequence in the editing template RNA. In such embodiments, the CRISPR nuclease is recruited to or brought into close proximity to the target sequence by binding to the nuclease binding sequence and the DNA binding sequence of the editing template RNA. Furthermore, in such embodiments, the reverse transcriptase is capable of and transcribes the reverse transcription template sequence described elsewhere herein into DNA.

[0121] In some embodiments, the CRISPR nuclease-reverse transcriptase fusion polypeptide transcribes the reverse transcription template sequence into the non-PAM strand of the target nucleic acid. In some embodiments, the CRISPR nuclease-reverse transcriptase fusion polypeptide transcribes the reverse transcription template sequence into the PAM strand of the target nucleic acid. In some embodiments, the CRISPR nuclease-reverse transcriptase fusion polypeptide transcribes the reverse transcription template sequence from 5' to 3', starting from the PBS (e.g., the 5' or 3' end of the PBS). In some embodiments, after hybridization of the PBS to the free 3' end of the non-PAM strand of the target nucleic acid, the CRISPR nuclease-reverse transcriptase fusion transcribes the reverse transcription template sequence from the 3' end of the non-PAM strand. In some embodiments, after hybridization of the PBS to the free 3' end of the PAM strand of the target nucleic acid, the CRISPR nuclease-reverse transcriptase fusion transcribes the reverse transcription template sequence from the 3' end of the PAM strand.

[0122] In some embodiments, the CRISPR nuclease is an RNA-guided CRISPR nuclease. In some embodiments, the CRISPR nuclease is a DNA-targeted nuclease.

[0123] In some embodiments, the CRISPR nuclease is Cas9 (e.g., Cas9 and nCas9), Casl2a / Cpf1, Casl2b / C2c1, Casl2c / C2c3, Casl2d / CasY, Casl2e / CasX, Casl2g, Casl2h, Casl2i, and Casl2j / CasPhi). Non-limiting examples of Cas enzymes include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas5d, Cas5t, Cas5h, Cas5a, Cas6, Cas7, Cas8, Cas8a, Cas8b, Cas8c, Cas9 (also known as Csn1 or Csxl2), Cas10, Cas10d, Casl2a / Cpfl, Casl2b / C2cl, Casl2c / C2c3, Casl2d / CasY, Casl2e / CasX, Casl2g, Casl2h, Casl2i, Casl2j / CasΦ, Cpf1, Csy1, Csy2, Csy3, Csy4, Csel, Cse2, Cse3, Cse4, Cse5e, Csc1, Csc2, Csxl2, Csxl3, Csxl4, Csxl5, Csxl6, Csxl7, Csxl8, Csxl9, Csxl10, Csxl11, Csxl12, Csxl13, Csxl14, Csxl15, Csxl16, Csxl17, Csxl18, Csxl19, Csxl21, Csxl19, Csxl22, Csxl19, Csxl23, Csxl19, Csxl24, Csxl25, Csxl26, Csxl27, Csxl28, Csxl29, Csxl31, Csxl21, Csxl32, Csxl33, Csxl4, Csxl10, Csxl11, Csxl21, Csxl12, Csxl13, Csxl14, Csxl15, Csxl16, Csxl17 sa5, Csnl, Csn2, Csm1, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl4, Csx10, Csxl6, CsaX, Csx3, Csx1, CsxlS, Csx11, Csf1, Csf2, CsO, Examples of CRISPR nucleases include Csf4, Csd1, Csd2, Cst1, Cst2, Csh1, Csh2, Csal, Csa2, Csa3, Csa4, Csa5, type II CRISPR nuclease, type V CRISPR nuclease, type VI CRISPR nuclease, CARF, DinG, homologs thereof, or modified or engineered versions thereof. Other CRISPR nucleases may not be specifically listed in this disclosure, but are also within the scope of this disclosure. See, for example, Makarova et al. "Classification and Nomenclature of CRISPR-Cas Systems: Where from Here?" CRISPR J. 1(5):325-36 (2018).

[0124] In some embodiments, the CRISPR nuclease is selected from the group consisting of WO2021 / 055874, WO2020 / 206036, WO2020 / 191102, WO2020 / 186213, WO2020 / 028555, WO2020 / 033601, WO2019 / 126762, WO2019 / 126774, WO2019 / 071048, WO2019 / 018423, WO2019 / 005866, WO2018 / 191388, WO2018 / 170333, WO2018 / 035388, WO2018 / 035387, WO2017 / 219 027, WO2017 / 189308, WO2017 / 184768, WO2017 / 106657, WO2016 / 205749, WO2017 / 070605, WO2016 / 205764, WO2016 / 205711, WO2016 / 028682, WO2015 / 0 89473, WO2014 / 093595, WO2015 / 089427, WO2014 / 204725, WO2015 / 070083, WO2014 / 093655, WO2014 / 093694, WO2014 / 093712, WO2014 / 093635, WO2021 / 133829, WO2021 / 007177, WO2020 / 197934, WO2020 / 181102, WO2020 / 181101, WO2020 / 041456, WO2020 / 023529, WO2020 / 005980, WO2019 / 104058, WO201 9 / 089820, WO2019 / 089808, WO2019 / 089804, WO2019 / 089796, WO2019 / 036185, WO2018 / 226855, WO2018 / 213351, WO2018 / 089664, WO2018 / 064371, WO2 018 / 064352, WO2017 / 106569, WO2017 / 048969, WO2016 / 196655, WO2016 / 106239, WO2016 / 036754, WO2015 / 103153, WO2015 / 089277, WO2014 / 150624, W O2013 / 176772, WO2021 / 119563, WO2021 / 118626, WO2020 / 247883, WO2020 / 247882, WO2020 / 223634, WO2020 / 142754, WO2020 / 086475, WO2020 / 028729,WO2019 / 241452, WO2019 / 173248, WO2018 / 236548, WO2018 / 183403, WO2017 / 027423, WO2018 / 106727, WO2018 / 071672, WO2017 / 096328, WO2017 / 070598, WO2016 / 201155, WO2014 / 150624, WO2013 / 098244, WO2021 / 113522, WO2021 / 050534, W WO2021 / 046442, WO2021 / 041569, WO2021 / 007563, WO2020 / 252378, WO2020 / 180699, WO2020 / 018142, WO2019 / 222555, WO2019 / 178428, WO2019 / 178427, or WO2019 / 006471, which are incorporated by reference for the subject matter and purposes referenced herein.

[0125] In some embodiments, the compositions of the invention comprise a type V CRISPR nuclease (e.g., a type V nuclease). In some embodiments, the type V nuclease is a Cas12 CRISPR nuclease. In some embodiments, the type V nuclease is a Cas12a (Cpf1), Cas12b (C2c1), Cas12c, Cas12d, Cas12e, Cas12f, Cas12h, Cas12i, or Cas12j (CasPhi) CRISPR nuclease. In some embodiments, the type V nuclease is a variant (e.g., a functional variant) of a Cas12a (Cpf1), Cas12b (C2c1), Cas12c, Cas12d, Cas12e, Cas12f, Cas12h, Cas12i, or Cas12j (CasPhi) CRISPR nuclease. In some embodiments, the type V nuclease comprises an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to a wild-type type V nuclease sequence (e.g., a wild-type amino acid sequence of Cas12a (Cpf1), Cas12b (C2c1), Cas12c, Cas12d, Cas12e, Cas12f, Cas12h, Cas12i, or Cas12j (CasPhi).

[0126] In some embodiments, the V-type nuclease of the present invention is a Cas12i CRISPR nuclease. In some embodiments, the Cas12i CRISPR nuclease is a Cas12i2 CRISPR nuclease comprising a nucleotide sequence, for example, SEQ ID NO: 1, or is encoded by a polypeptide comprising an amino acid sequence, for example, SEQ ID NO: 2. In some embodiments, the CRISPR nuclease of the present invention is a variant of a wild-type CRISPR nuclease, the wild-type comprising a nucleotide sequence, for example, SEQ ID NO: 1, or is encoded by a polypeptide comprising an amino acid sequence, for example, SEQ ID NO: 2. See Table 1.

[0127] In some embodiments, the type II nuclease of the present invention is a Cas9 CRISPR nuclease. In some embodiments, the Cas9 CRISPR nuclease is a SpCas9 CRISPR nuclease, and the SpCas9 CRISPR nuclease comprises an amino acid sequence, for example, SEQ ID NO: 120. In some embodiments, the Cas9 CRISPR nuclease is a nickase, for example, nSpCas9, and the nickase comprises an amino acid sequence, for example, SEQ ID NO: 121. In some embodiments, the CRISPR nuclease of the present invention is a different species of Cas9 CRISPR nuclease. In some embodiments, the Cas9 CRISPR nuclease is a SaCas9 CRISPR nuclease, and the SaCas9 CRISPR nuclease comprises an amino acid sequence, for example, SEQ ID NO: 122. See Table 1.

[0128] [Table 1-1]

[0129] [Table 1-2]

[0130] [Table 1-3]

[0131] [Table 1-4]

[0132] [Table 1-5]

[0133] [Table 1-6]

[0134] [Table 1-7]

[0135] [Table 1-8]

[0136] [Table 1-9]

[0137] The nucleic acid sequence encoding the CRISPR nuclease described herein can be substantially identical to a reference nucleic acid sequence, e.g., SEQ ID NO: 1. In some embodiments, the CRISPR nuclease is encoded by a nucleic acid comprising a sequence having at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 99.5% sequence identity to a reference nucleic acid sequence, e.g., a nucleic acid sequence encoding a wild-type polypeptide, e.g., SEQ ID NO: 1. The percent identity between two such nucleic acids may be determined manually by inspection of two optimally aligned nucleic acid sequences, or may be determined using standard parameters using a software program or algorithm (e.g., BLAST, ALIGN, CLUSTAL). One indication that two nucleic acid sequences are substantially identical is that the nucleic acid molecules hybridize under stringent conditions (eg, within moderate to high stringency), to the complementary sequence of the other.

[0138] In some embodiments, the CRISPR nuclease is encoded by a nucleic acid sequence having at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% or more, but not 100%, sequence identity to a reference nucleic acid sequence, e.g., a nucleic acid sequence encoding a CRISPR nuclease, e.g., SEQ ID NO:1.

[0139] In some embodiments, a CRISPR nuclease of the invention comprises a polypeptide sequence having 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 2. In some embodiments, a CRISPR nuclease of the invention comprises a sequence having more than 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% but not 100% identity to SEQ ID NO: 2.

[0140] In some embodiments, the present invention describes CRISPR nucleases that have a certain degree of amino acid sequence identity with one or more reference polypeptides, e.g., wild-type polypeptides, e.g., at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or even at least 99%, but not 100%, sequence identity with the amino acid sequence of SEQ ID NO: 2. Homology or identity can be determined by amino acid sequence alignment, e.g., using programs such as BLAST, ALIGN, or CLUSTAL, as described herein.

[0141] In some embodiments, the CRISPR nuclease is a variant Cas12i2 polypeptide described in WO / 2021 / 202800, the relevant disclosure of which is incorporated by reference for the subject matter and purposes referenced herein. In some embodiments, the variant Cas12i2 polypeptide comprises one or more of the amino acid substitutions listed in Table 2 of WO / 2021 / 202800. In some embodiments, the CRISPR nuclease is a variant Cas12i2 polypeptide having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:3 of PCT / US2021 / 025257. In some embodiments, the CRISPR nuclease is a variant Cas12i2 polypeptide having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 4 of PCT / US2021 / 025257. In some embodiments, the CRISPR nuclease is a variant Cas12i2 polypeptide having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 5 of PCT / US2021 / 025257. In some embodiments, the CRISPR nuclease is a variant Cas12i2 polypeptide having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:495 of PCT / US2021 / 025257.In some embodiments, the CRISPR nuclease is a variant Cas12i2 polypeptide having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 496 of PCT / US2021 / 025257. In some embodiments, the CRISPR nuclease is a variant Cas12i2 polypeptide having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to any one of SEQ ID NOs: 3-146 and 495-512 of WO / 2021 / 202800, which is incorporated by reference.

[0142] In some embodiments, the CRISPR nuclease is a Cas12i polypeptide. In some embodiments, the CRISPR nuclease is a Cas12i1 polypeptide. In some embodiments, the Cas12i1 polypeptide is a variant Cas12i1 polypeptide. In some embodiments, the variant Cas12i1 polypeptide of the invention comprises a polypeptide sequence having 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:8. In some embodiments, a variant Cas12i1 polypeptide of the invention comprises a polypeptide sequence having greater than 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:8.

[0143] In some embodiments, the CRISPR nuclease has a certain degree of amino acid sequence identity with one or more reference polypeptides, such as wild-type Casil polypeptides, for example, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 8. Homology or identity can be determined by amino acid sequence alignment, for example, using programs such as BLAST, ALIGN, or CLUSTAL, as described herein.

[0144] In some embodiments, a nucleic acid encoding a variant Cas12i1 polypeptide described herein encodes an amino acid sequence having at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 8. In some embodiments, a variant Cas12i1 polypeptide has a sequence that is greater than 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:8.

[0145] In some embodiments, a variant Cas12i1 polypeptide described herein having enzymatic activity, e.g., nuclease or endonuclease activity, comprises an amino acid sequence that differs from the amino acid sequence of any one of the CRISPR nucleases and SEQ ID NO:8 by 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 40, 35, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residues when aligned using any of the alignment methods described above.

[0146] In some embodiments, the Cas12i polypeptide is a Cas12i3 polypeptide. In some embodiments, the Cas12i3 polypeptide of the present invention comprises a polypeptide sequence having 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 11. In some embodiments, the Cas12i3 polypeptide of the present invention comprises a polypeptide sequence having 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more than 100% identity to SEQ ID NO: 11.

[0147] In some embodiments, the Cas12i3 polypeptide is a variant Cas12i3 polypeptide. In some embodiments, the variant Cas12i3 polypeptide has a certain degree of amino acid sequence identity with one or more reference polypeptides, e.g., wild-type polypeptides, e.g., at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 11. Homology or identity can be determined by amino acid sequence alignment, e.g., using programs such as BLAST, ALIGN, or CLUSTAL, as described herein.

[0148] In some embodiments, a nucleic acid encoding a variant Cas12i3 polypeptide described herein encodes an amino acid sequence having at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 11. In some embodiments, a variant Cas12i3 polypeptide has a sequence that is greater than 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 11.

[0149] In some embodiments, a variant Cas12i3 polypeptide described herein having enzymatic activity, e.g., nuclease or endonuclease activity, comprises an amino acid sequence that differs from the amino acid sequence of any one of the CRISPR nucleases and SEQ ID NO: 11 by 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 40, 35, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residues when aligned using any of the alignment methods described above.

[0150] In some embodiments, the Cas12i polypeptide is a Cas12i4 polypeptide. In some embodiments, the Cas12i4 polypeptide of the present invention comprises a polypeptide sequence having 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:9 or SEQ ID NO:10. In some embodiments, the Cas12i4 polypeptide of the present invention comprises a polypeptide sequence having 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more than 100% identity to SEQ ID NO:9 or SEQ ID NO:10.

[0151] In some embodiments, the Cas12i4 polypeptide is a variant Cas12i4 polypeptide. In some embodiments, the variant Cas12i4 polypeptide has a certain degree of amino acid sequence identity with one or more reference polypeptides, e.g., wild-type polypeptides, e.g., at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO:9 or SEQ ID NO:10. Homology or identity can be determined by amino acid sequence alignment, e.g., using programs such as BLAST, ALIGN, or CLUSTAL, as described herein.

[0152] In some embodiments, a nucleic acid encoding a variant Cas12i4 polypeptide described herein encodes an amino acid sequence having at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:9 or SEQ ID NO:10. In some embodiments, a variant Cas12i4 polypeptide has a sequence that is greater than 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:9 or SEQ ID NO:10.

[0153] In some embodiments, a variant Cas12i4 polypeptide described herein having enzymatic activity, e.g., nuclease or endonuclease activity, comprises an amino acid sequence that differs from the amino acid sequence of any one of a CRISPR nuclease and SEQ ID NO:9 or SEQ ID NO:10 by 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 40, 35, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residue when aligned using any of the alignment methods described above.

[0154] In some embodiments, the CRISPR nuclease is a type II CRISPR nuclease, such as a Cas9 nuclease. In some embodiments, the Cas9 nuclease is Cas9 from S. pyogenes or S. aureus, or a variant thereof. See, e.g., US2019 / 0136248, which is incorporated by reference in its entirety. In some embodiments, the Cas9 polypeptide is a nickase.

[0155] In some embodiments, the Cas9 polypeptides of the invention comprise a polypeptide sequence having 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to any one of SEQ ID NOs: 120-122. In some embodiments, the Cas9 polypeptides of the invention comprise a polypeptide sequence having 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater than 100% identity to any one of SEQ ID NOs: 120-122.

[0156] In some embodiments, the Cas9 polypeptide is a variant Cas9 polypeptide. In some embodiments, the variant Cas9 polypeptide has a certain degree of amino acid sequence identity with one or more reference polypeptides, e.g., a wild-type polypeptide, e.g., at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequence of any one of SEQ ID NOs: 120-122. Homology or identity may be determined by amino acid sequence alignment, e.g., using programs such as BLAST, ALIGN, or CLUSTAL, as described herein.

[0157] In some embodiments, a nucleic acid encoding a variant Cas9 polypeptide described herein encodes an amino acid sequence having at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to any one of SEQ ID NOs: 120-122. In some embodiments, a variant Cas9 polypeptide has a sequence that is greater than 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to any one of SEQ ID NOs: 120-122.

[0158] In some embodiments, a variant Cas9 polypeptide described herein having enzymatic activity, e.g., nuclease or endonuclease activity, comprises an amino acid sequence that differs from the amino acid sequence of a CRISPR nuclease and any one of SEQ ID NO:120 or SEQ ID NO:121 by 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 40, 35, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residue when aligned using any of the alignment methods described above.

[0159] In some embodiments, a CRISPR nuclease (e.g., a Type V nuclease, such as a Cas12i polypeptide, or a Type II nuclease) comprises an alteration at one or more (e.g., several) amino acids of a wild-type polypeptide, and comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, , 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 162, 164, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 193, 194, 195, 196, 197, 198, 199, or 200 or more have been modified.

[0160] In some embodiments, the CRISPR nuclease in any one of the embodiments described herein comprises crRNA processing activity. In some embodiments, the V-type nuclease (e.g., Cas12i polypeptide) is a variant lacking crRNA processing activity. For example, in some embodiments where the V-type nuclease is a variant Cas12i2 polypeptide, the variant Cas12i2 polypeptide comprises a H485 or H486 substitution. In some embodiments, the variant Cas12i2 polypeptide having at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to any one of SEQ ID NOs: 2-7 further comprises a H485 or H486 mutation. In some embodiments, the variant Cas12i2 polypeptide comprising the H485 or H486 mutation comprises reduced or no crRNA processing activity.

[0161] In some embodiments, the nucleotide sequence encoding the CRISPR nuclease described herein may be codon-optimized for use in a particular host cell or organism, or may be codon-optimized for a particular purpose, e.g., for expression. For example, the nucleic acid may be codon-optimized for any non-human eukaryote, including mouse, rat, rabbit, dog, livestock, or non-human primate. Codon usage tables are readily available, for example, in the "Codon Usage Database" available at www.kazusa.orjp / codon / , and these tables may be adapted in several ways. See Nakamura et al. Nucl. Acids Res. 28:292 (2000), which is incorporated herein by reference in its entirety. Also, computer algorithms are available for codon-optimizing a particular sequence for expression in a particular host cell, e.g., Gene Forge (Aptagen, Jacobus, PA). In some instances, a nucleic acid encoding a CRISPR nuclease (e.g., any of the Cas12i polypeptides, e.g., the Cas12i2 or Cas12i4 polypeptides disclosed herein), a reverse transcriptase, or any of their fusion polypeptides can be an mRNA molecule that can be codon-optimized. Also, in some instances, the RT template sequence in any of the editing template RNAs disclosed herein or a portion thereof can be codon-optimized.

[0162] The changes described herein can be one or more amino acid changes, but the changes to the CRISPR nuclease can also be structural or substantial in nature, for example, fusion of a polypeptide, such as an amino and / or carboxyl terminal extension. For example, the CRISPR nuclease can contain additional peptides, for example, one or more peptides. Examples of additional peptides can include epitope peptides for labeling, such as polyhistidine tags (His tags), Myc, and FLAG. In some embodiments, the CRISPR nuclease described herein can be fused to a detectable moiety, for example, a fluorescent protein (for example, green fluorescent protein (GFP) or yellow fluorescent protein (YFP)).

[0163] In some embodiments, the CRISPR nuclease in any one of the embodiments described herein comprises at least one (e.g., 2, 3, 4, 5, or 6 or more) nuclear localization signal (NLS). In some embodiments, the CRISPR nuclease comprises at least one (e.g., 2, 3, 4, 5, or 6 or more) nuclear export signal (NES). In some embodiments, the CRISPR nuclease comprises at least one (e.g., 2, 3, 4, 5, or 6 or more) NLS and at least one (e.g., 2, 3, 4, 5, or 6 or more) NES.

[0164] In some embodiments, the CRISPR nuclease comprises at least a RuvC domain, but less than the full CRISPR nuclease. In some embodiments, the CRISPR nuclease is a truncated CRISPR nuclease relative to wild-type CRISPR nuclease. In some embodiments, the truncated CRISPR nuclease comprises a RuvC domain. In some embodiments, the CRISPR nuclease comprises at least one functional domain of the full CRISPR nuclease. In some embodiments, the CRISPR nuclease comprises at least two RuvC domains or at least two RuvC motifs. In some embodiments, the CRISPR nuclease comprises at least three RuvC domains or at least three RuvC motifs. In some embodiments, the CRISPR nuclease comprises at least one catalytically inactive RuvC domain and at least one catalytically active RuvC domain. In some embodiments, the CRISPR nuclease comprises two RuvC domains from one or more type V or type II nucleases. In some embodiments, the CRISPR nuclease comprises at least a RuvC domain and a dimerization domain.

[0165] In some embodiments, the CRISPR nuclease in any one of the embodiments described herein is fused to a polymerase.In some embodiments, the CRISPR nuclease described in any one of the above embodiments is fused to a reverse transcriptase polypeptide.In some embodiments, the CRISPR nuclease comprises an N-terminal reverse transcriptase polypeptide.In some embodiments, the CRISPR nuclease comprises a C-terminal reverse transcriptase polypeptide.In some embodiments, the CRISPR nuclease comprises a reverse transcriptase polypeptide at an intramolecular location within the CRISPR nuclease (e.g., reverse transcriptase is within the loop of the CRISPR nuclease).

[0166] In some embodiments, the CRISPR nuclease in any one of the embodiments described herein interacts with the reverse transcriptase polypeptide (e.g., via electrostatic interaction). In some embodiments, the CRISPR nuclease comprises a dimerization domain. As used herein, the term "dimerization domain" refers to a polypeptide domain that is capable of specifically binding to a separate compatible polypeptide domain (e.g., a second compatible dimerization domain). In some embodiments, the dimer is formed by a non-covalent bond between a first dimerization domain and a second compatible dimerization domain. In some embodiments, the dimerization domain is a leucine zipper, a nanobody, or an antibody. In some embodiments, the dimerization domain recruits the reverse transcriptase polypeptide. In some embodiments, the CRISPR nuclease and the reverse transcriptase polypeptide interact via a coiled-coil peptide heterodimer.

[0167] In some embodiments, the CRISPR nuclease in any one of the embodiments described herein interacts with a ligase, an integrase, and / or a recombinase. In some embodiments, the CRISPR nuclease in any one of the embodiments described herein is fused to a ligase, an integrase, and / or a recombinase. In some embodiments, the ligase, integrase, and / or a recombinase is fused to the N-terminus or C-terminus of the CRISPR nuclease. In some embodiments, the ligase, integrase, and / or a recombinase is fused internally to the CRISPR nuclease. In some embodiments, the integrase is a serine integrase. In some embodiments, the integrase is a Bxb1, TP901, or PhiBT1 integrase. In some embodiments, the recombinase is a serine recombinase or a tyrosine recombinase. In some embodiments, the recombinase is a CRE recombinase. In some embodiments, the CRISPR nuclease that interacts with or is fused to a ligase, integrase, and / or recombinase is further interacting with or is further fused to a reverse transcriptase.

[0168] B. Reverse transcriptase In various embodiments, the compositions disclosed herein include a polymerase (e.g., a DNA-dependent DNA polymerase or an RNA-dependent DNA polymerase) or variant thereof, which may be provided as a fusion to a CRISPR nuclease. The polymerase may be a wild-type polymerase, a functional fragment, a variant, or a truncated variant, etc. The polymerase may include a wild-type polymerase from a eukaryotic, prokaryotic, archaeal, or viral organism, and / or the polymerase may be modified by genetic engineering, mutagenesis, or a process based on directed evolution.

[0169] Any of the CRISPR nuclease-RT fusion polypeptides, such as those disclosed herein (e.g., those shown in Tables 7 and 17), their encoding nucleic acids, vectors containing them, and methods of making them, are also within the scope of this disclosure.

[0170] In some embodiments, the polymerase is a reverse transcriptase. In some embodiments, the reverse transcriptase polypeptide is any wild-type reverse transcriptase, which may be obtained from any naturally occurring organism or virus, or from a commercially available or non-commercial source. The reverse transcriptase polypeptide may also be a variant reverse transcriptase polypeptide.

[0171] Reverse transcriptase polypeptide can be obtained from several different sources. For example, the gene can be obtained from a eukaryotic cell infected with a retrovirus, or from a plasmid that contains either a part or the entirety of a retroviral genome. In addition, RNA that contains the reverse transcriptase gene can be obtained from a retrovirus. In some embodiments, reverse transcriptase is expressed or otherwise provided as an individual component, i.e., not as a fusion protein with a CRISPR nuclease (e.g., Cas12i) polypeptide.

[0172] Reverse transcriptases are known in the art and include, but are not limited to, Moloney murine leukemia virus (MMLV) reverse transcriptase, human immunodeficiency virus (HIV) reverse transcriptase, and avian sarcoma leukemia virus (ASLV) reverse transcriptase, which include, but are not limited to, Rous sarcoma virus (RSV) reverse transcriptase, avian myeloblastosis virus (AMV) reverse transcriptase, avian erythroblastosis virus (AEV) helper virus MCAV reverse transcriptase, avian myelocytomatosis virus MC29 helper virus MCAV reverse transcriptase, avian reticuloendotheliosis virus (REV-T) helper virus REV-A reverse transcriptase, avian sarcoma virus UR2 helper virus UR2AV reverse transcriptase, avian sarcoma virus Y73 helper virus YAV reverse transcriptase, Rous associated virus (RAV) reverse transcriptase, and myeloblastosis associated virus (MAV) reverse transcriptase, which may be suitable for use in the compositions described herein, as will be recognized by those of skill in the art.

[0173] In some embodiments, the reverse transcriptase is MMLV-RT, Marathon RT from Eubacterium rectale, or RTX reverse transcriptase, or a variant of MMLV-RT, Marathon RT, or RTX reverse transcriptase. In some embodiments, the reverse transcriptase is a sequence shown in Table 2, a variant thereof, or an ortholog thereof.

[0174] [Table 2-1]

[0175] [Table 2-2]

[0176] [Table 2-3]

[0177] In some embodiments, the reverse transcriptase polypeptide is fused to the CRISPR nuclease in any one of the embodiments described herein.In some embodiments, the reverse transcriptase polypeptide comprises an N-terminal CRISPR nuclease.In some embodiments, the reverse transcriptase polypeptide comprises a C-terminal CRISPR nuclease.In some embodiments, the reverse transcriptase polypeptide comprises a CRISPR nuclease at an intramolecular location within the reverse transcriptase polypeptide (e.g., the CRISPR nuclease) is within the loop of the reverse transcriptase polypeptide.

[0178] In some embodiments, the reverse transcriptase polypeptide comprises a dimerization domain. In some embodiments, the dimerization domain is a leucine zipper, a nanobody, or an antibody. In some embodiments, the dimerization domain recruits a CRISPR nuclease (e.g., a V-type nuclease, e.g., a Cas12i polypeptide).

[0179] In some embodiments, the reverse transcriptase polypeptide is an "error-prone" reverse transcriptase variant. Any error-prone reverse transcriptase known and / or available in the art may be used. It will be understood that reverse transcriptase does not naturally have any proofreading function, and therefore the error rate of reverse transcriptase is generally higher than that of DNA polymerases that contain proofreading activity. In some embodiments, a reverse transcriptase is considered to be "error-prone" if it has an error rate of less than one error in about 15,000 nucleotides synthesized.

[0180] In some embodiments, the reverse transcriptase polypeptide has one or more mutations in the RNase H domain. In some embodiments, the reverse transcriptase polypeptide does not include an RNase H domain (e.g., the RNase H domain is removed from the reverse transcriptase polypeptide). In some embodiments, the RNase H domain is truncated in the reverse transcriptase polypeptide. In some embodiments, the reverse transcriptase polypeptide has one or more mutations in the RNA-dependent DNA polymerase domain. In some embodiments, the reverse transcriptase polypeptide is a variant with altered thermostability characteristics. The ability of reverse transcriptase to withstand high temperatures is an important aspect of cDNA synthesis. Elevated reaction temperatures aid in the denaturation of RNA with strong secondary structures and / or high GC content, allowing reverse transcriptase to read through the entire sequence. As a result, reverse transcription at higher temperatures allows full-length cDNA synthesis and higher yields. Wild-type M-MLV reverse transcriptase typically has an optimum temperature in the range of 37-48°C, however mutations can be introduced that allow reverse transcription activity at higher temperatures beyond 48°C, including 49°C, 50°C, 51°C, 52°C, 53°C, 54°C, 55°C, 56°C, 57°C, 58°C, 59°C, 60°C, 61°C, 62°C, 63°C, 64°C, 65°C, and 66°C or higher.

[0181] As used herein, a variant reverse transcriptase polypeptide can be at least about 20% identical, at least about 25% identical, at least about 30% identical, at least about 35% identical, at least about 40% identical, at least about 45% identical, at least about 50% identical, at least about 55% identical, at least about 60% identical, at least about 65% identical, at least about 70% identical, at least about 75% identical, at least about 80% identical, at least about 85% identical, at least about 90% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% identical to any reference reverse transcriptase polypeptide, where the reference reverse transcriptase polypeptide includes any wild-type reverse transcriptase, mutant reverse transcriptase, or fragment of reverse transcriptase, or other reverse transcriptase variant disclosed or contemplated herein or known in the art. In some embodiments, a reverse transcriptase variant can have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 21, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or up to 100, or up to 200, or up to 300, or up to 400, or up to 500 or more amino acid changes compared to a reference reverse transcriptase. In some embodiments, the reverse transcriptase variants include fragments of a reference reverse transcriptase such that the fragments are at least about 20% identical, at least about 25% identical, at least about 30% identical, at least about 35% identical, at least about 40% identical, at least about 45% identical, at least about 50% identical, at least about 55% identical, at least about 60% identical, at least about 65% identical, at least about 70% identical, at least about 75% identical, at least about 80% identical, at least about 85% identical, at least about 90% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% identical to the corresponding fragment of the reference reverse transcriptase.

[0182] Variant reverse transcriptases, including error-prone reverse transcriptases, thermostable reverse transcriptases, and reverse transcriptases with increased processivity, can be engineered by a variety of conventional strategies, including mutagenesis or evolutionary processes. In some cases, variants can be produced by introducing a single mutation. In other cases, variants may require two or more mutations. For those variants that contain two or more mutations, the effect of a given mutation can be evaluated by introducing the identified mutation into the wild-type gene by site-directed mutagenesis, in isolation from other mutations derived from the particular variant. Screening assays of the single mutants thus produced then allow for the determination of the effect of this mutation alone.

[0183] In some embodiments, the reverse transcriptase polypeptide comprises or is fused to a domain to improve the extension rate and / or efficiency of the reverse transcriptase. In some embodiments, the reverse transcriptase polypeptide is fused to an Sso7d polypeptide, e.g., an Sso7d polypeptide from Sulfolobus solfataricus. See, e.g., Wang et al., Nucleic Acids Res. 32(3):1197-207 (2004).

[0184] In some embodiments, the CRISPR nuclease-reverse transcriptase fusion polypeptide described elsewhere herein can bind to and bind to at least one nuclease binding sequence in the editing template RNA. In some embodiments, the CRISPR nuclease-reverse transcriptase fusion polypeptide can bind to and bind to the target sequence via at least one DNA binding sequence in the editing template RNA. In such embodiments, the CRISPR nuclease-reverse transcriptase fusion polypeptide is recruited to or brought into close proximity to the target sequence via the binding of the CRISPR nuclease via the nuclease binding sequence and DNA binding sequence of the editing template RNA.

[0185] In some embodiments, the reverse transcriptase transcribes the reverse transcription template sequence into the non-PAM strand of the target nucleic acid starting at the 5' end of the PBS. In some embodiments, the reverse transcriptase transcribes the reverse transcription template sequence into the non-PAM strand of the target nucleic acid starting at the 3' end of the PBS. In some embodiments, the reverse transcriptase transcribes the reverse transcription template sequence into the PAM strand of the target nucleic acid starting at the 5' end of the PBS. In some embodiments, the reverse transcriptase transcribes the reverse transcription template sequence into the PAM strand of the target nucleic acid starting at the 3' end of the PBS. In some embodiments, after binding of the PBS to the non-PAM strand of the target nucleic acid, the reverse transcriptase transcribes the reverse transcription template sequence from the free 3' end of the non-PAM strand. In some embodiments, after hybridization of the PBS to the PAM strand of the target nucleic acid, the reverse transcriptase transcribes the reverse transcription template sequence from the free 3' end of the PAM strand.

[0186] In some embodiments, the reverse transcriptase in any one of the embodiments described herein interacts with a ligase, an integrase, and / or a recombinase. In some embodiments, the reverse transcriptase in any one of the embodiments described herein is fused to a ligase, an integrase, and / or a recombinase. In some embodiments, the ligase, integrase, and / or a recombinase is fused to the N-terminus or C-terminus of the reverse transcriptase. In some embodiments, the ligase, integrase, and / or a recombinase is fused internally to the reverse transcriptase. In some embodiments, the integrase is a serine integrase. In some embodiments, the integrase is a Bxb1, TP901, or PhiBT1 integrase. In some embodiments, the recombinase is a serine recombinase or a tyrosine recombinase. In some embodiments, the recombinase is a CRE recombinase. In some embodiments, the reverse transcriptase that interacts with or is fused to a ligase, integrase, and / or recombinase is further interacting with or is further fused to a CRISPR nuclease.

[0187] C. Gene-editing RNA molecules Any of the gene editing systems disclosed herein may include an editing template RNA(s) (gene editing RNA), where the editing template RNA(s) includes an RNA guide and an RNA reverse transcriptase (RT) donor (RT donor RNA). The editing template RNA(s) assists in editing a target nucleic acid, e.g., a sequence at a desired genomic site. In some embodiments, the editing template RNA can be a single RNA molecule, where the single RNA molecule includes both an RNA guide (e.g., including a nuclease binding sequence and a DNA binding sequence) and an RT donor RNA. In other embodiments, the editing template RNA includes an RNA guide and an RT donor RNA as separate RNA molecules.

[0188] In some embodiments, the editing template RNA or any portion thereof is encoded in a vector. In some embodiments, the vector comprises a Pol II promoter or a Pol III promoter. In some embodiments, the editing template RNA disclosed herein does not comprise a tracrRNA component. Alternatively, the editing template RNA disclosed herein may comprise a tracrRNA component.

[0189] i.RNA guide In any of the gene editing systems disclosed herein, the editing template RNA comprises an RNA guide, and the RNA guide administers the cleavage of the target nucleic acid via the CRISPR nuclease also contained in the gene editing system. The RNA guide (or gRNA) comprises a nuclease binding sequence and a DNA binding sequence (spacer). The nuclease binding sequence can comprise one or more binding sites that can be recognized for binding by the CRISPR nuclease. In some cases, the gRNA is a single RNA molecule that comprises both the nuclease binding sequence and the spacer sequence. Alternatively, the gRNA can comprise the nuclease binding sequence and the spacer as two separate RNA molecules.

[0190] In some embodiments, the RNA guide comprises an RNA extension at the 5' end of the RNA guide, at the 3' end of the RNA guide, or at an intramolecular location within the RNA guide. In various embodiments, the RNA extension is at least 5 nucleotides, at least 6 nucleotides, at least 7 nucleotides, at least 8 nucleotides, at least 9 nucleotides, at least 10 nucleotides, at least 11 nucleotides, at least 12 nucleotides, at least 13 nucleotides, at least 14 nucleotides, at least 15 nucleotides, at least 16 nucleotides, at least 17 nucleotides, at least 18 nucleotides, at least 19 nucleotides, at least 20 nucleotides, at least 21 nucleotides, at least 22 nucleotides, at least 23 nucleotides, at least 24 nucleotides, at least 25 nucleotides, at least 26 nucleotides, at least 27 nucleotides, at least 28 nucleotides, at least 29 nucleotides, at least 30 nucleotides, at least 31 nucleotides, at least 32 nucleotides, at least 33 nucleotides, at least 34 nucleotides, at least 35 nucleotides, at least 36 nucleotides, at least 37 nucleotides, at least 38 nucleotides, at least 39 nucleotides, at least 40 nucleotides, at least 41 nucleotides, at least 42 nucleotides, at least 43 nucleotides, at least 44 nucleotides, at least 45 nucleotides, at least 46 nucleotides, at least 47 nucleotides, at least 48 nucleotides, at least 49 nucleotides, or at least 50 nucleotides in length. In some embodiments, the RNA extension is a reverse transcription donor RNA ("RT donor RNA") (e.g., an RNA guide is fused to the RT donor RNA). In some embodiments, the RT donor RNA comprises a primer binding site (PBS) and a reverse transcription template sequence as described herein.

[0191] Nuclease binding sequence In some embodiments, the compositions described herein comprise a nuclease binding sequence. In some embodiments, the nuclease binding sequence is a CRISPR nuclease binding sequence (e.g., the nuclease binding sequence is capable of binding to a type V nuclease or a type II nuclease). In some embodiments, the nuclease binding sequence is further a nucleic acid binding sequence (e.g., a DNA binding sequence).

[0192] In some embodiments, the nuclease binding sequence comprises an RNA guide. The RNA guide can bind with specific binding affinity to any one of the CRISPR nucleases described herein (e.g., type V nuclease or type II nuclease). In some embodiments, the RNA guide further comprises specific binding affinity to a target sequence. In some embodiments, the compositions described herein comprise two or more (e.g., 2, 3, 4, 5, 6, 7, 8, or 9 or more) RNA guides. In some embodiments, the nuclease binding sequence is encoded in a vector. In some embodiments, the vector comprises a Pol II promoter or a Pol III promoter.

[0193] In some embodiments, the nuclease binding sequence comprises a direct repeat sequence. In some embodiments, the nuclease binding sequence comprises a direct repeat sequence linked to a DNA binding sequence (e.g., a DNA target sequence or a spacer). In some embodiments, the nuclease binding sequence comprises a direct repeat sequence and a DNA binding sequence, or a direct repeat-DNA binding sequence-direct repeat sequence. In some embodiments, the nuclease binding sequence comprises a truncated direct repeat sequence and a DNA binding sequence, which is representative of the processed or mature crRNA.

[0194] In some embodiments, the nuclease binding sequence (e.g., a direct repeat sequence) is capable of binding to a Cas12a (Cpf1), Cas12b (C2c1), Cas12c, Cas12d, Cas12e, Cas12f, Cas12h, Cas12i, or Cas12j (CasPhi) polypeptide. In some embodiments, the direct repeat sequence is capable of binding to a Cas9 polypeptide.

[0195] In embodiments where the nuclease binding sequence is a direct repeat for a publicly available CRISPR nuclease, these direct repeat sequences are known in the art. In some embodiments, the direct repeat sequences capable of binding to a CRISPR nuclease are those described in WO2021 / 055874, WO2020 / 206036, WO2020 / 191102, WO2020 / 186213, WO2020 / 028555, WO2020 / 033601, WO2019 / 126762, WO2019 / 126774, WO2019 / 071048, WO2019 / 018423, WO2019 / 005866, WO2018 / 191388, WO2018 / 170333, WO2018 / 035388, WO2018 / 035387, WO2017 / 219027, WO2017 / 189308, WO2017 / 184768, WO2017 / 106657, WO2016 / 205749, WO2017 / 070605, WO2016 / 205764, WO2016 / 205711, WO2016 / 028682, WO2015 / 089473, WO2014 / 093595, WO2015 / 089427, WO2014 / 204725, WO2015 / 07 0083, WO2014 / 093655, WO2014 / 093694, WO2014 / 093712, WO2014 / 093635, WO2021 / 133829, WO2021 / 007177, WO2020 / 197934, WO2020 / 181 102, WO2020 / 181101, WO2020 / 041456, WO2020 / 023529, WO2020 / 005980, WO2019 / 104058, WO2019 / 089820, WO2019 / 089808, WO2019 / 0898 04, WO2019 / 089796, WO2019 / 036185, WO2018 / 226855, WO2018 / 213351, WO2018 / 089664, WO2018 / 064371, WO2018 / 064352, WO2017 / 10656 9, WO2017 / 048969, WO2016 / 196655, WO2016 / 106239, WO2016 / 036754, WO2015 / 103153, WO2015 / 089277, WO2014 / 150624, WO2013 / 176772,WO2021 / 119563, WO2021 / 118626, WO2020 / 247883, WO2020 / 247882, WO2020 / 2 23634, WO2020 / 142754, WO2020 / 086475, WO2020 / 028729, WO2019 / 241452, WO2 019 / 173248, WO2018 / 236548, WO2018 / 183403, WO2017 / 027423, WO2018 / 10672 7, WO2018 / 071672, WO2017 / 096328, WO2017 / 070598, WO2016 / 201155, WO2014 / 150624, WO2013 / 098244, WO2021 / 113522, WO2021 / 050534, WO2021 / 046442, WO2021 / 041569, WO2021 / 007563, WO2020 / 252378, WO2020 / 180699, WO2020 / 018142, WO2019 / 222555, WO2019 / 178428, WO2019 / 178427, or WO2019 / 006471, the relevant disclosures of which are incorporated by reference for the subject matter and for the purposes referenced herein.

[0196] In some embodiments where the CRISPR nuclease is a Cas12i polypeptide, the direct repeat sequence comprises at least 90% identity to any one of SEQ ID NOs: 12-24. In some embodiments where the CRISPR nuclease is a Cas12i polypeptide, the direct repeat sequence comprises at least 95% identity to any one of SEQ ID NOs: 12-24. In some embodiments where the CRISPR nuclease is a Cas12i polypeptide, the direct repeat sequence comprises any one of SEQ ID NOs: 12-24. In some embodiments, the direct repeat sequence comprises a portion of any one of SEQ ID NOs: 12-24.

[0197] [Table 3-1]

[0198] [Table 3-2]

[0199] [Table 3-3]

[0200] Nuclease binding sequences for other CRISPR nucleases, such as other V-type CRISPR nucleases, are known in the art and / or are provided in Tables 4-6 below.

[0201] DNA-binding polypeptides The RNA guide may also include a DNA binding sequence. In some embodiments, the DNA binding sequence is a DNA target sequence (e.g., a spacer). The spacer may have a length of about 7 nucleotides to about 100 nucleotides. For example, the spacer may have a length of about 7 nucleotides to about 80 nucleotides, about 7 nucleotides to about 50 nucleotides, about 7 nucleotides to about 40 nucleotides, about 7 nucleotides to about 30 nucleotides, about 7 nucleotides to about 25 nucleotides, about 7 nucleotides to about 20 nucleotides, or about 7 nucleotides to about 19 nucleotides. For example, the spacer may be about 7 nucleotides to about 20 nucleotides, about 7 nucleotides to about 25 nucleotides, about 7 nucleotides to about 30 nucleotides, about 7 nucleotides to about 35 nucleotides, about 7 nucleotides to about 40 nucleotides, about 7 nucleotides to about 45 nucleotides, about 7 nucleotides to about 50 nucleotides, about 7 nucleotides to about 60 nucleotides, about 7 nucleotides to about 70 nucleotides, about 7 nucleotides to about 80 nucleotides, about 7 nucleotides to about 90 nucleotides, about 7 nucleotides to about 100 nucleotides, or about 100 nucleotides. The nucleotide sequence can have a length of about 10 nucleotides to about 25 nucleotides, about 10 nucleotides to about 30 nucleotides, about 10 nucleotides to about 35 nucleotides, about 10 nucleotides to about 40 nucleotides, about 10 nucleotides to about 45 nucleotides, about 10 nucleotides to about 50 nucleotides, about 10 nucleotides to about 60 nucleotides, about 10 nucleotides to about 70 nucleotides, about 10 nucleotides to about 80 nucleotides, about 10 nucleotides to about 90 nucleotides, or about 10 nucleotides to about 100 nucleotides.

[0202] In some embodiments, the spacer in the RNA guide can be designed to have a length of generally 7-50 nucleotides or 15-35 nucleotides (e.g., 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides) and to be complementary to a particular target sequence. In some embodiments, the RNA guide can be designed to have a length of 18-22 nucleotides.

[0203] In some embodiments, the DNA binding sequence has at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 99.5% sequence identity to a target sequence described herein and is capable of binding to a complementary region of the target sequence via base pairing.

[0204] In some embodiments, the DNA binding sequence includes only RNA bases. In some embodiments, the DNA binding sequence includes DNA bases (e.g., the spacer includes at least one thymine). In some embodiments, the DNA binding sequence includes RNA and DNA bases (e.g., the DNA binding sequence includes at least one thymine and at least one uracil).

[0205] In some cases, the RNA guides disclosed herein may further comprise linker sequences, 5'-end and / or 3'-end protection fragments (see disclosure herein), or combinations thereof.

[0206] The spacer in any of the RNA guides disclosed herein can be specific to a target sequence, i.e., can bind to a complementary region of the target sequence via base pairing. In some cases, the target sequence can be within a genomic site of interest, e.g., within a genomic site where gene editing is required.

[0207] In some embodiments, the target sequence is adjacent to a PAM sequence.PAM sequences are known in the art.In some embodiments, the PAM sequence that can be recognized by CRISPR nuclease is described in WO2021 / 055874, WO2020 / 206036, WO2020 / 191102, WO2020 / 186213, WO2020 / 028555, WO2020 / 033601, WO2019 / 126762, WO2019 / 126774, WO2019 / 071048, WO2019 / 018423, WO2019 / 005866, WO2018 / 191388, WO2018 / 170333, WO20 18 / 035388, WO2018 / 035387, WO2017 / 219027, WO2017 / 189308, WO2017 / 184768, WO2017 / 106657, WO2016 / 205749, WO2017 / 070605, WO2016 / 205 764, WO2016 / 205711, WO2016 / 028682, WO2015 / 089473, WO2014 / 093595, WO2015 / 089427, WO2014 / 204725, WO2015 / 070083, WO2014 / 093655, WO2 014 / 093694, WO2014 / 093712, WO2014 / 093635, WO2021 / 133829, WO2021 / 007177, WO2020 / 197934, WO2020 / 181102, WO2020 / 181101, WO2020 / 04 1456, WO2020 / 023529, WO2020 / 005980, WO2019 / 104058, WO2019 / 089820, WO2019 / 089808, WO2019 / 089804, WO2019 / 089796, WO2019 / 036185, W O2018 / 226855, WO2018 / 213351, WO2018 / 089664, WO2018 / 064371, WO2018 / 064352, WO2017 / 106569, WO2017 / 048969, WO2016 / 196655, WO2016 / 106239, WO2016 / 036754, WO2015 / 103153, WO2015 / 089277, WO2014 / 150624, WO2013 / 176772, WO2021 / 119563, WO2021 / 118626, WO2020 / 247883,WO2020 / 247882, WO2020 / 223634, WO2020 / 142754, WO2020 / 086475, WO2020 / 028729, WO2019 / 241452, WO2019 / 173248, WO2018 / 236548, WO2 018 / 183403, WO2017 / 027423, WO2018 / 106727, WO2018 / 071672, WO2017 / 096328, WO2017 / 070598, WO2016 / 201155, WO2014 / 150624, WO2013 / No. 098244, WO2021 / 113522, WO2021 / 050534, WO2021 / 046442, WO2021 / 041569, WO2021 / 007563, WO2020 / 252378, WO2020 / 180699, WO2020 / 018142, WO2019 / 222555, WO2019 / 178428, WO2019 / 178427, or WO2019 / 006471, the relevant disclosures of each of which are incorporated herein for the subject matter and purposes referenced herein.

[0208] When the gene editing system comprises a Cas12i polypeptide, the PAM sequence comprises 5'-NTTN-3' (or 5'-TTN-3'), where N is any nucleotide (e.g., A, G, T, or C). The PAM sequence is upstream of the target sequence. PAM sequences associated with other CRISPR nucleases may comprise the sequence 5'-TTY-3' or 5'-TTB-3', where Y is C or T and B is G, T, or C. The PAM sequence may be immediately adjacent to the target sequence or may be within a small number of nucleotides (e.g., 1, 2, 3, 4, or 5) of the target sequence.

[0209] Tables 4 to 6 below provide exemplary V-type CRISPR nucleases known in the art, and their corresponding nuclease binding sequences and PAM sequences. These sequences allow those skilled in the art to design the editing template RNA described herein with different V-type CRISPR nucleases. [Table 4]

[0210] [Table 5]

[0211] See also Zetsche et al., Cell 163:759-771 (2015), the relevant disclosure of which is incorporated by reference for and purposes related to the subject matter referenced herein.

[0212] Table 6 below provides information about additional V-type CRISPR nucleases known in the art. [Table 6]

[0213] ii. RNA reverse transcriptase donor or RT donor RNA The editing template RNA in any of the gene editing systems disclosed herein may also include an RNA reverse transcriptase (RT) donor (RT donor RNA). The RT donor RNA may include (i) a primer binding site (PBS) and (ii) a reverse transcription template sequence. In some cases, the RT donor RNA may further include (iii) a nucleotide linker sequence, (iv) a 5'-end and / or a 3'-end protection fragment (see the disclosure herein), or a combination thereof. In some embodiments, the editing template RNA includes one or more RT donor RNAs. In some embodiments, the editing template RNA includes one or more PBSs, one or more reverse transcription template sequences, and / or one or more nucleotide linker sequences. In some embodiments, the first editing template RNA includes one or more PBSs and the second editing template RNA includes one or more reverse transcription template sequences.

[0214] In some embodiments, the RT donor RNA comprises an aptamer, hi some embodiments, the aptamer recruits a reverse transcriptase polypeptide.

[0215] Primer binding site (PBS) In some embodiments, the PBS in the RT donor RNA disclosed herein is an RNA sequence that can bind to a DNA strand via base pairing. The DNA strand is nicked or cut, or can be nicked or cut, by a CRISPR nuclease. In some embodiments, the PBS comprises an RNA sequence that can bind to a DNA strand (PBS target site) via base pairing. The DNA strand may have a free 3' free end, or the 3' free end may be generated via cleavage by a CRISPR nuclease contained in the same gene editing system. In some examples, the PBS target site may be located on the same DNA strand as the PAM sequence (PAM strand). In some examples, the PBS target site may be located on the complementary strand (non-PAM strand) of the PAM strand.

[0216] In some embodiments, the PBS is at least 3 nucleotides, at least 4 nucleotides, at least 5 nucleotides, at least 6 nucleotides, at least 7 nucleotides, at least 8 nucleotides, at least 9 nucleotides, at least 10 nucleotides, at least 11 nucleotides, at least 12 nucleotides, at least 13 nucleotides, at least 14 nucleotides, at least 15 nucleotides, at least 16 nucleotides, at least 17 nucleotides, at least 18 nucleotides, at least 19 nucleotides, at least 20 nucleotides, at least 30 nucleotides, at least 40 nucleotides, at least 50 nucleotides, at least 60 nucleotides, at least 70 nucleotides, at least 80 nucleotides, at least 90 nucleotides, at least 100 nucleotides, at least 200 nucleotides, at least 300 nucleotides, at least 400 nucleotides, or at least 500 nucleotides in length. In some embodiments, the PBS is about 3 nucleotides to about 200 nucleotides long (e.g., about 3 nucleotides, 5 nucleotides, 8 nucleotides, 10 nucleotides, 13 nucleotides, 15 nucleotides, 20 nucleotides, 30 nucleotides, 40 nucleotides, 50 nucleotides, 60 nucleotides, 70 nucleotides, 80 nucleotides, 90 nucleotides, 100 nucleotides, 110 nucleotides, 120 nucleotides, 130 nucleotides, 140 nucleotides, 150 nucleotides, 160 nucleotides, 170 nucleotides, 180 nucleotides, 190 nucleotides, 200 nucleotides long, or any length therebetween). In some embodiments, the PBS is about 3 nucleotides to about 100 nucleotides long (e.g., about 3 nucleotides, 5 nucleotides, 8 nucleotides, 10 nucleotides, 13 nucleotides, 15 nucleotides, 20 nucleotides, 30 nucleotides, 40 nucleotides, 50 nucleotides, 60 nucleotides, 70 nucleotides, 80 nucleotides, 90 nucleotides, or 100 nucleotides long, or any length therebetween).

[0217] In some embodiments, the PBS is about 10 nucleotides to about 50 nucleotides in length. In some embodiments, the PBS is about 10 nucleotides to about 40 nucleotides in length. In some embodiments, the PBS is about 10 nucleotides to about 30 nucleotides in length. In some embodiments, the PBS is about 10 nucleotides to about 20 nucleotides in length. In some embodiments, the PBS is about 10 nucleotides to about 15 nucleotides in length. In some embodiments, the PBS is about 11 nucleotides in length. In some embodiments, the PBS is about 12 nucleotides in length. In some embodiments, the PBS is about 13 nucleotides in length. In some embodiments, the PBS is about 14 nucleotides in length. In some embodiments, the PBS is about 30 nucleotides in length.

[0218] In a gene editing system including a Cas12i polypeptide (e.g., a Cas12i2 polypeptide as disclosed herein), the PBS in the RT donor RNA may bind to a region on the non-PAM strand (a PBS target site). In some cases, the PBS target site may be located upstream of the complementary region of the target sequence. For example, the PBS target site may be up to 20 nucleotides, e.g., up to 15 nucleotides, up to 10 nucleotides, or up to 5 nucleotides, upstream of the complementary region. In a specific example, the PBS target site may be about 3 nucleotides to about 10 nucleotides upstream of the complementary region. In specific examples, the PBS target site is 1 nucleotide, 1-2 nucleotides, 1-3 nucleotides, 1-4 nucleotides, 1-5 nucleotides, 1-6 nucleotides, 1-7 nucleotides, 1-8 nucleotides, 1-9 nucleotides, 1-10 nucleotides, 2-3 nucleotides, 2-4 nucleotides, 2-5 nucleotides, 2-6 nucleotides, 2-7 nucleotides, 2-8 nucleotides, 2-9 nucleotides, 2-10 nucleotides, 3-4 nucleotides, 3-5 nucleotides, 3-6 nucleotides, 3-7 nucleotides, 3-8 nucleotides, 3-9 nucleotides, 3-10 nucleotides, 3-4 nucleotides, 3-5 nucleotides, 3-6 nucleotides, 3-7 nucleotides, 3-8 ... The PBS target site may be 1 to 3 nucleotides, 3 to 9 nucleotides, 3 to 10 nucleotides, 4 to 5 nucleotides, 4 to 6 nucleotides, 4 to 7 nucleotides, 4 to 8 nucleotides, 4 to 9 nucleotides, 4 to 10 nucleotides, 5 to 6 nucleotides, 5 to 7 nucleotides, 5 to 8 nucleotides, 5 to 9 nucleotides, 5 to 10 nucleotides, 6 to 7 nucleotides, 6 to 8 nucleotides, 6 to 9 nucleotides, 6 to 10 nucleotides, 7 to 8 nucleotides, 7 to 9 nucleotides, 7 to 10 nucleotides, 8 to 9 nucleotides, 8 to 10 nucleotides, 9 to 10 nucleotides, or 10 nucleotides. In other cases, the PBS target site may overlap with the complementary region.When a free 3' end is generated within or near the target sequence and complementary region by the Cas12i polypeptide in the gene editing system, PBS binding to the non-PAM strand at a site upstream of or overlapping with the complementary region can efficiently facilitate DNA synthesis by the RT polypeptide in the gene editing system starting from the free 3' end generated in the non-PAM strand. Exemplary diagrams are provided in Figures 12A and 12B.

[0219] Reverse transcription template sequence A reverse transcription template sequence (template sequence) serves as a template for reverse transcription mediated by the RT polypeptide in the gene editing system disclosed herein. In some embodiments, the reverse transcription template sequence comprises a sequence having at least one encoded edit. In some embodiments, the reverse transcription template sequence comprises sequence homology with a target sequence having at least one encoded edit or its complementary region. In some embodiments, the reverse transcription template sequence is at least 3 nucleotides, at least 4 nucleotides, at least 5 nucleotides, at least 6 nucleotides, at least 7 nucleotides, at least 8 nucleotides, at least 9 nucleotides, at least 10 nucleotides, at least 11 nucleotides, at least 12 nucleotides, at least 13 nucleotides, at least 14 nucleotides, at least 15 nucleotides, at least 16 nucleotides, at least 17 nucleotides, at least 18 nucleotides, at least 19 nucleotides, at least 20 nucleotides, at least 30 nucleotides, at least 40 nucleotides, at least 50 nucleotides, at least 60 nucleotides, at least 70 nucleotides, at least 80 nucleotides, at least 90 nucleotides, at least 100 nucleotides, at least 200 nucleotides, at least 300 nucleotides, at least 400 nucleotides, or at least 500 nucleotides in length. In some embodiments, the reverse transcription template sequence is about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, or 120 nucleotides in length, or any length therebetween.

[0220] In some embodiments, the reverse transcription template sequence is about 25 nucleotides. In some embodiments, the reverse transcription template sequence is about 26 nucleotides. In some embodiments, the reverse transcription template sequence is about 27 nucleotides. In some embodiments, the reverse transcription template sequence is about 28 nucleotides. In some embodiments, the reverse transcription template sequence is about 29 nucleotides. In some embodiments, the reverse transcription template sequence is about 30 nucleotides. In some embodiments, the reverse transcription template sequence is about 31 nucleotides. In some embodiments, the reverse transcription template sequence is about 32 nucleotides. In some embodiments, the reverse transcription template sequence is about 33 nucleotides. In some embodiments, the reverse transcription template sequence is about 34 nucleotides. In some embodiments, the reverse transcription template sequence is about 35 nucleotides. In some embodiments, the reverse transcription template sequence is about 36 nucleotides. In some embodiments, the reverse transcription template sequence is about 37 nucleotides. In some embodiments, the reverse transcription template sequence is about 38 nucleotides. In some embodiments, the reverse transcription template sequence is about 39 nucleotides. In some embodiments, the reverse transcription template sequence is about 40 nucleotides. In some embodiments, the reverse transcription template sequence is about 41 nucleotides. In some embodiments, the reverse transcription template sequence is about 42 nucleotides. In some embodiments, the reverse transcription template sequence is about 43 nucleotides. In some embodiments, the reverse transcription template sequence is about 44 nucleotides. In some embodiments, the reverse transcription template sequence is about 45 nucleotides. In some embodiments, the reverse transcription template sequence is about 46 nucleotides. In some embodiments, the reverse transcription template sequence is about 47 nucleotides. In some embodiments, the reverse transcription template sequence is about 48 nucleotides. In some embodiments, the reverse transcription template sequence is about 49 nucleotides. In some embodiments, the reverse transcription template sequence is about 50 nucleotides.

[0221] In some embodiments, the reverse transcription template sequence comprises at least one coded edit relative to the target sequence. In other embodiments, the reverse transcription template sequence comprises at least one coded edit relative to the complementary region of the target sequence. In some embodiments, the at least one coded edit comprises at least one substitution, insertion, and / or deletion. In some embodiments, the edit in the target sequence comprises a substitution, insertion, and / or deletion relative to the sequence of the target sequence. In some embodiments, the reverse transcription template sequence comprises at least one LoxP site.

[0222] In some embodiments, the edits can be single or multiple nucleotide substitutions, for example, a G to T substitution, a G to A substitution, a G to C substitution, a T to G substitution, a T to A substitution, a T to C substitution, a C to G substitution, a C to T substitution, a C to A substitution, an A to T substitution, an A to G substitution, or an A to C substitution. In some embodiments, the changes in the sequence can convert a G:C base pair to a T:A base pair, a G:C base pair to an A:T base pair, a G:C base pair to a C:G base pair, a T:A base pair to a G:C base pair, a T:A base pair to an A:T base pair, a T:A base pair to a C:G base pair, a C:G base pair to a G:C base pair, a C:G base pair to a T:A base pair, a C:G base pair to an A:T base pair, an A:T base pair to a T:A base pair, an A:T base pair to a G:C base pair, or an A:T base pair to a C:G base pair.

[0223] In some embodiments, the single or multiple nucleotide substitutions comprise at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 200, at least 300, at least 400, or at least 500 nucleotides in length. In some embodiments, the substitutions are from 1 nucleotide to about 200 nucleotides in length, e.g., from 1 nucleotide to 5 nucleotides, from 5 nucleotides to 10 nucleotides, from 10 nucleotides to 15 nucleotides, from 15 nucleotides to 20 nucleotides, from 20 nucleotides to 25 nucleotides, from 25 nucleotides to 30 nucleotides, from 30 nucleotides to 35 nucleotides, from 35 nucleotides to 40 nucleotides, from 40 nucleotides to 45 nucleotides, from 45 nucleotides to 50 nucleotides, from 50 nucleotides to 55 nucleotides, from 55 nucleotides to 60 nucleotides, from 60 nucleotides to 65 nucleotides, from 65 nucleotides to 70 nucleotides, from 70 nucleotides to 75 nucleotides, from 75 nucleotides to 80 nucleotides, from 80 nucleotides to 85 nucleotides, from 85 nucleotides to 90 nucleotides, from 95 nucleotides to 10 ... nucleotides to 90 nucleotides, 90 nucleotides to 95 nucleotides, 95 nucleotides to 100 nucleotides, 100 nucleotides to 105 nucleotides, 105 nucleotides to 110 nucleotides, 110 nucleotides to 115 nucleotides, 115 nucleotides to 120 nucleotides, 120 nucleotides to 125 nucleotides, 125 nucleotides to 130 nucleotides, 130 nucleotides to 135 nucleotides, 135 nucleotides to 140 nucleotides, 140 nucleotides to 145 nucleotides, 145 nucleotides to 150 nucleotides, 150 nucleotides to 155 nucleotides, 155 nucleotides to 160 nucleotides, 160 nucleotides to 165 nucleotides, 165 nucleotides to 170 nucleotides, 170 nucleotides to 175 nucleotides,It is 175 nucleotides to 180 nucleotides, 180 nucleotides to 185 nucleotides, 185 nucleotides to 190 nucleotides, 190 nucleotides to 195 nucleotides, or 195 nucleotides to 200 nucleotides in length. In some embodiments, the substitutions are from 1 nucleotide to about 300 nucleotides in length, e.g., from 1 nucleotide to 5 nucleotides, from 5 nucleotides to 10 nucleotides, from 10 nucleotides to 15 nucleotides, from 15 nucleotides to 20 nucleotides, from 20 nucleotides to 25 nucleotides, from 25 nucleotides to 30 nucleotides, from 30 nucleotides to 35 nucleotides, from 35 nucleotides to 40 nucleotides, from 40 nucleotides to 45 nucleotides, from 45 nucleotides to 50 nucleotides, from 50 nucleotides to 55 nucleotides, from 55 nucleotides to 60 nucleotides, from 60 nucleotides to 65 nucleotides, from 65 nucleotides to 70 nucleotides, from 70 nucleotides to 75 nucleotides, from 75 nucleotides to 80 nucleotides, from 80 nucleotides to 85 nucleotides, from 85 nucleotides to 90 nucleotides, from 90 nucleotides to 95 nucleotides, from 95 nucleotides to 100 nucleotides, from 100 nucleotides to 105 nucleotides, from 105 nucleotides to 110 nucleotides, 110 nucleotides to 115 nucleotides, 115 nucleotides to 120 nucleotides, 120 nucleotides to 125 nucleotides, 125 nucleotides to 130 nucleotides, 130 nucleotides to 135 nucleotides, 135 nucleotides to 140 nucleotides, 140 nucleotides to 145 nucleotides, 145 nucleotides to 150 nucleotides, 150 nucleotides to 155 nucleotides, 155 nucleotides to 160 nucleotides, 160 nucleotides to 165 nucleotides, 165 nucleotides to 170 nucleotides, 170 nucleotides to 175 nucleotides, 175 nucleotides to 180 nucleotides, 180 nucleotides to 185 nucleotides, 185 nucleotides to 190 nucleotides, 190 nucleotides to 195 nucleotides, 195 nucleotides to 200 nucleotides, 200 nucleotides to 210 nucleotides, 210 nucleotides to 220 nucleotides, 220 nucleotides to 230 nucleotides, 230 nucleotides to 240 nucleotides,The length is 240 nucleotides to 250 nucleotides, 250 nucleotides to 260 nucleotides, 260 nucleotides to 270 nucleotides, 270 nucleotides to 280 nucleotides, 280 nucleotides to 290 nucleotides, or 290 nucleotides to 300 nucleotides. In some embodiments, the substitution is up to about 10,000 bases (10 kb) long. For example, in some embodiments, the substitution is 1 base, about 10 bases, about 20 bases, about 30 bases, about 40 bases, about 50 bases, about 60 bases, about 70 bases, about 80 bases, about 90 bases, about 100 bases, about 200 bases, about 300 bases, about 400 bases, about 500 bases, about 600 bases, about 700 bases, about 800 bases, about 900 bases, about 1 kb, about 1.1 kb, about 1.2 kb, about 1.5 kb, about 1.6 kb, about 1.7 kb, about 1.8 kb, about 1.9 kb, about 2.0 kb, about 2.5 kb, about 2.6 kb, about 2.7 kb, about 2.8 kb, about 2.9 kb, about 3.1 kb, about 3.2 kb, about 3.5 kb, about 3.6 kb, about 3.7 kb, about 3.8 kb, about 3.9 ... kb, about 1.3kb, about 1.4kb, about 1.5kb, about 1.6kb, about 1.7kb, about 1.8kb, about 1.9kb, about 2kb, about 2.1kb, about 2.2kb, about 2.3kb, about 2.4kb, about 2.5kb, about 2.6kb, about 2.7kb, about 2.8kb, about 2.9kb, 3kb, 4kb, 5kb, 6kb, 7kb, 8kb, 9kb, or 10kb long. ,

[0224] In some embodiments the edit comprises an insertion of single or multiple nucleotides, and the insertion of single or multiple nucleotides is at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 200, at least 300, at least 400, or at least 500 nucleotides in length. In some embodiments, the insertion of single or multiple nucleotides is from 1 nucleotide to about 200 nucleotides in length, e.g., from 1 nucleotide to 5 nucleotides, from 5 nucleotides to 10 nucleotides, from 10 nucleotides to 15 nucleotides, from 15 nucleotides to 20 nucleotides, from 20 nucleotides to 25 nucleotides, from 25 nucleotides to 30 nucleotides, from 30 nucleotides to 35 nucleotides, from 35 nucleotides to 40 nucleotides, from 40 nucleotides to 45 nucleotides, from 45 nucleotides to 50 nucleotides, from 50 nucleotides to 55 nucleotides, from 55 nucleotides to 60 nucleotides, from 60 nucleotides to 65 nucleotides, from 65 nucleotides to 70 nucleotides, from 70 nucleotides to 75 nucleotides, from 75 nucleotides to 80 nucleotides. nucleotides, 80 nucleotides to 85 nucleotides, 85 nucleotides to 90 nucleotides, 90 nucleotides to 95 nucleotides, 95 nucleotides to 100 nucleotides, 100 nucleotides to 105 nucleotides, 105 nucleotides to 110 nucleotides, 110 nucleotides to 115 nucleotides, 115 nucleotides to 120 nucleotides, 120 nucleotides to 125 nucleotides, 125 nucleotides to 130 nucleotides, 130 nucleotides to 135 nucleotides, 135 nucleotides to 140 nucleotides, 140 nucleotides to 145 nucleotides, 145 nucleotides to 150 nucleotides, 150 nucleotides to 155 nucleotides, 155 nucleotides to 160 nucleotides, 160 nucleotides to 165 nucleotides,It is 165 nucleotides to 170 nucleotides, 170 nucleotides to 175 nucleotides, 175 nucleotides to 180 nucleotides, 180 nucleotides to 185 nucleotides, 185 nucleotides to 190 nucleotides, 190 nucleotides to 195 nucleotides, or 195 nucleotides to 200 nucleotides in length. In some embodiments, the insertion of single or multiple nucleotides is from 1 nucleotide to about 300 nucleotides in length, e.g., from 1 nucleotide to 5 nucleotides, from 5 nucleotides to 10 nucleotides, from 10 nucleotides to 15 nucleotides, from 15 nucleotides to 20 nucleotides, from 20 nucleotides to 25 nucleotides, from 25 nucleotides to 30 nucleotides, from 30 nucleotides to 35 nucleotides, from 35 nucleotides to 40 nucleotides, from 40 nucleotides to 45 nucleotides, from 45 nucleotides to 50 nucleotides, from 50 nucleotides to 55 nucleotides, from 55 nucleotides to 60 nucleotides, from 60 nucleotides to 65 nucleotides, from 65 nucleotides to 70 nucleotides, from 70 nucleotides to 75 nucleotides, from 75 nucleotides to 80 nucleotides, from 80 nucleotides to 85 nucleotides, from 85 nucleotides to 90 nucleotides, from 90 nucleotides to 95 nucleotides, from 95 nucleotides to 100 nucleotides, from 100 nucleotides to 105 nucleotides, 105 nucleotides to 110 nucleotides, 110 nucleotides to 115 nucleotides, 115 nucleotides to 120 nucleotides, 120 nucleotides to 125 nucleotides, 125 nucleotides to 130 nucleotides, 130 nucleotides to 135 nucleotides, 135 nucleotides to 140 nucleotides, 140 nucleotides to 145 nucleotides, 145 nucleotides to 150 nucleotides, 150 nucleotides to 155 nucleotides, 155 nucleotides to 160 nucleotides, 160 nucleotides to 165 nucleotides, 165 nucleotides to 170 nucleotides, 170 nucleotides to 175 nucleotides, 175 nucleotides to 180 nucleotides, 180 nucleotides to 185 nucleotides, 185 nucleotides to 190 nucleotides, 190 nucleotides to 195 nucleotides, 195 nucleotides to 200 nucleotides, 200 nucleotides to 210 nucleotides, 210 nucleotides to 220 nucleotides,The length is 220 nucleotides to 230 nucleotides, 230 nucleotides to 240 nucleotides, 240 nucleotides to 250 nucleotides, 250 nucleotides to 260 nucleotides, 260 nucleotides to 270 nucleotides, 270 nucleotides to 280 nucleotides, 280 nucleotides to 290 nucleotides, or 290 nucleotides to 300 nucleotides. In some embodiments, the single or multiple nucleotide insertions are up to about 10,000 bases (10 kb) long. For example, in some embodiments, the insertions are 1 base, about 10 bases, about 20 bases, about 30 bases, about 40 bases, about 50 bases, about 60 bases, about 70 bases, about 80 bases, about 90 bases, about 100 bases, about 200 bases, about 300 bases, about 400 bases, about 500 bases, about 600 bases, about 700 bases, about 800 bases, about 900 bases, about 1 kb, about 1.1 kb, about 1.2 kb, about 1.5 kb, about 1.6 kb, about 1.7 kb, about 1.8 kb, about 1.9 kb, about 2.0 kb, about 2.5 kb, about 2.6 kb, about 2.7 kb, about 2.8 kb, about 2.9 kb, about 3.1 kb, about 3.2 kb, about 3.5 kb, about 3.6 kb, about 3.7 kb, about 3.8 kb, about 3.9 kb, about 3.9 kb, about 3.1 kb, about 3.2 kb, about 3.5 k kb, about 1.3kb, about 1.4kb, about 1.5kb, about 1.6kb, about 1.7kb, about 1.8kb, about 1.9kb, about 2kb, about 2.1kb, about 2.2kb, about 2.3kb, about 2.4kb, about 2.5kb, about 2.6kb, about 2.7kb, about 2.8kb, about 2.9kb, 3kb, 4kb, 5kb, 6kb, 7kb, 8kb, 9kb, or 10kb long. ,

[0225] In some embodiments, the edits comprise a deletion of single or multiple nucleotides, and the deletion of single or multiple nucleotides is at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 200, at least 300, at least 400, or at least 500 nucleotides in length. In some embodiments, the deletion of a single or multiple nucleotides is from 1 nucleotide to about 200 nucleotides in length, e.g., from 1 nucleotide to 5 nucleotides, from 5 nucleotides to 10 nucleotides, from 10 nucleotides to 15 nucleotides, from 15 nucleotides to 20 nucleotides, from 20 nucleotides to 25 nucleotides, from 25 nucleotides to 30 nucleotides, from 30 nucleotides to 35 nucleotides, from 35 nucleotides to 40 nucleotides, from 40 nucleotides to 45 nucleotides, from 45 nucleotides to 50 nucleotides, from 50 nucleotides to 55 nucleotides, from 55 nucleotides to 60 nucleotides, from 60 nucleotides to 65 nucleotides, from 65 nucleotides to 70 nucleotides, from 70 nucleotides to 75 nucleotides, from 75 nucleotides to 80 nucleotides. nucleotides, 80 nucleotides to 85 nucleotides, 85 nucleotides to 90 nucleotides, 90 nucleotides to 95 nucleotides, 95 nucleotides to 100 nucleotides, 100 nucleotides to 105 nucleotides, 105 nucleotides to 110 nucleotides, 110 nucleotides to 115 nucleotides, 115 nucleotides to 120 nucleotides, 120 nucleotides to 125 nucleotides, 125 nucleotides to 130 nucleotides, 130 nucleotides to 135 nucleotides, 135 nucleotides to 140 nucleotides, 140 nucleotides to 145 nucleotides, 145 nucleotides to 150 nucleotides, 150 nucleotides to 155 nucleotides, 155 nucleotides to 160 nucleotides, 160 nucleotides to 165 nucleotides,It is 165 nucleotides to 170 nucleotides, 170 nucleotides to 175 nucleotides, 175 nucleotides to 180 nucleotides, 180 nucleotides to 185 nucleotides, 185 nucleotides to 190 nucleotides, 190 nucleotides to 195 nucleotides, or 195 nucleotides to 200 nucleotides in length. In some embodiments, the deletion of a single or multiple nucleotides is from 1 nucleotide to about 300 nucleotides in length, e.g., from 1 nucleotide to 5 nucleotides, from 5 nucleotides to 10 nucleotides, from 10 nucleotides to 15 nucleotides, from 15 nucleotides to 20 nucleotides, from 20 nucleotides to 25 nucleotides, from 25 nucleotides to 30 nucleotides, from 30 nucleotides to 35 nucleotides, from 35 nucleotides to 40 nucleotides, from 40 nucleotides to 45 nucleotides, from 45 nucleotides to 50 nucleotides, from 50 nucleotides to 55 nucleotides, from 55 nucleotides to 60 nucleotides, from 60 nucleotides to 65 nucleotides, from 65 nucleotides to 70 nucleotides, from 70 nucleotides to 75 nucleotides, from 75 nucleotides to 80 nucleotides, from 80 nucleotides to 85 nucleotides, from 85 nucleotides to 90 nucleotides, from 90 nucleotides to 95 nucleotides, from 95 nucleotides to 100 nucleotides, from 100 nucleotides to 105 nucleotides, nucleotides, 105 nucleotides to 110 nucleotides, 110 nucleotides to 115 nucleotides, 115 nucleotides to 120 nucleotides, 120 nucleotides to 125 nucleotides, 125 nucleotides to 130 nucleotides, 130 nucleotides to 135 nucleotides, 135 nucleotides to 140 nucleotides, 140 nucleotides to 145 nucleotides, 145 nucleotides to 150 nucleotides, 150 nucleotides to 155 nucleotides, 155 nucleotides to 160 nucleotides, 160 nucleotides to 165 nucleotides, 165 nucleotides to 170 nucleotides, 170 nucleotides to 175 nucleotides, 175 nucleotides to 180 nucleotides, 180 nucleotides to 185 nucleotides, 185 nucleotides to 190 nucleotides, 190 nucleotides to 195 nucleotides, 195 nucleotides to 200 nucleotides, 200 nucleotides to 210 nucleotides, 210 nucleotides to 220 nucleotides,The deletion is 220 nucleotides to 230 nucleotides, 230 nucleotides to 240 nucleotides, 240 nucleotides to 250 nucleotides, 250 nucleotides to 260 nucleotides, 260 nucleotides to 270 nucleotides, 270 nucleotides to 280 nucleotides, 280 nucleotides to 290 nucleotides, or 290 nucleotides to 300 nucleotides in length. In some embodiments, the deletion is up to about 10,000 bases (10 kb) in length. For example, in some embodiments, the deletion is 1 base, about 10 bases, about 20 bases, about 30 bases, about 40 bases, about 50 bases, about 60 bases, about 70 bases, about 80 bases, about 90 bases, about 100 bases, about 200 bases, about 300 bases, about 400 bases, about 500 bases, about 600 bases, about 700 bases, about 800 bases, about 900 bases, about 1 kb, about 1.1 kb, about 1.2 kb, about 1.5 kb, about 1.6 kb, about 1.7 kb, about 1.8 kb, about 1.9 kb, about 2.0 kb, about 2.5 kb, about 2.6 kb, about 2.7 kb, about 2.8 kb, about 2.9 kb, about 3.1 kb, about 3.2 kb, about 3.5 kb, about 3.6 kb, about 3.7 kb, about 3.8 kb, about 3.9 kb, about 3.9 kb, about 3.1 kb, about 3.2 kb, about 3.5 kb, about kb, about 1.3kb, about 1.4kb, about 1.5kb, about 1.6kb, about 1.7kb, about 1.8kb, about 1.9kb, about 2kb, about 2.1kb, about 2.2kb, about 2.3kb, about 2.4kb, about 2.5kb, about 2.6kb, about 2.7kb, about 2.8kb, about 2.9kb, 3kb, 4kb, 5kb, 6kb, 7kb, 8kb, 9kb, or 10kb long. ,

[0226] In some embodiments, the reverse transcription template sequence comprises at least one encoded edit and is from about 5 nucleotides to about 10,000 nucleotides in length, e.g., from 5 nucleotides to 10 nucleotides, from 10 nucleotides to 15 nucleotides, from 15 nucleotides to 20 nucleotides, from 20 nucleotides to 25 nucleotides, from 25 nucleotides to 30 nucleotides, from 30 nucleotides to 35 nucleotides, from 35 nucleotides to 40 nucleotides, from 40 nucleotides to 45 nucleotides, from 45 nucleotides to 50 nucleotides, from 50 nucleotides to 55 nucleotides, from 55 nucleotides to 60 nucleotides, from 60 nucleotides to 65 nucleotides, from 65 nucleotides to 70 nucleotides, from 70 nucleotides to 75 nucleotides, from 75 nucleotides to 80 nucleotides, from 80 nucleotides to 85 nucleotides, from 85 nucleotides to 90 nucleotides, from 90 nucleotides to 95 nucleotides, from 95 nucleotides to 100 nucleotides, from 100 nucleotides to 105 nucleotides, from 105 nucleotides to 110 nucleotides, from 110 nucleotides to 115 nucleotides, from 115 nucleotides to 120 nucleotides, from 120 nucleotides to 130 nucleotides, from 130 nucleotides to 140 nucleotides, from 140 nucleotides to 150 nucleotides, from 150 nucleotides to 160 nucleotides, from 160 nucleotides to 170 nucleotides, from 170 nucleotides to 180 nucleotides, from 180 nucleotides to 19 nucleotides ~ 125 nucleotides, 125 nucleotides ~ 130 nucleotides, 130 nucleotides ~ 135 nucleotides, 135 nucleotides ~ 140 nucleotides, 140 nucleotides ~ 145 nucleotides, 145 nucleotides ~ 150 nucleotides, 150 nucleotides ~ 155 nucleotides, 155 nucleotides ~ 160 nucleotides, 160 nucleotides ~ 165 nucleotides, 165 nucleotides ~ 170 nucleotides, 170 nucleotides ~ 175 nucleotides, 175 nucleotides ~ 180 nucleotides, 180 nucleotides ~ 1 85 nucleotides, 185 nucleotides to 190 nucleotides, 190 nucleotides to 195 nucleotides, 195 nucleotides to 200 nucleotides, 200 nucleotides to 210 nucleotides, 210 nucleotides to 220 nucleotides, 220 nucleotides to 230 nucleotides, 230 nucleotides to 240 nucleotides, 240 nucleotides to 250 nucleotides, 250 nucleotides to 260 nucleotides, 260 nucleotides to 270 nucleotides, 270 nucleotides to 280 nucleotides, 280 nucleotides to 290 nucleotides,or 290 nucleotides to 300 nucleotides in length, or about 1 kilobase (kb), about 1.1 kb, about 1.2 kb, about 1.3 kb, about 1.4 kb, about 1.5 kb, about 1.6 kb, about 1.7 kb, about 1.8 kb, about 1.9 kb, about 2 kb, about 2.1 kb, about 2.2 kb, about 2.3 kb, about 2.4 kb, about 2.5 kb, about 2.6 kb, about 2.7 kb, about 2.8 kb, about 2.9 kb, 3 kb, 4 kb, 5 kb, 6 kb, 7 kb, 8 kb, 9 kb, or 10 kb in length.

[0227] The reverse transcription template sequence may be transcribed into DNA by the reverse transcriptase of the gene editing system described herein. In some embodiments, the reverse transcription template sequence is transcribed 5' to 3' into the DNA of the PAM strand. In some embodiments, the reverse transcription template sequence is transcribed 5' to 3' into the DNA of the non-PAM strand. In some embodiments, the reverse transcription template sequence is transcribed 5' to 3' into the DNA of the PAM strand. In some embodiments, the reverse transcription template sequence is transcribed 5' to 3' into the DNA of the non-PAM strand. In some embodiments, the reverse transcription template sequence is 5' of the PBS. In some embodiments, the reverse transcription template sequence is 3' of the PBS. In some embodiments, the reverse transcription template sequence is transcribed into the DNA of the PAM strand via a 3' extension from the PBS. In some embodiments, the reverse transcription template sequence is transcribed into the DNA of the non-PAM strand via a 3' extension from the PBS.

[0228] iii. Additional Elements In some embodiments, the editing template RNA may include one or more additional elements. For example, the editing template RNA, or its gRNA and / or RT donor RNA, may include one or more protection fragments at either or both ends of the RNA molecule. Alternatively or in addition, the editing template RNA, or its gRNA and / or RT donor RNA, may include additional elements inside the RNA molecule (e.g., between one or more of the sequences in the editing template RNA, e.g., between the PBS and the reverse transcription template sequence, e.g., the linker). In some embodiments, the editing template RNA includes additional elements between one or more sequences of the editing template RNA, e.g., between the RNA guide (nuclease binding sequence or DNA binding sequence) or the RT donor RNA (PBS or reverse transcription template sequence).

[0229] In some embodiments, the editing template RNA comprises additional elements, such as direct repeat sequences, at one or more ends. In some embodiments, the direct repeat sequences can recruit CRISPR nucleases (e.g., V-type nucleases, such as variant Cas12i2 polypeptides or variant Cas12i2-reverse transcriptase fusion polypeptides or Cas12i4-reverse transcriptase fusion polypeptides).

[0230] In some embodiments, the additional elements can be at least 3 nucleotides, at least 4 nucleotides, at least 5 nucleotides, at least 6 nucleotides, at least 7 nucleotides, at least 8 nucleotides, at least 9 nucleotides, at least 10 nucleotides, at least 11 nucleotides, at least 12 nucleotides, at least 13 nucleotides, at least 14 nucleotides, at least 15 nucleotides, at least 16 nucleotides, at least 17 nucleotides, at least 18 nucleotides, at least 19 nucleotides, at least 20 nucleotides, at least 30 nucleotides, at least 40 nucleotides, at least 50 nucleotides, at least 60 nucleotides, at least 70 nucleotides, at least 80 nucleotides, at least 90 nucleotides, at least 100 nucleotides, at least 200 nucleotides, at least 300 nucleotides, at least 400 nucleotides, or at least 500 nucleotides in length.

[0231] In some examples, the editing template RNA may include an optional nucleotide linker. Such optional nucleotide linker sequence may be at least 3 nucleotides, at least 4 nucleotides, at least 5 nucleotides, at least 6 nucleotides, at least 7 nucleotides, at least 8 nucleotides, at least 9 nucleotides, at least 10 nucleotides, at least 11 nucleotides, at least 12 nucleotides, at least 13 nucleotides, at least 14 nucleotides, at least 15 nucleotides, at least 16 nucleotides, at least 17 nucleotides, at least 18 nucleotides, at least 19 nucleotides, at least 20 nucleotides, at least 30 nucleotides, at least 40 nucleotides, at least 50 nucleotides, at least 60 nucleotides, at least 70 nucleotides, at least 80 nucleotides, at least 90 nucleotides, at least 100 nucleotides, at least 200 nucleotides, at least 300 nucleotides, at least 400 nucleotides, or at least 500 nucleotides long. In some embodiments, the optional nucleotide linker is between any of the nuclease binding sequence, the DNA binding sequence, the PBS, and / or the reverse transcription template sequence.

[0232] In some instances, the 5' and / or 3' ends of the editing template RNA, or its gRNA and / or RT donor RNA, may contain a protection fragment, which may enhance the resistance of the RNA molecule to exonuclease activity. See, for example, FIG. 11. In some instances, the end-protected fragment may include a nucleotide sequence capable of forming a secondary structure, such as a hairpin, a pseudoknot, or a triplex structure. In other instances, the end-protected fragment may include a sequence of an exoribonuclease-resistant RNA (xrRNA), a transfer RNA (tRNA), or a truncated tRNA. In some embodiments, the modification is a Zika-like pseudoknot, a murine leukemia virus pseudoknot (MLV-PK) sequence, a red clover necrotic mosaic virus (RCNMV) sequence, a sweet clover necrotic mosaic virus (SCNMV) sequence, a carnation ringspot virus (CRSV) sequence, a pre-Q sequence, or an RNA bacteriophage MS2 sequence. In a specific example, the end-protected fragment may include one or more CRISPR nuclease binding sites (e.g., binding sites for a Cas12i polypeptide, e.g., a Cas12i2 polypeptide), and may optionally include one or more segments (e.g., spacers) that do not share homology with any human sequence. In some cases, the one or more segments bind to a sequence that is 85% or less identical to any sequence in the human genome. See Figure 10, Figure 11, Figure 12A, and Figure 12B. Such end-protected fragments can recruit CRISPR nucleases contained within the same gene editing system to inhibit exoribonuclease activity without inducing off-target gene editing.

[0233] In some embodiments, the gene editing system disclosed herein comprises at least one editing template RNA (e.g., gene editing RNA) or a nucleotide sequence encoding the same. In some examples, the at least one editing template RNA is capable of binding to a CRISPR nuclease (e.g., a V-type CRISPR nuclease). In some examples, the at least one editing template RNA is further capable of binding to a nucleic acid (e.g., a DNA or a target nucleic acid). In some examples, the at least one editing template RNA comprises a nuclease binding sequence (e.g., one or more binding sites recognizable by a CRISPR nuclease) and a DNA binding sequence (e.g., a spacer). In some cases, the at least one editing template RNA comprises a gRNA (including a nuclease binding sequence and a spacer) and a RT donor RNA. In some embodiments, the editing template RNA comprises an RNA guide bound to a RT donor RNA. See, for example, FIG. 19B.

[0234] iv. Nucleic acid modification Any of the RNA components in the gene editing systems disclosed herein, for example, the editing template RNA, the RNA guide, the RT donor RNA, can contain one or more modifications.

[0235] Exemplary modifications can include any modification to the sugar, nucleobase, internucleoside linkage (e.g., phosphate linkage / phosphodiester linkage / phosphodiester backbone), and any combination thereof. Some of the exemplary modifications provided herein are described in detail below.

[0236] Any of the nucleic acid sequences encoding the RNA guide or components of the composition may include any useful modifications, such as modifications to the sugar, nucleobase, or internucleoside linkage (e.g., phosphate / phosphodiester linkage / phosphodiester backbone). One or more atoms of the pyrimidine nucleobase may be replaced or substituted with an optionally substituted amino, an optionally substituted thiol, an optionally substituted alkyl (e.g., methyl or ethyl), or a halo (e.g., chloro or fluoro). In certain embodiments, a modification (e.g., one or more modifications) is present in each of the sugar and the internucleoside linkage. The modification may be a modification from ribonucleic acid (RNA) to deoxyribonucleic acid (DNA), threose nucleic acid (TNA), glycol nucleic acid (GNA), peptide nucleic acid (PNA), locked nucleic acid (LNA), or a hybrid thereof). Additional modifications are described herein.

[0237] In some embodiments, the modification may include chemical or cell-induced modifications. For example, some non-limiting examples of intracellular RNA modifications are described by Lewis and Pan in "RNA modifications and structures cooperate to guide RNA-protein interactions" from Nat Reviews Mol Cell Biol, 2017, 18:202-210.

[0238] Different sugar modifications, nucleotide modifications, and / or internucleoside linkages (e.g., backbone structures) can be present at various positions in the sequence. It will be understood by those of skill in the art that the nucleotide analog or other modification(s) can be located at any position(s) of the sequence such that the function of the sequence is not substantially diminished. A sequence may contain from about 1% to about 100% modified nucleotides (either relative to the total nucleotide content or relative to one or more types of nucleotides, i.e., any one or more of A, G, U, or C), or any intervening percentage (e.g., 1%-20%>, 1%-25%, 1%-50%, 1%-60%, 1%-70%, 1%-80%, 1%-90%, 1%-95%, 10%-20%, 10%-25%, 10%-50%, 10%-60%, 10%-70%, 10%-80%, 10%-90%, 10%-95%). %, 10% to 100%, 20% to 25%, 20% to 50%, 20% to 60%, 20% to 70%, 20% to 80%, 20% to 90%, 20% to 95%, 20% to 100%, 50% to 60%, 50% to 70%, 50% to 80%, 50% to 90%, 50% to 95%, 50% to 100%, 70% to 80%, 70% to 90%, 70% to 95%, 70% to 100%, 80% to 90%, 80% to 95%, 80% to 100%, 90% to 95%, 90% to 100%, and 95% to 100%) of modified nucleotides.

[0239] In some embodiments, sugar modifications (e.g., at the 2' or 4' position) or sugar substitutions in one or more ribonucleotides of the sequence, and backbone modifications may include modification or substitution of phosphodiester bonds. Specific examples of sequences include, but are not limited to, sequences that include modified backbones or sequences that include internucleoside modifications, including non-natural internucleoside linkages, e.g., modified or substituted phosphodiester bonds. Sequences with modified backbones include, among others, those that do not have a phosphorus atom in the backbone. For the purposes of this application, and as often referred to in the art, modified RNAs that do not have a phosphorus atom in the internucleoside backbone may also be considered to be oligonucleosides. In certain embodiments, the sequence includes ribonucleotides that have a phosphorus atom in their internucleoside backbone.

[0240] Modified sequence backbones can include, for example, phosphorothioates; chiral phosphorothioates; phosphorodithioates; phosphotriesters; aminoalkyl phosphotriesters; methyl and other alkyl phosphonates, such as 3'-alkylene phosphonates and chiral phosphonates; phosphinates; phosphoramidates, such as 3'-amino phosphoramidates and aminoalkyl phosphoramidates; thionophosphoramidates; thionoalkyl phosphonates; thionoalkyl phosphotriesters; and boranophosphates with linear 3'-5' linkages, their 2'-5' linkage analogs, and those with reverse polarity, in which adjacent pairs of nucleoside units are linked 3'-5' to 5'-3' or 2'-5' to 5'-2'. Also included are various salts, mixed salts, and free acid forms. In some embodiments, sequences can have negative or positive charges.

[0241] Modified nucleotides that may be incorporated into a sequence may be modified on the internucleoside bond (e.g., phosphate backbone). In the context of polynucleotide backbone, the terms "phosphate" and "phosphodiester" are used interchangeably herein. The backbone phosphate group may be modified by replacing one or more of the oxygen atoms with different substituents. Furthermore, modified nucleosides and nucleotides may include a wide range of replacements of unmodified phosphate moieties with alternative internucleoside bond types as described herein. Examples of modified phosphate groups include, but are not limited to, phosphorothioates, phosphoroselenates, boranophosphates, boranophosphate esters, hydrogen phosphonates, phosphoramidates, phosphorodiamidates, alkyl or aryl phosphonates, and phosphotriesters. Phosphorodithioates have both non-linked oxygens replaced by sulfur. Phosphate linkers can also be modified by replacement of the linking oxygen at nitrogen (bridging phosphoramidates), sulfur (bridging phosphorothioates), and carbon (bridging methylene phosphonates).

[0242] The alpha-thio substituted phosphate moieties are provided to impart stability to RNA and DNA polymers through the unnatural phosphorothioate backbone linkages. Phosphorothioate DNA and RNA have increased nuclease resistance and subsequently longer half-lives in the cellular environment.

[0243] In specific embodiments, the modified nucleoside comprises an alpha-thio-nucleoside (e.g., 5'-O-(1-thiophosphate)-adenosine, 5'-O-(1-thiophosphate)-cytidine (a-thio-cytidine), 5'-O-(1-thiophosphate)-guanosine, 5'-O-(1-thiophosphate)-uridine, or 5'-O-(1-thiophosphate)-pseudouridine).

[0244] Other internucleoside linkages that can be used in accordance with the present invention are described herein, including internucleoside linkages that do not contain a phosphorus atom.

[0245] In some embodiments, the sequence may include one or more cytotoxic nucleosides. For example, cytotoxic nucleosides may be incorporated into the sequence, such as a bifunctional modification. Cytotoxic nucleosides may include, but are not limited to, adenosine arabinoside, 5-azacytidine, 4'-thio-aracytidine, cyclopentenylcytosine, cladribine, clofarabine, cytarabine, cytosine arabinoside, 1-(2-C-cyano-2-deoxy-beta-D-arabino-pentofuranosyl)-cytosine, decitabine, 5-fluorouracil, fludarabine, floxuridine, gemcitabine, a combination of tegafur and uracil, tegafur ((RS)-5-fluoro-1-(tetrahydrofuran-2-yl)pyrimidine-2,4(1H,3H)-dione), troxacitabine, tezacitabine, 2'-deoxy-2'-methylidenecytidine (DMDC), and 6-mercaptopurine. Additional examples include fludarabine phosphate, N4-behenoyl-1-beta-D-arabinofuranosylcytosine, N4-octadecyl-1-beta-D-arabinofuranosylcytosine, N4-palmitoyl-1-(2-C-cyano-2-deoxy-beta-D-arabino-pentofuranosyl)cytosine, and P-4055 (cytarabine 5'-elaidate).

[0246] In some embodiments, the sequence comprises one or more post-transcriptional modifications (e.g., capping, cleavage, polyadenylation, splicing, polyA sequences, methylation, acylation, phosphorylation, methylation of lysine and arginine residues, acetylation, and nitrosylation of thiol groups and tyrosine residues, etc.). The one or more post-transcriptional modifications can be any post-transcriptional modification, for example, any of the more than 100 different nucleoside modifications that have been identified in RNA (Rozenski, J, Crain, P, and McCloskey, J. (1999). The RNA Modification Database: 1999 update. Nucl Acids Res 27:196-197). In some embodiments, the first isolated nucleic acid comprises messenger RNA (mRNA). In some embodiments, the mRNA is selected from the group consisting of pyridin-4-one ribonucleosides, 5-aza-uridine, 2-thio-5-aza-uridine, 2-thiouridine, 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxyuridine, 3-methyluridine, 5-carboxymethyl-uridine, 1-carboxymethyl-pseudouridine, 5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinomethyluridine, 1-taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uridine, 1-taurinomethyl-4-thio-uridine, 5 In one embodiment, the nucleoside comprises at least one nucleoside selected from the group consisting of 1-methyl-uridine, 1-methyl-pseudouridine, 4-thio-1-methyl-pseudouridine, 2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-1-deaza-pseudouridine, dihydrouridine, dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxyuridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine, and 4-methoxy-2-thio-pseudouridine.In some embodiments, the mRNA is 5-aza-cytidine, pseudoisocytidine, 3-methyl-cytidine, N4-acetylcytidine, 5-formylcytidine, N4-methylcytidine, 5-hydroxymethylcytidine, 1-methyl-pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine, 2-thio-cytidine, 2-thio-5-methyl-cytidine, 4-thio-pseudoisocytidine, 4-thio-1-methyl-pseudoisocytidine, 4- The nucleoside comprises at least one nucleoside selected from the group consisting of thio-1-methyl-1-deaza-pseudoisocytidine, 1-methyl-1-deaza-pseudoisocytidine, zebularine, 5-aza-zebularine, 5-methyl-zebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine, 2-methoxy-cytidine, 2-methoxy-5-methyl-cytidine, 4-methoxy-pseudoisocytidine, and 4-methoxy-1-methyl-pseudoisocytidine. In some embodiments, the mRNA is 2-aminopurine, 2,6-diaminopurine, 7-deaza-adenine, 7-deaza-8-aza-adenine, 7-deaza-2-aminopurine, 7-deaza-8-aza-2-aminopurine, 7-deaza-2,6-diaminopurine, 7-deaza-8-aza-2,6-diaminopurine, 1-methyladenosine, N6-methyladenosine, N6-isopentenyladenosine, N6-(cis-hydroxyisobutyric acid), N6-isopropyl adenosine, ... The nucleoside comprises at least one nucleoside selected from the group consisting of N6-(cis-hydroxyisopentenyl)adenosine, 2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine, N6-glycinylcarbamoyladenosine, N6-threonylcarbamoyladenosine, 2-methylthio-N6-threonylcarbamoyladenosine, N6,N6-dimethyladenosine, 7-methyladenine, 2-methylthio-adenine, and 2-methoxy-adenine.In some embodiments, the mRNA comprises at least one nucleoside selected from the group consisting of inosine, 1-methyl-inosine, wyosine, wybutosine, 7-deaza-guanosine, 7-deaza-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deaza-guanosine, 6-thio-7-deaza-8-aza-guanosine, 7-methyl-guanosine, 6-thio-7-methyl-guanosine, 7-methylinosine, 6-methoxy-guanosine, 1-methylguanosine, N2-methylguanosine, N2,N2-dimethylguanosine, 8-oxo-guanosine, 7-methyl-8-oxo-guanosine, 1-methyl-6-thio-guanosine, N2-methyl-6-thio-guanosine, and N2,N2-dimethyl-6-thio-guanosine.

[0247] A sequence may be uniformly modified or unmodified along the entire length of the molecule. For example, one or more or all types of nucleotides (e.g., naturally occurring nucleotides, purines, or pyrimidines, or any one or more or all of A, G, U, C, I, pU) may be uniformly modified or unmodified in a sequence or within a given predetermined sequence region thereof. In some embodiments, the sequence includes pseudouridine. In some embodiments, the sequence includes inosine, which may aid the immune system in characterizing the sequence as endogenous versus viral RNA. Incorporation of inosine may also mediate improved RNA stability / reduced degradation. See, e.g., Yu, Z. et al. (2015) RNA editing by ADAR1 marks dsRNA as “self”. Cell Res. 25, 1283-1284, which is incorporated by reference in its entirety.

[0248] In some embodiments, any RNA sequence described herein, e.g., an edited template RNA, can include a terminal modification (e.g., a 5'-end modification or a 3'-end modification). In some embodiments, the terminal modification is a chemical modification. In some embodiments, the terminal modification is a structural modification. See the disclosure herein.

[0249] When the gene editing systems disclosed herein include nucleic acids, e.g., mRNA molecules, encoding CRISPR nucleases and / or RT polypeptides, such nucleic acid molecules may contain any of the modifications disclosed herein, if applicable.

[0250] D. Exemplary Gene Editing Systems The exemplary gene editing systems described herein are intended to be exemplary only.

[0251] In some embodiments, an exemplary gene editing system is shown in FIG. 1A and FIG. 1B. In these exemplary designs, the RNA guide may include a 3' fusion partner, which may include a RT donor RNA (including a PBS and a reverse transcription template sequence), any of the additional elements disclosed herein, or a combination thereof. In some cases, the PBS is about 3 to about 24 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 nucleotides) in length. Alternatively or additionally, the PBS may have at least about 75% complementarity with a corresponding PBS target site that may be located on the PAM strand. In some embodiments, the reverse transcription template sequence is about 10 nucleotides to about 100 nucleotides (e.g., about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 nucleotides) in length. In some embodiments, a linker is present between the DNA binding sequence (spacer) in the RNA guide and the reverse transcription template sequence. In some examples, the linker comprises one or more hairpins. For example, the hairpins can reduce annealing between the PBS and the DNA binding sequence.

[0252] In some cases, the CRISPR nuclease (e.g., a V-type nuclease, e.g., a Cas12i polypeptide) in the exemplary gene editing system may include an N- or C-terminal fusion partner. In some embodiments, the N- or C-terminal fusion partner includes a reverse transcriptase polypeptide.

[0253] In other embodiments, an exemplary gene editing system disclosed herein is shown in FIG. 2. In these exemplary designs, the RNA guide can include a 5' fusion partner, which can include one or more of the following: RT donor RNA (including the PBS and the reverse transcription template sequence), additional elements, or a combination thereof. In some embodiments, the reverse transcription template sequence is about 10 nucleotides to about 100 nucleotides (e.g., about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 nucleotides) in length. In some embodiments, the PBS is about 3 nucleotides to about 24 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 nucleotides) in length. Alternatively or additionally, the PBS has at least about 75% complementarity with the corresponding PBS target site that may be located on the PAM strand. In some embodiments, a linker is present between the DNA binding sequence of the RNA guide and the PBS. In some instances, the linker comprises one or more hairpins. For example, the hairpins can reduce annealing between the PBS and the DNA binding sequence.

[0254] In some cases, the CRISPR nuclease (e.g., a V-type nuclease, e.g., a Cas12i polypeptide) in the exemplary gene editing system may include an N- or C-terminal fusion partner. In some embodiments, the N- or C-terminal fusion partner includes a reverse transcriptase polypeptide.

[0255] The exemplary gene editing systems shown in Figures 1A, 1B, and 2 can be used to edit the PAM strand of a target nucleic acid (e.g., a genomic site of interest). Without wishing to be bound by theory, using these exemplary gene editing systems of Figures 1A, 1B, and 2, during cleavage by a CRISPR nuclease (e.g., a V-type nuclease, e.g., a Cas12i polypeptide), the free 3' end of the PAM strand can base-pair with the PBS, can be extended using a reverse transcription template sequence as a template, and can strand-exchange to base-pair again with the complementary genomic strand, resulting in an edited integration.

[0256] In yet another embodiment, an exemplary gene editing system disclosed herein is shown in FIG. 3. Such an exemplary gene editing system includes two RNA molecules: an RNA guide that includes a nuclease binding sequence and a DNA binding sequence (spacer) and an RT donor RNA. The RT donor RNA can include a PBS and a reverse transcription template sequence. In some examples, the reverse transcription template sequence does not code for an edit. In other examples, the RT donor RNA includes a PBS and a reverse transcription template sequence that codes for an edit. In some embodiments, the reverse transcription template sequence or a portion thereof can bind to a target nucleic acid via base pairing.

[0257] In some cases, the PBS is up to about 100 nucleotides in length. In some embodiments, the PBS is about 3 nucleotides to about 100 nucleotides in length. In some embodiments, the reverse transcription template sequence is about 10 nucleotides to about 100 nucleotides in length. In some embodiments, the reverse transcription template sequence of the RT donor RNA comprises an aptamer at the 5' end. In some embodiments, the aptamer recruits a reverse transcriptase polypeptide. In some embodiments, the PBS of the RT donor RNA is not complementary to any other portion of the editing template RNA (e.g., a nuclease binding sequence and / or a DNA binding sequence).

[0258] The exemplary gene editing system shown in Figure 3 can include either one or two protein components. For example, the exemplary gene editing system can include a CRISPR nuclease (e.g., a V-type nuclease, e.g., a Cas12i polypeptide), which has an N- or C-terminal fusion partner, and the N- or C-terminal fusion partner can include a reverse transcriptase polypeptide. Alternatively, the gene editing system can include a CRISPR nuclease (e.g., a V-type nuclease, e.g., a Cas12i polypeptide) and a reverse transcriptase polypeptide as two separate polypeptides.

[0259] The exemplary gene editing system shown in Figure 3 can be used to edit either the PAM strand or the non-PAM strand of a target nucleic acid (e.g., a genomic site of interest). Without wishing to be bound by theory, using such an exemplary gene editing system, after a CRISPR nuclease (e.g., a V-type nuclease, e.g., a Cas12i polypeptide) is released from the target nucleic acid, the free 3' end of the PAM strand or the non-PAM strand can base-pair with PBS, can be extended using a reverse transcription template sequence as a template, and can strand-exchange and rehybridize with a complementary genomic strand, resulting in the incorporation of an edit from the RT donor RNA. The exemplary gene editing system can be used to edit at the PAM-distal region of a target nucleic acid.

[0260] In yet another embodiment, an exemplary gene editing system disclosed herein is shown in FIG. 4. Such an exemplary gene editing system may include two RNA molecules, an RNA guide and an RT donor RNA, as two separate RNA molecules. An exemplary gene editing system may include any of one or two protein components disclosed herein. For example, an exemplary gene editing system may include a CRISPR nuclease (e.g., a V-type nuclease, e.g., a Cas12i polypeptide), which has an N- or C-terminal fusion partner, and which includes a reverse transcriptase polypeptide. Alternatively, the CRISPR nuclease (e.g., a V-type nuclease, e.g., a Cas12i polypeptide) and the reverse transcriptase polypeptide are not fused to each other (are two separate polypeptides).

[0261] The exemplary gene editing system shown in Figure 4 can be used to edit either the PAM strand or the non-PAM strand. Without wishing to be bound by theory, using the exemplary gene editing system, the free 3' end of the PAM strand or the non-PAM strand can be base-paired with the PBS of the RT donor RNA in the same gene editing system, extended using the reverse transcription template sequence as a template, strand-exchanged and re-hybridized with the complementary genomic strand, resulting in the incorporation of an edit from the RT donor RNA.

[0262] In some embodiments, an exemplary gene editing system disclosed herein is shown in Figure 5. In such an exemplary gene editing system, the RNA guide can include a 3' fusion partner, which can include a RT donor RNA (including a reverse transcription template sequence and a PBS). In some cases, the PBS binds to a site on the non-PAM strand upstream of the complementary region of the target sequence.

[0263] In some instances, the PBS is about 3 nucleotides to about 100 nucleotides (e.g., about 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 nucleotides) in length. In some embodiments, the DNA binding sequence (spacer) is about 20 nucleotides to about 25 nucleotides in length. In some embodiments, the DNA binding sequence includes at least one edit, and the at least one edit is incorporated about 10 nucleotides to about 25 nucleotides from the PAM sequence.

[0264] In some examples, the exemplary gene editing system may include a CRISPR nuclease (e.g., a V-type nuclease, e.g., a Cas12i polypeptide), which includes a 5'-fusion or 3'-fusion partner. The 5'-fusion or 3'-fusion partner may include a reverse transcriptase polypeptide. In some embodiments, the CRISPR nuclease (e.g., a V-type nuclease, e.g., a Cas12i polypeptide) lacks crRNA processing activity.

[0265] The exemplary gene editing system shown in Figure 5 can be used to edit the non-PAM strand of a target nucleic acid (e.g., a genomic site of interest). Without wishing to be bound by theory, using such an exemplary gene editing system, during cleavage by a CRISPR nuclease (e.g., a V-type nuclease, e.g., a Cas12i polypeptide), the free 3' end of the non-PAM strand can base-pair with the PBS and can be extended using the DNA binding sequence as a template. The RT extension on the non-PAM strand strand-exchanges and base-pairs again with the complementary genomic strand, resulting in the incorporation of the edit from the RT donor RNA.

[0266] In some embodiments, an exemplary gene editing system is shown in FIG. 6A and FIG. 6B. In such an exemplary gene editing system, the RNA guide can include a 3' fusion partner, which can include a RT donor RNA (including a reverse transcription template sequence and a PBS). In some embodiments, the PBS is complementary to a region in the non-PAM strand that is upstream of the complementary region of the target sequence on the PAM strand. In some examples, a hairpin is present between the DNA binding sequence of the RNA guide and the reverse transcription template sequence. In some embodiments, the hairpin is present within the reverse transcription template sequence. In some embodiments, an edit in the template sequence can create a hairpin in the target nucleic acid where the edit is incorporated.

[0267] In some examples, the exemplary gene editing system can include a CRISPR nuclease (e.g., a V-type nuclease, e.g., a Cas12i polypeptide), which includes an N- or C-terminal fusion partner. The N- or C-terminal fusion partner can include a reverse transcriptase polypeptide.

[0268] In some embodiments, an exemplary gene editing system is shown in FIG. 7. In such an exemplary gene editing system, the RNA guide may include a 5' fusion partner, which may include a RT donor RNA (including a PBS and a reverse transcription template sequence). In some embodiments, the PBS is about 5 nucleotides to about 20 nucleotides (e.g., about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides) long. Alternatively or additionally, the PBS has at least about 75% complementarity with a region on the non-PAM strand (the corresponding PBS target site). In some cases, a linker is present between the nuclease binding sequence of the RNA guide and the PBS of the RT donor RNA. Alternatively or additionally, a hairpin may be present between the DNA binding sequence of the RNA guide and the revia transcription template sequence of the RT donor RNA. In some embodiments, the hairpin is present within the reverse transcription template sequence. In some embodiments, edits in the template sequence can create a hairpin in the target nucleic acid into which the edit is incorporated.

[0269] In some cases, the CRISPR nuclease (e.g., a V-type nuclease, e.g., a Cas12i polypeptide) in the exemplary gene editing system may include an N- or C-terminal fusion partner, which may include a reverse transcriptase polypeptide. In some embodiments, the CRISPR nuclease (e.g., a V-type nuclease, e.g., a Cas12i polypeptide) lacks crRNA processing activity (e.g., as disclosed herein).

[0270] The exemplary gene editing system shown in Figure 6A, Figure 6B, or Figure 7 can be used to edit the non-PAM strand of a target nucleic acid (e.g., a genomic site of interest). Without wishing to be bound by theory, using the exemplary gene editing system, during cleavage by a CRISPR nuclease (e.g., a V-type nuclease, e.g., a Cas12i polypeptide), the free 3' end of the non-PAM strand can be base-paired with the PBS and extended using the reverse transcription template sequence as a template. The RT extension on the non-PAM strand strand-exchanges and re-base-pairs with the complementary genomic strand, resulting in the incorporation of at least one edit from the RT donor RNA.

[0271] Exemplary gene editing systems disclosed herein, such as those shown in Figures 6A, 6B, and 7, can be used to incorporate at least one PAM-proximal edit into a region on the non-PAM strand that is complementary to a target sequence on the PAM strand. In some examples, the exemplary gene editing system can be used to modify the PAM sequence and / or the sequence upstream of the PAM sequence (e.g., via the introduction of diversity into the region complementary to the PAM sequence and / or the upstream sequence). Such exemplary gene editing systems can be used to prevent retargeting of the resulting modified locus by the same CRISPR nuclease (e.g., a V-type nuclease, e.g., a Cas12i polypeptide).

[0272] In some embodiments, an exemplary gene editing system disclosed herein is shown in FIG. 10. In such an exemplary gene editing system, the RNA guide may include a 3' fusion partner, which may include a RT donor RNA (including a PBS and a reverse transcription template sequence). Alternatively, the RNA guide may include a 5' fusion partner, which may include a RT donor RNA (including a reverse transcription template sequence and a PBS). The length of the PBS may be variable. For example, the PBS length may be about 3 nucleotides to about 16 nucleotides long. In some examples, the PBS may bind to a region on the PAM strand of the target nucleic acid (e.g., a genomic site of interest), e.g., a region that overlaps with the target sequence. In some examples, a hairpin is present between the DNA binding sequence of the RNA guide and the reverse transcription template sequence of the RT donor RNA. One or both ends of the RNA guide-reverse transcription template sequence may include a protection fragment, e.g., one disclosed herein, to prevent exonuclease or endonuclease activity.

[0273] An exemplary gene editing system may include a CRISPR nuclease (e.g., a V-type nuclease, e.g., a Cas12i polypeptide), which may include an N- or C-terminal fusion partner. In some examples, the N- or C-terminal fusion partner includes a reverse transcriptase polypeptide. In some examples, the CRISPR nuclease (e.g., a V-type nuclease, e.g., a Cas12i polypeptide) lacks crRNA processing activity. In some examples, the CRISPR nuclease is a nickase. In some examples, edits are incorporated into the PAM strand of a target nucleic acid using the exemplary gene editing system shown in FIG. 10.

[0274] The exemplary editing template RNAs shown in Figures 1-7, 8A-8C, and 10 that include either an RT donor RNA sequence fused to the 3' end of an RNA guide sequence or an RT donor RNA sequence fused to the 5' end of an RNA guide sequence can instead include an RT donor RNA sequence fused to an internal position of the RNA guide sequence, or vice versa. For example, the RT donor RNA can be fused to an internal position of the RNA guide, sgRNA, or RNA guide-tracrRNA (e.g., sgRNA).

[0275] The extended RNA guide end (e.g., via 5' or 3' extension with the RT donor RNA) may be vulnerable to exonuclease and / or endonuclease activity, which reduces the reverse transcription template sequence concentration along with the efficiency of editing incorporation. In some embodiments, the RNA guide-RT donor RNA fusion further comprises an additional secondary structure to inhibit or prevent exonuclease activity. In some embodiments, the additional secondary structure is a triplex structure, a pseudoknot, an xrRNA, a circular RNA, a tRNA, or a truncated tRNA. In some embodiments, the additional secondary structure is a Zika-like pseudoknot, a murine leukemia virus pseudoknot (MLV-PK) sequence, a red clover necrotic mosaic virus (RCNMV) sequence, a sweet clover necrotic mosaic virus (SCNMV) sequence, a carnation ringspot virus (CRSV) sequence, a pre-Q sequence, or an RNA bacteriophage MS2 sequence. In some embodiments, the additional secondary structure is via base stacking or 3' end base pairing. In other embodiments, the additional secondary structure is a nuclease binding sequence, or a nuclease binding sequence and a DNA binding sequence. See Figure 10, Figure 11, Figure 12A, and Figure 12B. In some embodiments, the additional DNA binding sequence is directed to a non-mammalian target. In some embodiments, the additional DNA binding sequence is directed to a non-human target. In some embodiments, the added DNA binding sequence is not found in the human genome. In some embodiments, the additional DNA binding sequence is 85% or less identical to any sequence in the human genome. See Example 2.

[0276] Without wishing to be bound by theory, the addition of nuclease binding sequences and DNA binding sequences can recruit CRISPR nucleases or CRISPR nuclease-reverse transcriptase fusions. Through protein-RNA interactions, the bound CRISPR nuclease can provide resistance to endogenous exonucleases and endonucleases. In some embodiments, the additional nuclease binding sequences and DNA binding sequences recruit CRISPR nucleases that lack RNA processing activity. In some embodiments, the secondary structure is an aptamer (e.g., an RNA aptamer), and the composition further comprises a protein that interacts with the aptamer. In some embodiments, the composition comprising the aptamer and the aptamer-interacting protein inhibits endogenous exonuclease and / or endonuclease activity.

[0277] Additional exemplary gene editing systems disclosed herein are provided below for illustrative purposes only.

[0278] In some embodiments, the gene editing system disclosed herein comprises at least one RNA guide (or guide RNA, as used interchangeably herein) and at least one RT donor RNA. In some examples, the at least one RNA guide comprises a nuclease binding sequence and a DNA binding sequence (spacer). The RNA guide may be capable of binding to a CRISPR nuclease (e.g., a V-type CRISPR nuclease). In some examples, the at least one RNA guide is further capable of binding to a target nucleic acid, for example, via a spacer region. In some examples, the RT donor RNA comprises at least one primer binding site (PBS) and at least one reverse transcription template sequence. The PBS may bind to one strand of the target nucleic acid, and the one strand may be either the sense strand or the antisense strand. The region to which the PBS binds is described herein as a PBS target site. The at least one reverse transcription template sequence may comprise a sequence having at least one nucleotide diversity (coded edit) relative to the corresponding sequence of the target nucleic acid. In some cases, at least one of the encoded edits is an insertion, substitution, and / or deletion.

[0279] In some embodiments, the gene editing system disclosed herein comprises at least one RNA guide, at least one RT donor RNA, and at least one other sequence. In some embodiments, the at least one RNA guide comprises a nuclease binding sequence and a DNA binding sequence. In some embodiments, the RNA guide is capable of binding to a CRISPR nuclease (e.g., a V-type CRISPR nuclease). In some embodiments, the at least one RNA guide is further capable of binding to a target nucleic acid. In some embodiments, the PBS of the at least one RT donor RNA is capable of binding to a non-PAM strand of the target nucleic acid. In some embodiments, the PBS of the at least one RT donor RNA is capable of binding to a PAM strand of the target nucleic acid.

[0280] In some embodiments, the gene editing system disclosed herein may include at least one of a CRISPR nuclease, a reverse transcriptase, and an editing template RNA, and the editing template RNA may include an RNA guide and a RT donor RNA. In some examples, at least one of a CRISPR nuclease, a reverse transcriptase, and an editing template RNA is provided in an individual composition. In some embodiments, at least one of a CRISPR nuclease, a reverse transcriptase, an RNA guide, and an RT donor RNA is provided in an individual composition. In some embodiments, one or more of a CRISPR nuclease, a reverse transcriptase, and at least one of an editing template RNA is provided in a separate composition. In some embodiments, a composition comprising a CRISPR nuclease and a reverse transcriptase is provided separately from a composition comprising an editing template RNA. In some embodiments, one or more of a CRISPR nuclease, a reverse transcriptase, an RNA guide, and at least one of an RT donor RNA is provided in a separate composition. In some embodiments, the composition comprising the CRISPR nuclease and reverse transcriptase is provided separately from the composition comprising the RNA guide and the RT donor RNA.

[0281] In some embodiments, the gene editing system provided herein may be capable of binding to a target nucleic acid, and the target nucleic acid may be a genomic site where gene editing is required. In some embodiments, one or more components of the composition, such as an editing template RNA, bind to the target nucleic acid. In some embodiments, one or more components of the composition, such as an RNA guide and an RT donor RNA, bind to the target nucleic acid. In some embodiments, the target nucleic acid is DNA. In some embodiments, the composition of the invention modifies or is capable of modifying the target nucleic acid. In some embodiments, one or more of the components of the composition, such as a CRISPR nuclease and a reverse transcriptase, modify the target nucleic acid. In some embodiments, the composition of the invention introduces a substitution, insertion, or deletion into the target nucleic acid. In some embodiments, the composition of the invention is capable of introducing a substitution, insertion, or deletion into the non-PAM strand of the target nucleic acid. In some embodiments, the gene editing system disclosed herein is capable of introducing a substitution, insertion, or deletion into the PAM strand of the target nucleic acid.

[0282] In some embodiments, the gene editing system disclosed herein may include a CRISPR nuclease, an RT polypeptide, or both protein components. Alternatively, the gene editing system may include one or more nucleic acids (e.g., vectors, e.g., viral vectors) encoding the protein components. In some examples, the gene editing system may include one vector encoding both the CRISPR nuclease and the RT polypeptide. Alternatively or in addition, the gene editing system disclosed herein may include a gene editing RNA, a guide RNA, or both RNA components. Alternatively, the gene editing system may include one or more nucleic acids (vectors) encoding the RNA components. For example, the gene editing system may include one vector (e.g., viral vectors, e.g., AAV vectors, e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrhlO, AAV11, and AAV12) encoding both the gene editing RNA and the guide RNA.

[0283] In some examples, the gene editing system disclosed herein may include a CRISPR nuclease, an RT polypeptide, or both protein components, and an RNA component of a gene editing RNA and an RNA guide. In other examples, the gene editing system disclosed herein may include a CRISPR nuclease, an RT polypeptide, or both protein components, and one or more nucleic acids encoding the RNA component of a gene editing RNA and an RNA guide. In yet other examples, the gene editing system disclosed herein may include one or more nucleic acids encoding the CRISPR nuclease, an RT polypeptide, or both protein components, and an RNA component of a gene editing RNA and an RNA guide. Alternatively, the gene editing system disclosed herein may include one or more nucleic acids encoding the CRISPR nuclease, an RT polypeptide, or both protein components, and one of more nucleic acids encoding the RNA component of a gene editing RNA and an RNA guide. In some cases, the gene editing system may include one vector encoding multiple components of the gene editing system. In some cases, the nucleic acid(s) encoding the CRISPR nuclease, RT polypeptide, and / or fusion polypeptide thereof can be one or more mRNA molecules. In some examples, the mRNA molecule(s) can be codon optimized.

[0284] In some embodiments, the gene editing systems disclosed herein include one or more lipid nanoparticles (LNPs), which include one or more of the protein and / or RNA components of the gene editing system or their encoding nucleic acids. In other embodiments, the gene editing system includes a portion of the components, which may include one or more LNPs, and one or more vectors encoding the remaining components.

[0285] II. Preparation of gene editing system components The protein components, RNA components, or their encoding nucleic acids (eg, vectors or mRNA) may be prepared by conventional methods according to the methods disclosed herein.

[0286] In some embodiments, the CRISPR nuclease (e.g., a type V nuclease, e.g., a Cas12i polypeptide), reverse transcriptase, or CRISPR nuclease-reverse transcriptase fusion can be prepared by (a) culturing a host cell, e.g., a bacterial cell or a mammalian cell, capable of producing the protein, isolating the protein thus produced, and optionally purifying the protein. The CRISPR nuclease, reverse transcriptase, or fusion protein thus prepared can form a complex with an editing template RNA.

[0287] CRISPR nuclease and reverse transcriptase can also be prepared by (b) known genetic engineering techniques, specifically by isolating the gene encoding the CRISPR nuclease and reverse transcriptase of the present invention from bacteria, constructing a recombinant expression vector, and then transferring the vector into a suitable host cell, the host cell expresses the edited template RNA for expression of recombinant protein, and the recombinant protein forms a complex with the edited template RNA in the host cell. Alternatively, CRISPR nuclease and reverse transcriptase can be prepared by (c) in vitro coupling transcription-translation system, and then complexing with the edited template RNA. The bacteria that can be used for preparing the CRISPR nuclease and reverse transcriptase of the present invention are not particularly limited, as long as they can produce the CRISPR nuclease and reverse transcriptase of the present invention. Some non-limiting examples of bacteria include E.coli cells described herein.

[0288] Unless otherwise noted, all compositions and complexes and polypeptides provided herein are made with respect to the activity level of the composition or complex or polypeptide, and exclude impurities such as residual solvents or by-products that may be present in commercial sources. Enzymatic component weights are based on total active protein. All percentages and ratios are calculated by weight unless otherwise indicated. All percentages and ratios are calculated based on total composition unless otherwise indicated. In exemplary compositions, enzyme levels are expressed by pure enzyme by weight of total composition, and components are expressed by weight of total composition unless otherwise indicated.

[0289] A. Vector The present disclosure provides one or more vectors for expressing the CRISPR nuclease, reverse transcriptase, or fusion polypeptides thereof described herein, or the nucleic acid encoding the components described herein may be incorporated into a vector. In some embodiments, the vectors disclosed herein include a nucleotide sequence encoding a CRISPR nuclease, reverse transcriptase, or fusion polypeptide. The present disclosure also provides one or more vectors encoding an editing template RNA, or any portion thereof, such as an RNA guide or RT donor RNA. In some embodiments, the vector includes a Pol II promoter or a Pol III promoter.

[0290] Expression of natural or synthetic polynucleotides is typically achieved by operably linking a polynucleotide encoding a gene of interest, such as a nucleotide sequence encoding a CRISPR nuclease, reverse transcriptase, or fusion polypeptide, and / or an editing template RNA, to a promoter and incorporating the construct into an expression vector. The expression vector is not particularly limited, as long as it contains a polynucleotide encoding the CRISPR nuclease and reverse transcriptase and / or an editing template RNA of the present invention, and can be suitable for replication and integration in eukaryotic cells.

[0291] A typical expression vector comprises transcription and translation terminators, initiation sequences, and promoters useful for expressing a desired polynucleotide. For example, a plasmid vector (pSP64, pBluescript, etc.) carrying a recognition sequence for RNA polymerase can be used. Vectors, including those derived from retroviruses, such as lentiviruses, are suitable tools for achieving long-term gene transfer, since they allow the long-term stable integration of transgenes and their propagation in daughter cells. Examples of vectors include expression vectors, replication vectors, probe generation vectors, and sequencing vectors. An expression vector can be provided to cells in the form of a viral vector.

[0292] Viral vector technology is well known in the art and described in various virology and molecular biology manuals.Viruses that are useful as vectors include, but are not limited to, phage virus, retrovirus, adenovirus, adeno-associated virus, herpes virus, and lentivirus.Generally, suitable vectors contain a replication origin that is functional in at least one organism, a promoter sequence, convenient restriction endonuclease sites, and one or more selectable markers.

[0293] The type of vector is not particularly limited, and a vector that can be expressed in a host cell can be appropriately selected. More specifically, depending on the type of host cell, a promoter sequence for ensuring expression of a polypeptide (or polypeptides) from a polynucleotide is appropriately selected, and the promoter sequence and polynucleotide are inserted into any of various plasmids for preparing an expression vector.

[0294] Additional promoter elements, e.g. enhancing sequences, regulate the frequency of transcription initiation. Typically, these are located in the region 30-110 bp upstream of the start site, but some promoters have recently been shown to contain functional elements downstream of the start site as well. Depending on the promoter, individual elements appear to be able to function either cooperatively or independently to activate transcription.

[0295] Furthermore, the present disclosure should not be limited to the use of constitutive promoters. Inducible promoters are also contemplated as part of the present disclosure. The use of inducible promoters provides a molecular switch that can turn on expression of the polynucleotide sequence to which it is operably linked when such expression is desired, or turn off expression when expression is not desired. Examples of inducible promoters include, but are not limited to, metallothionine promoters, glucocorticoid promoters, progesterone promoters, and tetracycline promoters.

[0296] The introduced expression vector may also contain either or both of a selectable marker gene or a reporter gene to facilitate identification and selection of expressing cells from a population of cells intended for transfection or infection via a viral vector. In other embodiments, the selectable marker may be carried on a separate piece of DNA and used in a co-transfection procedure. Both the selectable marker and the reporter gene may be flanked by appropriate transcription control sequences to allow expression in the host cell. Examples of such markers include the dihydrofolate reductase gene and the neomycin resistance gene for eukaryotic cell culture; and the tetracycline resistance gene and the ampirilin resistance gene for E. coli and other bacterial culture. By using such a selectable marker, it can be confirmed whether the polynucleotide encoding the polypeptide(s) of the present invention has been transferred into the host cell and then expressed without failure.

[0297] The method for preparing the recombinant expression vector is not particularly limited, and examples thereof include methods using a plasmid, a phage, or a cosmid.

[0298] B. Methods of Expression The present disclosure includes a method for protein expression comprising translating a CRISPR nuclease and a reverse transcriptase and expressing an editing template RNA described herein.

[0299] In some embodiments, the host cells described herein are used to express CRISPR nuclease and reverse transcriptase and / or edited template RNA. The host cell is not particularly limited, and various known cells can be preferably used. Specific examples of host cells include bacteria, such as E. coli, yeast (budding yeast Saccharomyces cerevisiae and fission yeast Schizosaccharomyces pombe), nematodes (Caenorhabditis elegans), Xenopus laevis oocytes, and animal cells (e.g., CHO cells, COS cells, and HEK293 cells). The method for transferring the above expression vector into the host cell, i.e., the transformation method, is not particularly limited, and known methods, such as electroporation, calcium phosphate method, liposome method, and DEAE dextran method, can be used.

[0300] After the host is transformed with the expression vector, the host cells may be cultured, cultivated, or propagated for the production of the CRISPR nuclease, reverse transcriptase, and / or edited template RNA. After expression of the CRISPR nuclease, reverse transcriptase, and / or edited template RNA, the host cells may be harvested, and the CRISPR nuclease, reverse transcriptase, and / or edited template RNA may be purified from the culture, etc., by conventional methods (e.g., filtration, centrifugation, cell disruption, gel filtration chromatography, ion exchange chromatography, etc.).

[0301] In some embodiments, methods for CRISPR nuclease and reverse transcriptase expression include translation of at least 5 amino acids, at least 10 amino acids, at least 15 amino acids, at least 20 amino acids, at least 50 amino acids, at least 100 amino acids, at least 150 amino acids, at least 200 amino acids, at least 250 amino acids, at least 300 amino acids, at least 400 amino acids, at least 500 amino acids, at least 600 amino acids, at least 700 amino acids, at least 800 amino acids, at least 900 amino acids, or at least 1000 amino acids of a polypeptide(s). In some embodiments, methods for protein expression include translation of about 5 amino acids, about 10 amino acids, about 15 amino acids, about 20 amino acids, about 50 amino acids, about 100 amino acids, about 150 amino acids, about 200 amino acids, about 250 amino acids, about 300 amino acids, about 400 amino acids, about 500 amino acids, about 600 amino acids, about 700 amino acids, about 800 amino acids, about 900 amino acids, or about 1000 amino acids or more of the CRISPR nuclease and reverse transcriptase.

[0302] Various methods can be used to determine the level of production of mature CRISPR nuclease, reverse transcriptase, and / or edited template RNA in host cells. Such methods include, but are not limited to, for example, using polyclonal or monoclonal antibodies specific for the protein, or any of the labeling tags described elsewhere herein. Exemplary methods include, but are not limited to, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (MA), fluorescent immunoassay (FIA), and fluorescence-activated cell sorting (FACS). These and other assays are well known in the art (see, for example, Maddox et al., J. Exp. Med. 158:1211

[1983] ).

[0303] The disclosure provides a method for in vivo expression of a CRISPR nuclease and reverse transcriptase and / or an editing template RNA in a cell, the method comprising providing a polyribonucleotide encoding the CRISPR nuclease, reverse transcriptase, and / or editing template RNA to a host cell, where the polyribonucleotide encodes the CRISPR nuclease, reverse transcriptase, and / or editing template RNA, expressing the CRISPR nuclease, reverse transcriptase, and / or editing template RNA in the cell, and obtaining the CRISPR nuclease, reverse transcriptase, and / or editing template RNA from the cell.

[0304] III. Methods for Gene Editing Any of the gene editing systems can be used to genetically modify (edit) a target nucleic acid, which can be a genetic site of interest, such as a genetic site where gene editing is required, e.g., to repair a genetic mutation, to introduce a protective mutation, to introduce a modification to regulate expression of a gene, etc.

[0305] The gene editing systems and compositions disclosed herein are applicable to editing and introducing edits into various target sequences. In some embodiments, the target sequence is a DNA molecule, e.g., a DNA locus (referred to herein as a target sequence or an on-target sequence). In some embodiments, the target sequence is RNA, e.g., an RNA locus or an mRNA. In some embodiments, the target sequence is single-stranded (e.g., single-stranded DNA). In some embodiments, the target sequence is double-stranded (e.g., double-stranded DNA). In some embodiments, the target sequence includes both single-stranded and double-stranded regions. In some embodiments, the target sequence is linear. In some embodiments, the target sequence is circular. In some embodiments, the target sequence includes one or more modified nucleotides, e.g., methylated nucleotides, damaged nucleotides, or nucleotide analogs. In some embodiments, the target sequence is unmodified. In some embodiments, a single-stranded target sequence does not require a PAM sequence.

[0306] The target sequence may be of any length, for example, at least any one of about 100bp, 200bp, 500bp, 1000bp, 2000bp, 5000bp, 10kb, 20kb, 50kb, 100kb, 200kb, 500kb, or 1Mb or more. The target sequence may also include any sequence. In some embodiments, the target sequence is GC-rich, for example, at least any one of about 40%, 45%, 50%, 55%, 60%, or 65% or more GC content. In some embodiments, the target sequence has at least about 70% or 80% or more GC content. In some embodiments, the target sequence is a GC-rich fragment of a non-GC-rich target sequence. In some embodiments, the target sequence is not GC-rich. In some embodiments, the target sequence has one or more secondary or higher order structures. In some embodiments, the target sequence is not in a condensed state, such as chromatin, that renders the target sequence inaccessible by ribonucleoproteins.

[0307] In some embodiments, the target nucleic acid is a genomic site in a cell. In some cases, the target nucleic acid where gene editing occurs can be in a protein coding region. Alternatively, the target nucleic acid can be in a regulatory region, such as a promoter, enhancer, 5' or 3' untranslated region. In other cases, the target nucleic acid can be in a non-coding gene, such as a transposon, miRNA, tRNA, ribosomal RNA, ribozyme, or lincRNA.

[0308] A. Exemplary Genes for Gene Editing Any of the gene editing systems disclosed herein can be used to edit a target gene of interest, for example, a gene involved in a disease (e.g., a genetic disease). In some embodiments, the target gene can be involved in the immune response in a subject. For example, the target gene can be an immune checkpoint gene.

[0309] Exemplary target genes include BCL11A intronic erythroid enhancer, CD3, beta 2 microglobulin (B2M), T cell receptor alpha constant (TRAC), programmed cell death 1 (PDCD1), T cell receptor alpha, T cell receptor beta, B cell lymphoma / leukemia 11A (BCL11A), cytotoxic T lymphocyte antigen 4 (CTLA-4), chemokine (CC motif) receptor 5 (gene / pseudogene) (CCR5), CXCR4 gene, C D160 molecule (CD160), adenosine A2a receptor (ADORA), CD276, B7-H3, B7-H4, BTLA, nicotinamide adenine dinucleotide phosphate NADPH oxidase isoform 2 (NOX2), V-domain Ig inhibitor of T cell activation (VISTA), sialic acid-binding immunoglobulin-type lectin 7 (SIGLEC7), sialic acid-binding immunoglobulin-type lectin 9 (SIGLEC9), SIGLEC10, V-set domain Indoleamine 2,3-dioxygenase (IDO), indoleamine 2,3-dioxygenase 1 (IDO1), killer cell immunoglobulin-like receptor (KIR), killer cell immunoglobulin-like receptor three domain long cytoplasmic tail 1 (KIR3DL1), lymphocyte activation gene 3 (LAG3), T cell immunoglobulin domain and mucin domain 3 (TIM3 ), Hepatitis A virus cell receptor 2 (HAVCR2), Natural killer cell receptor 2B4 (CD244), Hypoxanthine phosphoribosyltransferase 1 (HPRT), T cell immunoreceptor with Ig and ITIM domains (TIGIT), CD96 molecule (CD96), Cytotoxic and regulatory T cell molecule (CRTAM), Leukocyte-associated immunoglobulin-like receptor 1 (LAIR1), Adeno-associated virus integration site 1 (AAVS1), AAVS 2, AAVS3, AAVS4, AAVS5, AAVS6, AAVS7, AAVS8, Transforming growth factor beta receptor II (TGFBRII), Transforming growth factor beta receptor I (TGFBR1), SMAD family member 2 (SMAD2), SMAD family member 3 (SMAD3), SMAD family member 4 (SMAD4), SKI proto-oncogene (SKI), SKI-like proto-oncogene (SKIL),egl-9 family hypoxia inducible factor 1 (EGLN1), egl-9 family hypoxia inducible factor 2 (EGLN2), egl-9 family hypoxia inducible factor 3 (EGLN3), protein phosphatase 1 regulatory subunit 12C (PPP1R12C), TGFB-inducible factor homeobox 1 (TGIF1), tumor necrosis factor receptor superfamily member, tumor necrosis factor receptor superfamily member 10b (TNFRSF10B), tumor necrosis factor receptor superfamily member Family member 10a (TNFRSF10A), BY55, B7H5, caspase 8 (CASP8), caspase 10 (CASP10), caspase 3 (CASP3), caspase 6 (CASP6), caspase 7 (CASP7), Fas-associated death domain-mediated (FADD), Fas cell surface death receptor (FAS), interleukin-10 receptor subunit alpha (IL10RA), interleukin-10 receptor subunit beta (IL10RB), heme oxygenase hemoglobinase 2 (HMOX2), interleukin 6 receptor (IL6R), interleukin 6 signal transducer and activator (IL6ST), c-src tyrosine kinase (CSK), phosphoprotein membrane anchor 1 with glycosphingolipid microdomains (PAG1), guanylate cyclase 1 soluble beta 3 (GUCY1B3), signaling threshold regulating transmembrane adaptor 1 (SIT1), forkhead box P3 (FOXP3), PR domain 1 (PRDM1), basic The modified genes include, but are not limited to, leucine zipper transcription factor, ATF-like (BATF), guanylate cyclase 1 soluble alpha 2 (GUCY1A2), guanylate cyclase 1 soluble alpha 3 (GUCY1A3), guanylate cyclase 1 soluble beta 2 (GUCY1B2), prolyl hydroxylase domain (PHD1, PHD2, PHD3) protein family, CD27, CD28, CD40, CD122, CD137, OX40, GITR, and ICOS. In some embodiments, the modified genes include, but are not limited to, programmed death ligand 1 (PD-L1), class II major histocompatibility complex transactivator (CIITA), citramalyl-CoA lyase (CLYBL), transthyretin (TTR), lactate dehydrogenase-A (LDHA), dihydroxyacid oxidase 1 (HAO1),Alanine-glyoxylate and serine-pyruvate aminotransferase (AGXT), glyoxylate reductase / hydroxypyruvate reductase (GRHPR), 4-hydroxy-2-oxoglutarate aldolase (HOGA), polypyrimidine tract binding protein 1 (PTBP1), stathmin 2 (STMN2), or actin beta (ACTB).

[0310] The present disclosure provides methods for gene editing any of the target genes disclosed herein using the gene editing system also disclosed herein.

[0311] B. Editing In some aspects, methods are provided herein for introducing at least one edit into a target nucleic acid (e.g., a genomic site of interest, e.g., a genomic site in any of the target genes disclosed herein) using the gene editing system described herein. In some embodiments, the edit may include a substitution, an insertion, a deletion, or a combination thereof into the target nucleic acid. In some examples, the edit may be a single nucleotide substitution, such as a G to T substitution, a G to A substitution, a G to C substitution, a T to G substitution, a T to A substitution, a T to C substitution, a C to G substitution, a C to T substitution, a C to A substitution, an A to T substitution, an A to G substitution, or an A to C substitution. In some examples, the edits can convert a G:C base pair to a T:A base pair, a G:C base pair to an A:T base pair, a G:C base pair to a C:G base pair, a T:A base pair to a G:C base pair, a T:A base pair to an A:T base pair, a T:A base pair to a C:G base pair, a C:G base pair to a G:C base pair, a C:G base pair to a T:A base pair, a C:G base pair to an A:T base pair, an A:T base pair to a T:A base pair, an A:T base pair to a G:C base pair, or an A:T base pair to a C:G base pair.

[0312] In some embodiments, methods are described for introducing at least one edit into a target nucleic acid, wherein the edit is at least one substitution, at least one insertion, and / or at least one deletion. In some embodiments, the edit comprises at least one substitution, insertion, or deletion. In some embodiments, the substitution, insertion, or deletion is at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 200, at least 300, at least 400, or at least 500 nucleotides in length. In some embodiments, the substitutions, insertions, or deletions are from 1 nucleotide to about 200 nucleotides in length, e.g., from 1 nucleotide to 5 nucleotides, from 5 nucleotides to 10 nucleotides, from 10 nucleotides to 15 nucleotides, from 15 nucleotides to 20 nucleotides, from 20 nucleotides to 25 nucleotides, from 25 nucleotides to 30 nucleotides, from 30 nucleotides to 35 nucleotides, from 35 nucleotides to 40 nucleotides, from 40 nucleotides to 45 nucleotides, from 45 nucleotides to 50 nucleotides, from 50 nucleotides to 55 nucleotides, from 55 nucleotides to 60 nucleotides, from 60 nucleotides to 65 nucleotides, from 65 nucleotides to 70 nucleotides, from 75 nucleotides to 80 nucleotides, from 75 nucleotides to 85 ... nucleotides to 70 nucleotides, 70 nucleotides to 75 nucleotides, 75 nucleotides to 80 nucleotides, 80 nucleotides to 85 nucleotides, 85 nucleotides to 90 nucleotides, 90 nucleotides to 95 nucleotides, 95 nucleotides to 100 nucleotides, 100 nucleotides to 105 nucleotides, 105 nucleotides to 110 nucleotides, 110 nucleotides to 115 nucleotides, 115 nucleotides to 120 nucleotides, 120 nucleotides to 125 nucleotides, 125 nucleotides to 130 nucleotides, 130 nucleotides to 135 nucleotides, 135 nucleotides to 140 nucleotides,It is 140 nucleotides to 145 nucleotides, 145 nucleotides to 150 nucleotides, 150 nucleotides to 155 nucleotides, 155 nucleotides to 160 nucleotides, 160 nucleotides to 165 nucleotides, 165 nucleotides to 170 nucleotides, 170 nucleotides to 175 nucleotides, 175 nucleotides to 180 nucleotides, 180 nucleotides to 185 nucleotides, 185 nucleotides to 190 nucleotides, 190 nucleotides to 195 nucleotides, or 195 nucleotides to 200 nucleotides. In some embodiments, the substitutions, insertions, or deletions are from 1 nucleotide to about 300 nucleotides in length, e.g., from 1 nucleotide to 5 nucleotides, from 5 nucleotides to 10 nucleotides, from 10 nucleotides to 15 nucleotides, from 15 nucleotides to 20 nucleotides, from 20 nucleotides to 25 nucleotides, from 25 nucleotides to 30 nucleotides, from 30 nucleotides to 35 nucleotides, from 35 nucleotides to 40 nucleotides, from 40 nucleotides to 45 nucleotides, from 45 nucleotides to 50 nucleotides, from 50 nucleotides to 55 nucleotides, from 55 nucleotides to 60 nucleotides, from 60 nucleotides to 65 nucleotides, from 65 nucleotides to 70 nucleotides, from 70 nucleotides to 75 nucleotides, from 75 nucleotides to 80 nucleotides, from 80 nucleotides to 85 nucleotides, from 85 nucleotides to 90 nucleotides, from 90 nucleotides to 100 nucleotides, nucleotides to 95 nucleotides, 95 nucleotides to 100 nucleotides, 100 nucleotides to 105 nucleotides, 105 nucleotides to 110 nucleotides, 110 nucleotides to 115 nucleotides, 115 nucleotides to 120 nucleotides, 120 nucleotides to 125 nucleotides, 125 nucleotides to 130 nucleotides, 130 nucleotides to 135 nucleotides, 135 nucleotides to 140 nucleotides, 140 nucleotides to 145 nucleotides, 145 nucleotides to 150 nucleotides, 150 nucleotides to 155 nucleotides, 155 nucleotides to 160 nucleotides, 160 nucleotides to 165 nucleotides, 165 nucleotides to 170 nucleotides, 170 nucleotides to 175 nucleotides, 175 nucleotides to 180 nucleotides, 180 nucleotides to 185 nucleotides,185 nucleotides to 190 nucleotides, 190 nucleotides to 195 nucleotides, 195 nucleotides to 200 nucleotides, 200 nucleotides to 210 nucleotides, 210 nucleotides to 220 nucleotides, 220 nucleotides to 230 nucleotides, 230 nucleotides to 240 nucleotides, 240 nucleotides to 250 nucleotides, 250 nucleotides to 260 nucleotides, 260 nucleotides to 270 nucleotides, 270 nucleotides to 280 nucleotides, 280 nucleotides to 290 nucleotides, or 290 nucleotides to 300 nucleotides. In some embodiments, the substitutions, insertions, or deletions are up to about 10,000 base pairs (10 kb) in length. For example, in some embodiments, the substitution, insertion, or deletion is at least 1 base pair, about 10 base pairs, about 20 base pairs, about 30 base pairs, about 40 base pairs, about 50 base pairs, about 60 base pairs, about 70 base pairs, about 80 base pairs, about 90 base pairs, about 100 base pairs, about 200 base pairs, about 300 base pairs, about 400 base pairs, about 500 base pairs, about 600 base pairs, about 700 base pairs, about 800 base pairs, about 900 base pairs, about 1 base pair, about 2 base pairs, about 3 base pairs, about 4 base pairs, about 5 base pairs, about 6 base pairs, about 7 base pairs, about 8 base pairs, about 9 base pairs, about 1 ...0 base pairs, about 20 base pairs, about 30 base pairs, about 40 base pairs, about 50 base pairs, about 600 base pairs, about 700 base pairs, about 800 base pairs, about 900 base pairs, about 10 base pairs, about 10 base pairs, about 20 base pairs, about 30 base pairs, about 40 base pairs, about 50 base pairs, about kb, about 1.1kb, about 1.2kb, about 1.3kb, about 1.4kb, about 1.5kb, about 1.6kb, about 1.7kb, about 1.8kb, about 1.9kb, about 2kb, about 2.1kb, about 2.2kb, about 2.3 kb, about 2.4 kb, about 2.5 kb, about 2.6 kb, about 2.7 kb, about 2.8 kb, about 2.9 kb, 3 kb, 4 kb, 5 kb, 6 kb, 7 kb, 8 kb, 9 kb, or 10 kb long. ,

[0313] In some embodiments, the insertion is or includes a hairpin. For example, the reverse transcriptase can transcribe the hairpin and the hairpin can be incorporated into the target nucleic acid. In other embodiments, the reverse transcription template sequence includes a hairpin structure and the reverse transcriptase stops transcription of the reverse transcription template sequence at the hairpin.

[0314] In some embodiments, editing occurs within about 500 nucleotides of a type II PAM sequence (e.g., 5'-NGG-3' for SpCas9) or a type V PAM sequence (e.g., 5'-NTTN-3' for a Cas12i polypeptide). In some embodiments, editing occurs adjacent to the PAM sequence, e.g., within about 500 nucleotides upstream or downstream of the PAM sequence. In some embodiments, editing occurs within about 400 nucleotides of the PAM sequence. In some embodiments, editing occurs within about 400 nucleotides upstream or downstream of the PAM sequence. In some embodiments, editing occurs within about 300 nucleotides of the PAM sequence. In some embodiments, editing occurs within about 300 nucleotides upstream or downstream of the PAM sequence. In some embodiments, editing occurs within about 200 nucleotides of the PAM sequence. In some In embodiments, the editing occurs within about 200 nucleotides upstream or downstream of the PAM sequence. In some embodiments, the editing occurs within about 100 nucleotides of the PAM sequence. In some embodiments, the editing occurs within about 100 nucleotides upstream or downstream of the PAM sequence. In some embodiments, the editing occurs within about 50 nucleotides of the PAM sequence. In some embodiments, the editing occurs within about 50 nucleotides upstream or downstream of the PAM sequence. In some embodiments, the editing occurs within about 30 nucleotides upstream or downstream of the PAM sequence. In some embodiments, the editing occurs within about 30 nucleotides upstream or downstream of the PAM sequence. In some embodiments, the editing occurs within about 20 nucleotides of the PAM sequence. In some embodiments, the editing occurs within about 20 nucleotides upstream or downstream of the PAM sequence.

[0315] In some embodiments, editing begins within about 300 nucleotides upstream of the PAM sequence. In some embodiments, editing begins within about 290 nucleotides upstream of the PAM sequence. In some embodiments, editing begins within about 280 nucleotides upstream of the PAM sequence. In some embodiments, editing begins within about 270 nucleotides upstream of the PAM sequence. In some embodiments, editing begins within about 260 nucleotides upstream of the PAM sequence. In some embodiments, editing begins within about 250 nucleotides upstream of the PAM sequence. In some embodiments, editing begins within about 240 nucleotides upstream of the PAM sequence. In some embodiments, editing begins within about 230 nucleotides upstream of the PAM sequence. In some embodiments, editing begins within about 2020 nucleotides upstream of the PAM sequence. In some embodiments, editing begins within about 210 nucleotides upstream of the PAM sequence. In some embodiments, editing begins within about 200 nucleotides upstream of the PAM sequence. In some embodiments, editing begins within about 190 nucleotides upstream of the PAM sequence. In some embodiments, editing begins within about 180 nucleotides upstream of the PAM sequence. In some embodiments, editing begins within about 170 nucleotides upstream of the PAM sequence. In some embodiments, editing begins within about 160 nucleotides upstream of the PAM sequence. In some embodiments, editing begins within about 150 nucleotides upstream of the PAM sequence. In some embodiments, editing begins within about 140 nucleotides upstream of the PAM sequence. In some embodiments, editing begins within about 130 nucleotides upstream of the PAM sequence. In some embodiments, editing begins within about 120 nucleotides upstream of the PAM sequence. In some embodiments, editing begins within about 110 nucleotides upstream of the PAM sequence. In some embodiments, editing begins within about 100 nucleotides upstream of the PAM sequence.In some embodiments, editing begins within about 90 nucleotides upstream of the PAM sequence. In some embodiments, editing begins within about 80 nucleotides upstream of the PAM sequence. In some embodiments, editing begins within about 70 nucleotides upstream of the PAM sequence. In some embodiments, editing begins within about 60 nucleotides upstream of the PAM sequence. In some embodiments, editing begins within about 50 nucleotides upstream of the PAM sequence. In some embodiments, editing begins within about 40 nucleotides upstream of the PAM sequence. In some embodiments, editing begins within about 30 nucleotides upstream of the PAM sequence. In some embodiments, editing begins within about 20 nucleotides upstream of the PAM sequence. In some embodiments, editing begins within about 10 nucleotides upstream of the PAM sequence. In some embodiments, editing begins within about 9 nucleotides upstream of the PAM sequence. In some embodiments, editing begins within about 8 nucleotides upstream of the PAM sequence. In some embodiments, editing begins within about 7 nucleotides upstream of the PAM sequence. In some embodiments, editing begins within about 6 nucleotides upstream of the PAM sequence. In some embodiments, editing begins within about 5 nucleotides upstream of the PAM sequence. In some embodiments, editing begins within about 4 nucleotides upstream of the PAM sequence. In some embodiments, editing begins within about 3 nucleotides upstream of the PAM sequence. In some embodiments, editing begins within about 2 nucleotides upstream of the PAM sequence. In some embodiments, editing begins within about 1 nucleotide upstream of the PAM sequence.

[0316] In some embodiments, editing begins at the PAM sequence. In some embodiments, editing begins within about 1 nucleotide downstream of the PAM. In some embodiments, editing begins within about 2 nucleotides downstream of the PAM. In some embodiments, editing begins within about 3 nucleotides downstream of the PAM. In some embodiments, editing begins within about 4 nucleotides downstream of the PAM. In some embodiments, editing begins within about 5 nucleotides downstream of the PAM. In some embodiments, editing begins within about 6 nucleotides downstream of the PAM. In some embodiments, editing begins within about 7 nucleotides downstream of the PAM. In some embodiments, editing begins within about 8 nucleotides downstream of the PAM. In some embodiments, editing begins within about 9 nucleotides downstream of the PAM. In some embodiments, editing begins within about 10 nucleotides downstream of the PAM. In some embodiments, editing begins within about 11 nucleotides downstream of the PAM. In some embodiments, editing begins within about 12 nucleotides downstream of the PAM. In some embodiments, editing begins within about 13 nucleotides downstream of the PAM. In some embodiments, editing begins within about 14 nucleotides downstream of the PAM. In some embodiments, editing begins within about 15 nucleotides downstream of the PAM. In some embodiments, editing begins within about 16 nucleotides downstream of the PAM. In some embodiments, editing begins within about 17 nucleotides downstream of the PAM. In some embodiments, editing begins within about 18 nucleotides downstream of the PAM. In some embodiments, editing begins within about 19 nucleotides downstream of the PAM. In some embodiments, editing begins within about 20 nucleotides downstream of the PAM. In some embodiments, editing begins within about 21 nucleotides downstream of the PAM. In some embodiments, editing begins within about 22 nucleotides downstream of the PAM.In some embodiments, editing begins within about 23 nucleotides downstream of the PAM. In some embodiments, editing begins within about 24 nucleotides downstream of the PAM. In some embodiments, editing begins within about 25 nucleotides downstream of the PAM. In some embodiments, editing begins within about 26 nucleotides downstream of the PAM. In some embodiments, editing begins within about 27 nucleotides downstream of the PAM. In some embodiments, editing begins within about 28 nucleotides downstream of the PAM. In some embodiments, editing begins within about 29 nucleotides downstream of the PAM. In some embodiments, editing begins within about 30 nucleotides downstream of the PAM.

[0317] In some embodiments, the editing ends within about 300 nucleotides upstream of the PAM sequence. In some embodiments, the editing ends within about 290 nucleotides upstream of the PAM sequence. In some embodiments, the editing ends within about 280 nucleotides upstream of the PAM sequence. In some embodiments, the editing ends within about 270 nucleotides upstream of the PAM sequence. In some embodiments, the editing ends within about 260 nucleotides upstream of the PAM sequence. In some embodiments, the editing ends within about 250 nucleotides upstream of the PAM sequence. In some embodiments, the editing ends within about 240 nucleotides upstream of the PAM sequence. In some embodiments, the editing ends within about 230 nucleotides upstream of the PAM sequence. In some embodiments, the editing ends within about 2020 nucleotides upstream of the PAM sequence. In some embodiments, the editing ends within about 210 nucleotides upstream of the PAM sequence. In some embodiments, the editing ends within about 200 nucleotides upstream of the PAM sequence. In some embodiments, the editing ends within about 190 nucleotides upstream of the PAM sequence. In some embodiments, the editing ends within about 180 nucleotides upstream of the PAM sequence. In some embodiments, the editing ends within about 170 nucleotides upstream of the PAM sequence. In some embodiments, the editing ends within about 160 nucleotides upstream of the PAM sequence. In some embodiments, the editing ends within about 150 nucleotides upstream of the PAM sequence. In some embodiments, the editing ends within about 140 nucleotides upstream of the PAM sequence. In some embodiments, the editing ends within about 130 nucleotides upstream of the PAM sequence. In some embodiments, the editing ends within about 120 nucleotides upstream of the PAM sequence. In some embodiments, the editing ends within about 110 nucleotides upstream of the PAM sequence. In some embodiments, the editing ends within about 100 nucleotides upstream of the PAM sequence.In some embodiments, the editing ends within about 90 nucleotides upstream of the PAM sequence. In some embodiments, the editing ends within about 80 nucleotides upstream of the PAM sequence. In some embodiments, the editing ends within about 70 nucleotides upstream of the PAM sequence. In some embodiments, the editing ends within about 60 nucleotides upstream of the PAM sequence. In some embodiments, the editing ends within about 50 nucleotides upstream of the PAM sequence. In some embodiments, the editing ends within about 40 nucleotides upstream of the PAM sequence. In some embodiments, the editing ends within about 30 nucleotides upstream of the PAM sequence. In some embodiments, the editing ends within about 20 nucleotides upstream of the PAM sequence. In some embodiments, the editing ends within about 10 nucleotides upstream of the PAM sequence. In some embodiments, the editing ends within about 9 nucleotides upstream of the PAM sequence. In some embodiments, the editing ends within about 8 nucleotides upstream of the PAM sequence. In some embodiments, the editing ends within about 7 nucleotides upstream of the PAM sequence. In some embodiments, the editing ends within about 6 nucleotides upstream of the PAM sequence. In some embodiments, the editing ends within about 5 nucleotides upstream of the PAM sequence. In some embodiments, the editing ends within about 4 nucleotides upstream of the PAM sequence. In some embodiments, the editing ends within about 3 nucleotides upstream of the PAM sequence. In some embodiments, the editing ends within about 2 nucleotides upstream of the PAM sequence. In some embodiments, the editing ends within about 1 nucleotide upstream of the PAM sequence.

[0318] In some embodiments, the edits end at the PAM sequence. In some embodiments, the edits end within about 1 nucleotide downstream of the PAM. In some embodiments, the edits end within about 2 nucleotides downstream of the PAM. In some embodiments, the edits end within about 3 nucleotides downstream of the PAM. In some embodiments, the edits end within about 4 nucleotides downstream of the PAM. In some embodiments, the edits end within about 5 nucleotides downstream of the PAM. In some embodiments, the edits end within about 6 nucleotides downstream of the PAM. In some embodiments, the edits end within about 7 nucleotides downstream of the PAM. In some embodiments, the edits end within about 8 nucleotides downstream of the PAM. In some embodiments, the edits end within about 9 nucleotides downstream of the PAM. In some embodiments, the edits end within about 10 nucleotides downstream of the PAM. In some embodiments, the edits end within about 11 nucleotides downstream of the PAM. In some embodiments, the edits end within about 12 nucleotides downstream of the PAM. In some embodiments, the edits end within about 13 nucleotides downstream of the PAM. In some embodiments, the edits end within about 14 nucleotides downstream of the PAM. In some embodiments, the edits end within about 15 nucleotides downstream of the PAM. In some embodiments, the edits end within about 16 nucleotides downstream of the PAM. In some embodiments, the edits end within about 17 nucleotides downstream of the PAM. In some embodiments, the edits end within about 18 nucleotides downstream of the PAM. In some embodiments, the edits end within about 19 nucleotides downstream of the PAM. In some embodiments, the edits end within about 20 nucleotides downstream of the PAM. In some embodiments, the edits end within about 21 nucleotides downstream of the PAM. In some embodiments, the edits end within about 22 nucleotides downstream of the PAM.In some embodiments, the edits end within about 23 nucleotides downstream of the PAM. In some embodiments, the edits end within about 24 nucleotides downstream of the PAM. In some embodiments, the edits end within about 25 nucleotides downstream of the PAM. In some embodiments, the edits end within about 26 nucleotides downstream of the PAM. In some embodiments, the edits end within about 27 nucleotides downstream of the PAM. In some embodiments, the edits end within about 28 nucleotides downstream of the PAM. In some embodiments, the edits end within about 29 nucleotides downstream of the PAM. In some embodiments, the edits end within about 30 nucleotides downstream of the PAM.

[0319] C. Non-PAM chain editing In some embodiments, methods are provided herein for introducing at least one edit into the non-PAM strand of a target nucleic acid using a suitable gene editing system disclosed herein, such as those shown in Figure 5, Figure 6A, Figure 6B, Figure 7, Figure 8A, Figure 12A, or Figure 12B. At least one edit can be introduced into the non-PAM strand first using a reverse transcription template sequence contained in the gene editing system. Through cellular DNA repair mechanisms, at least one edit is eventually introduced into both strands of the target nucleic acid. The gene editing system can include an editing template RNA that targets the non-PAM strand, the editing template RNA including (a) a CRISPR nuclease binding sequence, (b) a DNA binding sequence, and (c) and a RT donor RNA. In some embodiments, the RT donor RNA includes a PBS and a reverse transcription template sequence.

[0320] In some embodiments, methods and gene editing systems or compositions are described for introducing at least one edit into the non-PAM strand of a target nucleic acid via 5' to 3' transcription of a reverse transcription template sequence of an RT donor RNA. In some embodiments, methods and compositions are described for introducing at least one edit into the non-PAM strand of a target nucleic acid via 5' to 3' transcription of a reverse transcription template sequence.

[0321] In some embodiments, the PBS of the RT donor RNA (e.g., the RT donor RNA of the editing template RNA) binds to a region on the non-PAM strand (the PBS target site). The reverse transcription template sequence contains an edit that is incorporated into the non-PAM strand. In some examples, the reverse transcription template contains sequence similarity with the PAM strand. In some examples, the reverse transcription template contains an edit to the sequence of the PAM strand. In some embodiments, the non-PAM strand binds to the PBS of the RT donor RNA through base pairing, and the reverse transcriptase (e.g., a CRISPR nuclease-reverse transcriptase fusion) copies the reverse transcription template sequence. After strand exchange and re-base pairing with the complementary genomic strand, the edit is incorporated into the target nucleic acid.

[0322] In some embodiments, the editing template RNA targeting the non-PAM strand includes the following components from 5' to 3': a CRISPR nuclease binding sequence, a DNA binding sequence, a reverse transcription template sequence, and a PBS (see, e.g., Figures 5, 6A, 6B, 8A, and 12A). In some embodiments, the editing template RNA targeting the non-PAM strand includes the following components from 5' to 3': a reverse transcription template sequence, a PBS, a CRISPR nuclease binding sequence, and a DNA binding sequence (spacer), or includes the following components from 5' to 3': a reverse transcription template sequence, a PBS, a linker, a CRISPR nuclease binding sequence, and a DNA binding sequence (Figures 7 and 12B).

[0323] In some embodiments, the CRISPR nuclease binding sequence is adjacent to the DNA binding sequence. In some embodiments, the CRISPR nuclease binding sequence is a 5' extension of the DNA binding sequence (Figures 5, 6A, 6B, 8A, and 12A). In some embodiments, the CRISPR nuclease binding sequence is adjacent to the DNA binding sequence and the PBS. In some embodiments, the CRISPR nuclease binding sequence is a 3' extension of the PBS (Figures 7 and 12B). In some embodiments, the CRISPR nuclease binding sequence binds to a type II CRISPR nuclease. In some embodiments, the CRISPR nuclease binding sequence binds to a type V CRISPR nuclease (e.g., a Cas12i polypeptide, e.g., a Cas12i1, Cas12i2, Cas12i3, or Cas12i4 polypeptide). In some embodiments, the CRISPR nuclease binding sequence binds to a CRISPR nuclease that lacks crRNA processing activity. In some embodiments, the CRISPR nuclease binding sequence is a direct repeat sequence (e.g., a Cas9 direct repeat sequence or a Cas12i direct repeat sequence).

[0324] In some embodiments, the DNA binding sequence is adjacent to the CRISPR nuclease binding sequence and the PBS. In some embodiments, the DNA binding sequence is a 3' extension of the CRISPR nuclease binding sequence (Figures 5, 6A, 6B, 7, 8A, 12A, and 12B). In some embodiments, the DNA binding sequence may comprise an RNA sequence, a DNA sequence, or an RNA / DNA hybrid sequence. In some embodiments, the DNA binding sequence comprises from about 10 nucleotides to about 50 nucleotides in length. In some embodiments, the DNA binding sequence comprises from about 15 nucleotides to about 35 nucleotides in length.

[0325] In some embodiments, the PBS flanks the reverse transcription template sequence. In some embodiments, the PBS is a 3' extension of the reverse transcription template sequence (FIGS. 5, 6A, 6B, 7, 8A, 12A, and 12B). In some embodiments, the PBS flanks the reverse transcription template sequence and the CRISPR nuclease binding sequence. In some embodiments, the PBS is about 3 nucleotides to about 200 nucleotides in length. In some embodiments, the PBS is about 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, or 110 nucleotides in length. In some embodiments, the DNA binding sequence and the PBS bind to the same strand (e.g., the non-PAM strand) of the target nucleic acid.

[0326] In some embodiments, the reverse transcription template sequence is flanked by a PBS and a DNA binding sequence. In some embodiments, the reverse transcription template sequence is a 5' extension of the PBS (Figures 5, 6A, 6B, 8A, and 12A). In some embodiments, the reverse transcription template sequence is a 3' extension of the DNA target sequence (Figures 5, 6A, 6B, 8A, and 12A). In some embodiments, the reverse transcription template sequence is a 5' extension of the PBS (Figures 7 and 12B). In some embodiments, the reverse transcription template sequence is about 10 nucleotides to about 300 nucleotides in length. In some embodiments, the reverse transcription template sequence is about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, or 120 nucleotides in length.

[0327] In some embodiments, the editing template RNA targeted to the non-PAM strand comprises a loop of unpaired nucleotides when the DNA binding sequence and the PBS are bound to the target nucleic acid. See FIG. 6A, FIG. 6B, FIG. 8A, and FIG. 12A. In some embodiments, the editing template RNA targeted to the non-PAM strand comprises a loop adjacent to the PBS. See FIG. 7 and FIG. 12B. In some embodiments, the loop comprises the reverse transcription template sequence followed by the PBS. In some embodiments, the PBS comprises complementarity to the non-PAM strand of the target nucleic acid. In some embodiments, the sequence of the loop comprises sequence similarity to the PAM strand. In some embodiments, the loop comprises an edit relative to the sequence of the PAM strand. In some embodiments, the edit is a substitution, insertion, or deletion. In some embodiments, the loop comprises a hairpin.

[0328] D.PAM ChainEdit In some embodiments, methods are provided herein for introducing at least one edit into a PAM strand of a target nucleic acid (e.g., a genomic site of interest) using a suitable gene editing system disclosed herein, such as those shown in Figure 1A, Figure 1B, Figure 2, Figure 3, Figure 4, or Figure 10. Such methods may include the use of an editing template RNA that targets the PAM strand, and the editing template RNA may include (a) a CRISPR nuclease binding sequence, (b) a DNA binding sequence, and (c) an RT donor RNA (Figure 1A, Figure 1B, Figure 2, and Figure 10). In some examples, the composition that targets the PAM strand includes an RNA guide and an RT donor RNA (Figure 3 and Figure 4). In some examples, the RT donor RNA includes a PBS and a reverse transcription template sequence.

[0329] In some embodiments, methods and compositions are described for introducing at least one edit into the PAM strand of a target nucleic acid via 5' to 3' transcription of a reverse transcription template sequence.In some embodiments, methods and compositions are described for introducing at least one edit into the PAM strand of a target nucleic acid via 5' to 3' transcription of a reverse transcription template sequence.

[0330] In some cases, the PBS of the RT donor RNA (e.g., the RT donor RNA of the editing template RNA) binds to the PAM strand. The reverse transcription template sequence of the RT donor RNA contains an edit that is incorporated into the PAM strand. In some examples, the reverse transcription template contains sequence similarity with the non-PAM strand. In some embodiments, the reverse transcription template contains an edit relative to the sequence of the non-PAM strand. In some embodiments, the PAM strand can bind to the PBS of the RT donor RNA through base pairing, and the reverse transcriptase (e.g., a CRISPR nuclease-reverse transcriptase fusion) copies the reverse transcription template sequence. After strand exchange and re-base pairing with the complementary genomic strand, the edit is incorporated into the target nucleic acid.

[0331] In some embodiments, the editing template RNA targeted to the PAM strand includes the following components from 5' to 3': a CRISPR nuclease binding sequence, a DNA binding sequence, a reverse transcription template sequence, and a PBS (Figure 1A, Figure 1B, and Figure 10). In some embodiments, the editing template RNA targeted to the PAM strand includes the following components from 5' to 3': a reverse transcription template sequence, a PBS, a CRISPR nuclease binding sequence, and a DNA binding sequence, or includes the following components from 5' to 3': a reverse transcription template sequence, a PBS, a linker, a CRISPR nuclease binding sequence, and a DNA binding sequence (Figure 2).

[0332] In some embodiments, the CRISPR nuclease binding sequence is adjacent to a DNA binding sequence. In some embodiments, the DNA binding sequence is a 3' extension of the CRISPR nuclease binding sequence (Figure 1A, Figure 1B, Figure 2, and Figure 10). In some embodiments, the CRISPR nuclease binding sequence is adjacent to a DNA binding sequence and a PBS (Figure 2). In some embodiments, the DNA binding sequence is a 3' extension of the PBS (Figure 2). In some embodiments, the CRISPR nuclease binding sequence binds to a type II CRISPR nuclease. In some embodiments, the CRISPR nuclease binding sequence binds to a type V CRISPR nuclease (e.g., a Cas12i polypeptide, e.g., a Cas12i1, Cas12i2, Cas12i3, or Cas12i4 polypeptide). In some embodiments, the CRISPR nuclease binding sequence binds to a CRISPR nuclease that lacks crRNA processing activity. In some embodiments, the CRISPR nuclease binding sequence is a direct repeat sequence (e.g., a Cas9 direct repeat sequence or a Cas12i direct repeat sequence).

[0333] In some embodiments, the DNA binding sequence is adjacent to the CRISPR nuclease binding sequence. In some embodiments, the DNA binding sequence is a 3' extension of the CRISPR nuclease binding sequence (FIG. 1A, FIG. 1B, FIG. 2, and FIG. 10). In some embodiments, the DNA binding sequence is adjacent to the CRISPR nuclease binding sequence and the reverse transcription template sequence. In some embodiments, the reverse transcription template sequence is a 3' extension of the DNA binding sequence (FIG. 10). In some embodiments, the DNA binding sequence is an RNA sequence, a DNA sequence, or an RNA / DNA hybrid sequence. In some embodiments, the DNA binding sequence comprises about 10 nucleotides to about 50 nucleotides in length. In some embodiments, the DNA binding sequence comprises about 15 nucleotides to about 35 nucleotides in length. In some embodiments, the DNA binding sequence is a spacer sequence.

[0334] In some embodiments, the PBS is adjacent to the reverse transcription template sequence. In some embodiments, the PBS is a 3' extension of the reverse transcription template sequence (FIG. 1A, FIG. 2, FIG. 1B, and FIG. 10). In some embodiments, the PBS is adjacent to the CRISPR nuclease binding sequence. In some embodiments, the CRISPR nuclease binding sequence is a 3' extension of the PBS (FIG. 2). In some embodiments, the PBS is about 3 nucleotides to about 200 nucleotides in length. In some embodiments, the PBS is about 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, or 110 nucleotides in length. In some embodiments, the DNA binding sequence and the PBS bind to different strands of the target nucleic acid (e.g., the DNA binding sequence binds to the target strand and the PBS binds to the PAM strand).

[0335] In some embodiments, the reverse transcription template sequence is adjacent to a DNA binding sequence. In some embodiments, the reverse transcription template sequence is a 3' extension of the DNA binding sequence (FIGS. 1A, 1B, and 10). In some embodiments, the reverse transcription template sequence is adjacent to a PBS. In some embodiments, the reverse transcription template sequence is a 5' extension of the PBS (FIGS. 1A, 1B, 2). In some embodiments, the PBS is a 3' extension of the reverse transcription template sequence (FIG. 10). In some embodiments, the reverse transcription template sequence is about 10 nucleotides to about 300 nucleotides in length. In some embodiments, the reverse transcription template sequence is about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, or 120 nucleotides in length.

[0336] E. Gene editing in cells In some embodiments, methods are provided herein for editing a genomic site of interest in a cell (e.g., a target gene disclosed herein) using a suitable gene editing system also disclosed herein. To carry out this method, the gene editing system may be delivered to or introduced into a population of cells. In some cases, cells containing the desired gene edits may be collected and, optionally, cultured and propagated in vitro.

[0337] The cells described herein can be a variety of cells. In some embodiments, the cells are isolated cells. In some embodiments, the cells are in cell culture or a co-culture of two or more cell types. In some embodiments, the cells are ex vivo. In some embodiments, the cells are obtained from a living organism and maintained in cell culture. In some embodiments, the cells are single-cell organisms.

[0338] In some embodiments, the cell is a prokaryotic cell. In some embodiments, the cell is a bacterial cell or is derived from a bacterial cell. In some embodiments, the cell is an archaeal cell or is derived from an archaeal cell.

[0339] In some embodiments, the cell is a eukaryotic cell. In some embodiments, the cell is or is derived from a plant cell. In some embodiments, the cell is or is derived from a fungal cell. In some embodiments, the cell is or is derived from an animal cell. In some embodiments, the cell is or is derived from an invertebrate cell. In some embodiments, the cell is or is derived from a vertebrate cell. In some embodiments, the cell is or is derived from a mammalian cell. In some embodiments, the cell is a human cell. In some embodiments, the cell is a zebrafish cell. In some embodiments, the cell is a primate cell. In some embodiments, the cell is a rodent cell. In some embodiments, the cell is synthetically produced, often referred to as an artificial cell.

[0340] In some embodiments, the cell is derived from a cell line. A wide variety of cell lines for tissue culture are known in the art. Examples of cell lines include, but are not limited to, 293T, MF7, K562, HeLa, CHO, and transgenic variants thereof. Cell lines are available from a variety of sources known to those of skill in the art (see, for example, American Type Culture Collection (ATCC) (Manassas, Va.)). In some embodiments, the cell is an immortal or immortalized cell. In some embodiments, the cell is a stem cell, such as a totipotent stem cell (e.g., allopotent), pluripotent stem cell, multipotent stem cell, oligopotent stem cell, or unipotent stem cell. In some embodiments, the cell is an induced pluripotent stem cell (iPSC) or is derived from an iPSC. In some embodiments, the cell is a mesenchymal stem cell. In some embodiments, the cell is an embryonic stem cell. In some embodiments, the cell is a hematopoietic stem cell. In some embodiments, the cell is a differentiated cell. For example, in some embodiments, the differentiated cell is a muscle cell (e.g., a myocyte), a fat cell (e.g., an adipocyte), a bone cell (e.g., an osteoblast, an osteocyte, an osteoclast), a blood cell (e.g., a monocyte, a lymphocyte, a neutrophil, an eosinophil, a basophil, a macrophage, a red blood cell, or a platelet), a nerve cell (e.g., a neuron), an epithelial cell, an immune cell (e.g., a lymphocyte, a neutrophil, a monocyte, or a macrophage), a liver cell (e.g., a hepatocyte), a fibroblast, or a sex cell. In some embodiments, the cell is a terminally differentiated cell. For example, in some embodiments, the terminally differentiated cell is a neuronal cell, an adipocyte, a cardiac muscle cell, a skeletal muscle cell, an epidermal cell, or an intestinal cell. In some embodiments, the cell is a glial cell. In some embodiments, the cell is a pancreatic islet cell, and the pancreatic islet cell comprises an alpha cell, a beta cell, a delta cell, or an enterochromaffin cell. In some embodiments, the cell is an immune cell. In some embodiments, the immune cell is a T cell. In some embodiments, the immune cell is a B cell.In some embodiments, the immune cell is a natural killer (NK) cell. In some embodiments, the immune cell is a tumor infiltrating lymphocyte (TIL). In some embodiments, the cell is a mammalian cell, e.g., a human cell or a primate cell or a mouse cell. In some embodiments, the mouse cell is from a wild type mouse, an immunosuppressed mouse, or a disease-specific mouse model. In some embodiments, the cell is a cell within a living tissue, organ, or organism.

[0341] In some embodiments, the cells are primary cells. For example, a culture of primary cells can be passaged 0, 1, 2, 4, 5, 10, or 15 or more times. In some embodiments, the primary cells are harvested from an individual by any known method. For example, white blood cells can be harvested by apheresis, leukapheresis, density gradient separation, and the like. Cells from tissues, such as skin, muscle, bone marrow, spleen, liver, pancreas, lung, intestinal tract, stomach, and the like, can be harvested by biopsy. An appropriate solution can be used for dispersion or suspension of the harvested cells. Such solutions can generally be balanced salt solutions (e.g., normal saline, phosphate buffered saline (PBS), Hank's balanced salt solution, and the like), conveniently supplemented with fetal bovine serum or other naturally occurring factors with an acceptable buffer at low concentrations. Buffers can include HEPES, phosphate buffer, lactate buffer, and the like. The cells can be used immediately or stored (e.g., by freezing). Frozen cells may be thawed and may be able to be reused. Cells may be frozen in DMSO, serum, media buffer (e.g., 10% DMSO, 50% serum, 40% buffered media), and / or some other common solution used to store cells at freezing temperatures.

[0342] In embodiments where the gene editing system disclosed herein is introduced into a plurality of cells, at least about 0.5% of the cells contain the desired edit. In some embodiments, at least about 1% of the cells contain the desired edit. In some embodiments, at least about 2% of the cells contain the desired edit. In some embodiments, at least about 3% of the cells contain the desired edit. In some embodiments, at least about 4% of the cells contain the desired edit. In some embodiments, at least about 5% of the cells contain the desired edit. In some embodiments, at least about 10% of the cells contain the desired edit. In some embodiments, at least about 20% of the cells contain the desired edit. In some embodiments, at least about 30% of the cells contain the desired edit. In some embodiments, at least about 40% of the cells contain the desired edit. In some embodiments, at least about 50% of the cells contain the desired edit.

[0343] Cells carrying the desired gene edits, for example, cells produced by the methods disclosed herein using any of the gene editing systems also disclosed herein, are also within the scope of the present disclosure. In some cases, cells modified with the CRISPR nuclease, reverse transcriptase, and edited template RNA described herein may be useful as expression systems for producing biomolecules. For example, the modified cells may be useful for producing biomolecules, for example, proteins (e.g., cytokines, antibodies, antibody-based molecules), peptides, lipids, carbohydrates, nucleic acids, amino acids, and vitamins. In other embodiments, the modified cells may be useful in producing viral vectors, for example, lentiviruses, adenoviruses, adeno-associated viruses, and oncolytic viral vectors. In some embodiments, the modified cells may be useful in cytotoxicity studies. In some embodiments, the modified cells may be useful as disease models. In some embodiments, the modified cells may be useful in vaccine production. In some embodiments, the modified cells may be useful in therapeutics. For example, in some embodiments, the modified cells may be useful in cell therapy, for example, infusions and transplants.

[0344] In some embodiments, cells modified by the CRISPR nuclease, reverse transcriptase, and edited template RNA described herein can be useful for establishing new cell lines containing modified genomic sequences. In some embodiments, the modified cells of the present disclosure are modified stem cells (e.g., modified totipotent / pluripotent stem cells, modified pluripotent stem cells, modified multipotent stem cells, modified oligopotent stem cells, or modified unipotent stem cells), which differentiate into one or more cell lineages that contain deletions of the modified stem cells. The present disclosure further provides organisms (e.g., animals, plants, or fungi) that contain or are produced from the modified cells of the present disclosure.

[0345] F. Delivery of the gene editing system into cells In some embodiments, any of the gene editing systems disclosed herein or components thereof can be formulated, for example, to include a carrier, such as a carrier and / or a polymeric carrier, for example, a liposome or lipid nanoparticle, and delivered to cells (e.g., prokaryotes, eukaryotes, plants, mammals, etc.) by known methods, including, but not limited to, transfection (e.g., lipid-mediated, cationic polymers, calcium phosphate, dendrimers), electroporation or other methods of membrane disruption (e.g., nucleofection), viral delivery (e.g., lentivirus, retrovirus, adenovirus, AAV), microinjection, biolistic projectile ("gene gun"), fugene, direct sonic loading, cell squeezing, phototransfection, protoplast fusion, imparefection, magnetofection, exome-mediated transfer, lipid nanoparticle-mediated transfer, and any combination thereof.

[0346] In some embodiments, the method includes delivering one or more nucleic acids (e.g., nucleic acids encoding CRISPR nucleases, reverse transcriptases, editing template RNAs (e.g., RNA guides and RT donor RNAs), etc.), one or more transcripts thereof, and / or preformed ribonucleoproteins to a cell. Exemplary intracellular delivery methods include, but are not limited to, viruses or virus-like agents; chemical-based transfection methods, such as those using calcium phosphate, dendrimers, liposomes, or cationic polymers (e.g., DEAE-dextran or polyethyleneimine); non-chemical methods, such as microinjection, electroporation, cell squeezing, sonoporation, phototransfection, imparefection, protoplast fusion, bacterial conjugation, delivery of plasmids or transposons; particle-based methods, such as gene guns, magnetofection or magnetically assisted transfection, using particle guns; and hybrid methods, such as nucleofection. In some embodiments, the present application further provides cells produced by such methods, and organisms (e.g., animals, plants, or fungi) that contain or are produced from such cells. In some embodiments, the compositions of the present invention are further delivered with an agent (e.g., a compound, molecule, or biomolecule) that affects DNA repair or DNA repair mechanisms. In some embodiments, the compositions of the present invention are further delivered with an agent (e.g., a compound, molecule, or biomolecule) that affects the cell cycle.

[0347] In some embodiments, a first composition comprising a CRISPR nuclease or comprising a CRISPR nuclease and a reverse transcriptase (e.g., a CRISPR nuclease-reverse transcriptase fusion) is delivered to the cell. In some embodiments, a second composition comprising an RNA guide or comprising an RNA guide and a RT donor RNA (e.g., an editing template RNA) is delivered to the cell. In some embodiments, the first composition contacts the cell before the second composition contacts the cell. In some embodiments, the first composition contacts the cell at the same time that the second composition contacts the cell. In some embodiments, the first composition contacts the cell after the second composition contacts the cell. In some embodiments, the first composition is delivered by a first delivery method and the second composition is delivered by a second delivery method. In some embodiments, the first delivery method is the same as the second delivery method. For example, in some embodiments, the first composition and the second composition are delivered via viral delivery. In some embodiments, the first delivery method is different from the second delivery method. For example, in some embodiments, a first composition is delivered by viral delivery and a second composition is delivered by lipid nanoparticle-mediated transfer and the second composition is delivered by viral delivery, or a first composition is delivered by lipid nanoparticle-mediated transfer and a second composition is delivered by viral delivery.

[0348] IV. Therapeutic uses Any of the gene editing systems disclosed herein or modified cells generated using such gene editing systems can be used to treat diseases that may benefit from gene editing introduced by the gene editing system or carried by the modified cells. For example, the disease may be a genetic disease and the gene editing repairs a genetic mutation associated with the genetic disease. Alternatively, the disease may be associated with abnormal expression of a gene and the gene editing rescues such abnormal expression.

[0349] In some embodiments, provided herein is a method for treating a disease, comprising administering any of the gene editing systems disclosed herein to a subject (e.g., a human patient) in need of treatment. The gene editing system can be delivered to a specific tissue or specific cell type where gene editing is required. The gene editing system can include a LNP, which includes one or more of the components, one or more vectors (e.g., viral vectors) encoding one or more of the components, or a combination thereof. The components of the gene editing system can be formulated to form a pharmaceutical composition, which can further include one or more pharma- ceutically acceptable carriers.

[0350] In some embodiments, modified cells produced using any of the gene editing systems disclosed herein can be administered to a subject (e.g., a human patient) in need of treatment. The modified cells can include substitutions, insertions, and / or deletions as described herein. In some examples, the modified cells can include cell lines modified with CRISPR nucleases, reverse transcriptase polypeptides, and edited template RNAs (e.g., RNA guides and RT donor RNAs). In some cases, the modified cells can be a heterogeneous population, where the heterogeneous population includes cells with different types of gene edits. Alternatively, the modified cells can include a substantially homogeneous cell population (e.g., at least 80% of the cells in the total population), where the substantially homogeneous cell population includes one particular gene edit. In some examples, the cells can be suspended in a suitable medium.

[0351] In some embodiments, compositions comprising gene editing systems or components thereof, or modified cells are provided herein. Such compositions can be pharmaceutical compositions. Useful pharmaceutical compositions can be prepared, packaged, or sold in formulations suitable for oral, rectal, vaginal, parenteral, topical, pulmonary, intranasal, intralesional, buccal, ocular, intravenous, intravisceral, or another route of administration. Pharmaceutical compositions of the present disclosure can be prepared, packaged, or sold in bulk, as a single unit dose, or as a plurality of single unit doses. As used herein, a "unit dose" is a discrete amount of pharmaceutical composition that contains a predetermined number of cells. The number of cells is generally equal to the dose of cells administered to a subject, or a convenient fraction of such a dose, for example, one-half or one-third of such a dose.

[0352] A formulation of a pharmaceutical composition suitable for parenteral administration may include an active agent (e.g., a gene editing system or its components, or modified cells) combined with a pharma- ceutically acceptable carrier, such as sterile water or sterile isotonic saline. Such a formulation may be prepared, packaged, or sold in a form suitable for bolus administration or continuous administration. Some injectable formulations may be prepared, packaged, or sold in unit dosage form, for example, in ampoules or multi-dose containers containing a preservative. Some formulations for parenteral administration include, but are not limited to, suspensions, solutions, emulsions in oily or aqueous vehicles, pastes, and implantable sustained-release or biodegradable formulations. Some formulations may further include one or more additional ingredients, including, but not limited to, suspending agents, stabilizing agents, or dispersing agents.

[0353] The pharmaceutical composition may be in the form of a sterile injectable aqueous or oily suspension or solution. The suspension or solution may be formulated by known techniques and may contain, in addition to the cells, additional components such as dispersing agents, wetting agents, or suspending agents described herein. Such sterile injectable formulations may be prepared using non-toxic parenterally acceptable diluents or solvents, such as water or saline. Other acceptable diluents and solvents include, but are not limited to, Ringer's solution, isotonic saline, and fixed oils, such as synthetic mono- or diglycerides. Other parentally administrable formulations that are useful include those that may contain the cells in packaged form, in liposomal preparations, or as a component of a biodegradable polymer system. Some compositions for sustained release or implantation may contain pharma-ceutically acceptable polymers or hydrophobic materials, such as emulsions, ion exchange resins, poorly soluble polymers, or poorly soluble salts.

[0354] V. KITS AND THEIR USES The present disclosure also provides kits or systems that can be used, for example, to carry out the methods described herein. In some embodiments, the kits or systems include a CRISPR nuclease (e.g., a V-type nuclease, e.g., a Cas12i polypeptide) and a reverse transcriptase. In some embodiments, the kits or systems include a polynucleotide encoding a CRISPR nuclease (e.g., a V-type nuclease, e.g., a Cas12i polypeptide) and a reverse transcriptase, optionally contained within a vector, e.g., a vector described herein. In some embodiments, the kits or systems include a V-type nuclease-reverse transcriptase fusion polypeptide (e.g., a Cas12i-reverse transcriptase fusion polypeptide, e.g., a Cas12i2-RT fusion or a Cas12i4-RT fusion). The kits or systems can also include a reverse transcriptase as described herein and an editing template RNA (e.g., an RNA guide and a RT donor RNA) as described herein. The RNA guide and / or RT donor RNA of the kits or systems of the present invention can be designed to target a sequence of interest. The CRISPR nuclease (e.g., a V-type nuclease, e.g., a Cas12i polypeptide), reverse transcriptase, and editing template RNA (e.g., an RNA guide and a RT donor RNA) may be packaged in the same vial or other container within the kit or system, or may be packaged in separate vials or other containers, and the contents thereof may be mixed prior to use. In addition, the kit or system may optionally include buffers and / or instructions for use of the CRISPR nuclease (e.g., a V-type nuclease, e.g., a Cas12i polypeptide) and reverse transcriptase with the editing template RNA (e.g., an RNA guide and a RT donor RNA).

[0355] In some embodiments, the kit comprises a first composition, the first composition comprises a CRISPR nuclease, or the first composition comprises a CRISPR nuclease and a reverse transcriptase (e.g., a CRISPR nuclease-reverse transcriptase fusion). In some embodiments, the kit comprises a second composition, the second composition comprises an RNA guide, or the second composition comprises an RNA guide and a RT donor RNA (e.g., an editing template RNA). In some embodiments, the first composition and the second composition are packaged in the same vial. In some embodiments, the first composition and the second composition are packaged in different vials.

[0356] In some embodiments, the kits may be useful for research purposes, for example, in some embodiments, the kits may be useful for studying gene function.

[0357] All references and publications cited herein are hereby incorporated by reference.

[0358] Additional Embodiments Additional embodiments are provided below and are also within the scope of the present disclosure.

[0359] Embodiment 1: (a) a type V CRISPR nuclease polypeptide or a nucleic acid encoding a type V CRISPR nuclease polypeptide, wherein the type V CRISPR nuclease polypeptide is optionally a Cas12 polypeptide; (b) an RNA guide or a nucleic acid encoding an RNA guide, wherein the RNA guide comprises a V-type nuclease binding sequence (e.g., a direct repeat sequence) and a DNA binding sequence (e.g., a spacer sequence); (c) a reverse transcriptase polypeptide or a nucleic acid encoding a reverse transcriptase polypeptide; and (d) a reverse transcription donor RNA (RT donor RNA) comprising a primer binding.

[0360] In embodiment 1, the V-type CRISPR nuclease can be Cas12a (Cpf1), Cas12b (C2c1), Cas12c, Cas12d, Cas12e, Cas12f, Cas12h, Cas12i, or Cas12j (CasPhi) polypeptide. In some examples, the V-type CRISPR nuclease polypeptide is a Cas12i polypeptide, and the Cas12i polypeptide optionally comprises a Cas12i1 polypeptide or a variant Cas12i1 polypeptide, a Cas12i2 polypeptide or a variant Cas12i2 polypeptide, a Cas12i3 polypeptide or a variant Cas12i3 polypeptide, or a Cas12i4 polypeptide or a variant Cas12i4 polypeptide.

[0361]

[0036] Embodiment 2: The composition of embodiment 1 may comprise a Cas12i polypeptide, the Cas12i polypeptide being: (a) the Cas12i1 polypeptide comprises an amino acid sequence having at least 80% identity to SEQ ID NO:8, and optionally at least 95% identity to SEQ ID NO:8; (b) the Cas12i2 polypeptide comprises an amino acid sequence having at least 80% identity to any one of SEQ ID NOs: 2-7, and optionally at least 95% identity to any one of SEQ ID NOs: 2-7; (c) the Cas12i3 polypeptide comprises an amino acid sequence having at least 80% identity to SEQ ID NO:11, and optionally, at least 95% identity to SEQ ID NO:11; and (d) the Cas12i4 polypeptide can be one of: comprising an amino acid sequence having at least 80% identity to SEQ ID NO:9 or at least 80% to SEQ ID NO:10, and optionally having at least 95% identity to SEQ ID NO:9 or at least 95% to SEQ ID NO:10.

[0362] In a specific example, the composition of embodiment 2 comprises: (a) the Cas12i1 polypeptide comprises the amino acid sequence set forth in SEQ ID NO:8; (b) the Cas12i2 polypeptide comprises an amino acid sequence set forth in any one of SEQ ID NOs: 2 to 7; (c) the Cas12i3 polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 11; and (d) the Cas12i4 polypeptide comprises one of the amino acid sequences set forth in SEQ ID NO: 9 or SEQ ID NO: 10.

[0363] Any of the compositions of embodiment 2 disclosed herein may comprise a type V CRISPR nuclease polypeptide, wherein the type V CRISPR nuclease polypeptide has reduced or no crRNA processing activity. For example, the type V CRISPR nuclease polypeptide is a Cas12i2 polypeptide, wherein the Cas12i2 polypeptide comprises a substitution at H485 or H486. In some cases, the Cas12i2 polypeptide comprises at least 80% identity with any one of SEQ ID NOs: 2-7, wherein the Cas12i2 polypeptide comprises a substitution at H485 or H486. In some examples, the Cas12i2 polypeptide comprises at least 95% identity with any one of SEQ ID NOs: 2-7, wherein the Cas12i2 polypeptide comprises a substitution at H485 or H486.

[0364] Any of the compositions of embodiment 2 disclosed herein may comprise a Type V CRISPR nuclease polypeptide, wherein the Type V CRISPR nuclease polypeptide comprises at least one of an epitope peptide, a nuclear localization signal, and a nuclear export signal.

[0365] In some examples, the composition of embodiment 2 comprises: (a) the Cas12i1 polypeptide comprises an amino acid sequence having at least 80% (e.g., at least 95%) identity to SEQ ID NO: 8, and the direct repeat sequence comprises a nucleotide sequence having at least 90% identity to any one of SEQ ID NOs: 12 to 14; (b) the Cas12i2 polypeptide comprises an amino acid sequence having at least 80% (e.g., at least 95%) identity to any one of SEQ ID NOs: 2 to 7, and the direct repeat sequence comprises a nucleotide sequence having at least 90% identity to any one of SEQ ID NOs: 15 to 17; (c) the Cas12i3 polypeptide comprises an amino acid sequence having at least 80% (e.g., at least 95%) identity to SEQ ID NO: 11, and the direct repeat sequence comprises a nucleotide sequence having at least 90% identity to any one of SEQ ID NOs: 18 to 20; and (d) the Cas12i4 polypeptide comprises an amino acid sequence having at least 80% identity, for example at least 95%, to SEQ ID NO: 9 or SEQ ID NO: 10, and the direct repeat sequence comprises a nucleotide sequence having at least 90% identity to any one of SEQ ID NOs: 21 to 24.

[0366] In some examples, the composition of embodiment 2 comprises: (a) the Cas12i1 polypeptide comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 8, and the direct repeat sequence comprises a nucleotide sequence having at least 95% identity to any one of SEQ ID NOs: 12 to 14; (b) the Cas12i2 polypeptide comprises an amino acid sequence having at least 95% identity to any one of SEQ ID NOs: 2 to 7, and the direct repeat sequence comprises a nucleotide sequence having at least 95% identity to any one of SEQ ID NOs: 15 to 17; (c) the Cas12i3 polypeptide comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 11, and the direct repeat sequence comprises a nucleotide sequence having at least 95% identity to any one of SEQ ID NOs: 18 to 20; and (d) the Cas12i4 polypeptide comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 9 or SEQ ID NO: 10, and the direct repeat sequence comprises a nucleotide sequence having at least 95% identity to any one of SEQ ID NOs: 21 to 24.

[0367] Embodiment 3: The spacer sequence of any of the compositions of embodiment 1 or embodiment 2 disclosed herein comprises a length of about 10 nucleotides to about 50 nucleotides. In some examples, the spacer sequence comprises a length of about 15 nucleotides to about 35 nucleotides. In some examples, the spacer sequence is substantially complementary to a target strand of a target nucleic acid (e.g., a complementary sequence of the target sequence). In some examples, the target sequence is adjacent to a protospacer adjacent motif (PAM) sequence on a non-target strand.

[0368] Embodiment 4: Any of the compositions of embodiments 1, 2, or 3 may comprise a type V nuclease, wherein the type V nuclease is a Cas12i polypeptide and the PAM sequence comprises a sequence set forth as 5'-NTTN-3', wherein N is any nucleotide.

[0369] Embodiment 5: In any of the compositions of any of the above embodiments, the reverse transcriptase polypeptide comprises MMLV-RT, MMTV-RT, Marathon-RT, or RTX reverse transcriptase.

[0370] Embodiment 6: In any of the compositions of any of embodiments 1-5, the reverse transcriptase polypeptide is fused to a type V CRISPR nuclease polypeptide. In some examples, the reverse transcriptase polypeptide is fused to the N-terminus of the type V CRISPR nuclease polypeptide. In other examples, the reverse transcriptase polypeptide is fused to the C-terminus of the type V CRISPR nuclease polypeptide. In yet other examples, the reverse transcriptase polypeptide is inserted into a loop of the type V CRISPR nuclease polypeptide.

[0371] Embodiment 7: In any of the compositions of any of embodiments 1 to 5, the reverse transcriptase polypeptide and the Type V CRISPR nuclease polypeptide form a complex via a leucine zipper, a nanobody, an antibody, or a coiled-coil domain.

[0372] Embodiment 8: In any of the compositions of any of embodiments 1-7, the RT donor RNA can be fused to the RNA guide. In some examples, the RT donor RNA is fused to the 5' end of the RNA guide. In other examples, the RT donor RNA is fused to the 3' end of the RNA guide. In some cases, the spacer sequence of the RNA guide is adjacent to the reverse transcription template sequence in the RT donor RNA. Alternatively, the spacer sequence of the RNA guide is adjacent to the PBS in the RT donor RNA. In other cases, the direct repeat sequence of the RNA guide is adjacent to the reverse transcription template sequence in the RT RNA donor. Alternatively, the direct repeat sequence of the RNA guide is adjacent to the PBS in the RT donor RNA.

[0373] In some cases, the RT donor RNA-RNA guide fusion polynucleotide may further comprise a linker. In some cases, the linker is between the direct repeat sequence and the PBS. In other cases, the linker is between the spacer sequence in and the reverse transcription template sequence. The linker may be about 1 nucleotide to about 200 nucleotides in length. In some cases, the linker comprises a hairpin.

[0374] Embodiment 9: In any of the compositions of any one of embodiments 1-8, the PBS can be about 3 nucleotides to about 200 nucleotides in length. For example, the PBS is about 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, or 110 nucleotides in length. In some cases, the PBS hybridizes (binds via base pairing) with the free 3' end of the non-target strand (PAM strand). In other cases, the PBS hybridizes with the free 3' end of the target strand (non-PAM strand).

[0375] Embodiment 10: In any of the compositions of any one of embodiments 1-9, the reverse transcription template sequence is about 10 nucleotides to about 300 nucleotides in length. For example, the reverse transcription template sequence is about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, or 120 nucleotides in length.

[0376] Embodiment 11: In any of the compositions of any one of embodiments 1-10, the PBS has substantial complementarity with a target strand or a non-target strand of a target nucleic acid (which is double-stranded). For example, the PBS comprises at least about 75% complementarity with a target strand or a non-target strand of the target nucleic acid. In another example, the PBS comprises at least about 85% complementarity with a target strand or a non-target strand of the target nucleic acid. In another example, the PBS comprises at least about 95% complementarity with a target strand or a non-target strand of the target nucleic acid.

[0377]

[0036] Embodiment 12: In any of the compositions of any one of embodiments 1 to 11, the reverse transcription template sequence comprises an aptamer. In some cases, the aptamer recruits a reverse transcriptase polypeptide.

[0378] Embodiment 13: In any of the compositions of any one of embodiments 1 to 11, the reverse transcription template comprises a modification, for example, a modification at the 5' end or the 3' end. In some examples, the modification is a chemical modification. In other examples, the modification is a nucleic acid sequence comprising a secondary structure. In specific examples, the modification is a hairpin, a pseudoknot, a triplex structure, an exoribonuclease-resistant RNA (xrRNA), a transfer RNA (tRNA), or a truncated tRNA. In other specific examples, the modification comprises a nuclease binding sequence (e.g., one or more direct repeat sequences), or comprises a nuclease binding sequence and a DNA binding sequence (spacer).

[0379] Any of the compositions of any one of embodiments 1-13 may introduce an edit into the target strand or the non-target strand. In some cases, the edit is a substitution, an insertion, or a deletion. In some cases, the edit is a substitution of 1 nucleotide to about 200 nucleotides. In some cases, the edit is a substitution of 1 nucleotide to about 120 nucleotides. In some cases, the edit is a substitution of 1 nucleotide to about 20 nucleotides. In other cases, the edit is an insertion of 1 nucleotide to about 200 nucleotides, e.g., an insertion of 1 nucleotide to about 120 nucleotides, an insertion of 1 nucleotide to about 20 nucleotides. In some cases, the insertion comprises a hairpin. In still other cases, the edit is a deletion of 1 nucleotide to about 100 nucleotides. For example, the edit is a deletion of 1 nucleotide to about 120 nucleotides, or a deletion of 1 nucleotide to about 20 nucleotides.

[0380] In some examples, the editing occurs within about 200 nucleotides of the PAM sequence. In one example, the editing occurs within about 100 nucleotides of the PAM sequence. In another example, the editing occurs within about 50 nucleotides of the PAM sequence. In yet another example, the editing occurs within about 30 nucleotides of the PAM sequence. In yet another example, the editing occurs within about 20 nucleotides of the PAM sequence.

[0381] In some examples, editing begins and / or ends within about 200 nucleotides upstream of the PAM sequence, e.g., begins and / or ends within about 100 nucleotides upstream of the PAM sequence, begins and / or ends within about 50 nucleotides upstream of the PAM sequence, begins and / or ends within about 30 nucleotides upstream of the PAM sequence, begins and / or ends within about 20 nucleotides upstream of the PAM sequence, begins and / or ends within about 10 nucleotides upstream of the PAM sequence, begins and / or ends within about 5 nucleotides upstream of the PAM sequence, or begins and / or ends within about 5 nucleotides downstream of the PAM sequence.

[0382] In other examples, editing begins and / or ends within about 10 nucleotides downstream of the PAM sequence, for example, beginning and / or ending within about 25 nucleotides downstream of the PAM sequence.

[0383] In some instances, the edit removes or alters the PAM sequence. In some instances, the edit prevents retargeting by a type V CRISPR nuclease polypeptide (e.g., prevents binding of a type V CRISPR nuclease to a target sequence).

[0384] Embodiment 14: In any of the compositions of embodiments 1-13, the target sequence is present intracellularly.

[0385] Embodiment 15: Any of the compositions of embodiments 1-14 can be formulated for delivery to cells. In some examples, the cells are mammalian cells, e.g., human cells. In one example, the cells are liver cells (e.g., hepatocytes).

[0386] In some examples, the Type V CRISPR nuclease polypeptide or a nucleic acid encoding a Type V CRISPR nuclease polypeptide, the RNA guide or a nucleic acid encoding an RNA guide, the reverse transcriptase polypeptide or a nucleic acid encoding a reverse transcriptase polypeptide, and the RT donor RNA are formulated within a single delivery vehicle.

[0387] In other examples, the Type V CRISPR nuclease polypeptide or a nucleic acid encoding a Type V CRISPR nuclease polypeptide, the RNA guide or a nucleic acid encoding an RNA guide, the reverse transcriptase polypeptide or a nucleic acid encoding a reverse transcriptase polypeptide, and the RT donor RNA are formulated in two or more delivery vehicles.

[0388] In yet other examples, the type V CRISPR nuclease polypeptide or a nucleic acid encoding a type V CRISPR nuclease polypeptide and the reverse transcriptase polypeptide or a nucleic acid encoding a reverse transcriptase polypeptide are formulated within a single delivery vehicle.

[0389] In some cases, the RNA guide and the RT donor RNA are formulated in a single delivery vehicle. In some cases, the V-type CRISPR nuclease polypeptide or a nucleic acid encoding a V-type CRISPR nuclease polypeptide and the reverse transcriptase polypeptide or a nucleic acid encoding a reverse transcriptase polypeptide are formulated in a first delivery vehicle, and the RNA guide and the RT donor RNA are formulated in a second delivery vehicle.

[0390] Embodiment 16: In any of the compositions of any one of embodiments 1 to 15, where applicable, the Type V CRISPR nuclease polypeptide, reverse transcriptase polypeptide, RNA guide, and / or RT donor RNA are encoded in one or more vectors, e.g., one or more expression vectors.

[0391] Embodiment 17: A vector comprising a sequence encoding a type V CRISPR nuclease polypeptide, a reverse transcriptase polypeptide, an RNA guide, and / or an RT donor RNA of any of the compositions of embodiments 1 to 16.

[0392] Embodiment 18: A cell comprising the composition of any one of embodiments 1 to 16 or the vector of embodiment 17. In some examples, the cell is a mammalian cell, e.g., a human cell. In one example, the cell is a liver cell (e.g., a hepatocyte).

[0393] Embodiment 19: A method for expressing the vector of embodiment 17.

[0394] Embodiment 20: A method of producing the composition of any one of embodiments 1 to 17.

[0395] Embodiment 21: A method of delivering the composition of any one of embodiments 1-16.

[0396] In some examples, the Type V CRISPR nuclease polypeptide or a nucleic acid encoding a Type V CRISPR nuclease polypeptide, the RNA guide or a nucleic acid encoding an RNA guide, the reverse transcriptase polypeptide or a nucleic acid encoding a reverse transcriptase polypeptide, and the RT donor RNA are delivered within a single delivery vehicle.

[0397] In other examples, the Type V CRISPR nuclease polypeptide or a nucleic acid encoding a Type V CRISPR nuclease polypeptide, the RNA guide or a nucleic acid encoding an RNA guide, the reverse transcriptase polypeptide or a nucleic acid encoding a reverse transcriptase polypeptide, and the RT donor RNA are delivered in two or more delivery vehicles.

[0398] In yet other examples, the type V CRISPR nuclease polypeptide or a nucleic acid encoding a type V CRISPR nuclease polypeptide and the reverse transcriptase polypeptide or a nucleic acid encoding a reverse transcriptase polypeptide are delivered within a single delivery vehicle.

[0399] In some cases, the RNA guide and RT donor RNA are delivered within a single delivery vehicle.

[0400] In a specific example, the Type V CRISPR nuclease polypeptide or a nucleic acid encoding a Type V CRISPR nuclease polypeptide and the reverse transcriptase polypeptide or a nucleic acid encoding a reverse transcriptase polypeptide are delivered in a first delivery vehicle, and the RNA guide and RT donor RNA are delivered in a second delivery vehicle.

[0401] Embodiment 22: A method of binding the composition of any one of embodiments 1 to 16 to a target nucleic acid. In some examples, the target nucleic acid is present in a cell, e.g., a mammalian cell, e.g., a human cell. In one example, the cell is a liver cell (e.g., a hepatocyte).

[0402] Embodiment 23: A method of introducing an edit into a target nucleic acid, comprising contacting the target nucleic acid with a composition of any one of embodiments 1 to 16. In some instances, the composition introduces an edit into a target strand or a non-target strand of the target nucleic acid. In some instances, the edit is a substitution, insertion, or deletion.

[0403] In specific examples, the editing is a substitution of 1 nucleotide to about 200 nucleotides, for example, a substitution of 1 nucleotide to about 120 nucleotides, or a substitution of 1 nucleotide to about 20 nucleotides. In other specific examples, the editing is an insertion of 1 nucleotide to about 200 nucleotides, for example, an insertion of 1 nucleotide to about 120 nucleotides, or an insertion of 1 nucleotide to about 20 nucleotides. In some cases, the insertion comprises a hairpin. In yet other specific examples, the editing is a deletion of 1 nucleotide to about 100 nucleotides, for example, a deletion of 1 nucleotide to about 120 nucleotides, or a deletion of 1 nucleotide to about 20 nucleotides.

[0404] In some cases, editin...

Claims

1. (a) a type V CRISPR nuclease polypeptide or a first nucleic acid encoding said type V CRISPR nuclease polypeptide; (b) a reverse transcriptase (RT) polypeptide or a second nucleic acid encoding said RT polypeptide; (c) a guide RNA (gRNA) or a third nucleic acid encoding said gRNA, wherein said gRNA comprises one or more binding sites (CRISPR nuclease binding sites) recognizable by said type V CRISPR nuclease and a spacer sequence specific for a target sequence within a target genomic site, said target sequence being adjacent to a protospacer adjacent motif (PAM), gRNA or third nucleic acid; (d) a reverse transcription donor RNA (RT donor RNA) or a fourth nucleic acid encoding said RT donor RNA, wherein said RT donor RNA comprises a primer binding site (PBS) and a template sequence, RT donor RNA or fourth nucleic acid, a gene editing system comprising: The type V CRISPR nuclease polypeptide is a Cas12i2 polypeptide, and said Cas12i2 polypeptide comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 2 and comprises one or more mutations at positions H485, H486, D581, G624, F626, P868, I926, V1030, E1035, and / or S1046 of SEQ ID NO: 2, a gene editing system.

2. said Cas12i2 polypeptide being: (i) mutations at positions D581, D911, I926, and V1030; (ii) mutations at positions D581, I926, and V1030; (iii) mutations at positions D581, I926, V1030, and S1046; (iv) mutations at positions D581, G624, F626, I926, V1030, E1035, and S1046, or (v) mutations at positions D581, G624, F626, P868, I926, V1030, E1035, and S1046, and / or said Cas12i2 polypeptide comprises a mutation of H485 and / or H486, The gene editing system according to claim 1.

3. said Cas12i2 polypeptide being: (i) amino acid substitutions of D581R, D911R, I926R, and V1030G; (ii) amino acid substitutions of D581R, I926R, and V1030G; (iii) amino acid substitutions of D581R, I926R, V1030G, and S1046G; (iv) amino acid substitutions of D581R, G624R, F626R, I926R, V1030G, E1035R, and S1046G, or (v) including amino acid substitutions of D581R, G624R, F626R, P868T, I926R, V1030G, E1035R, and S1046G, and / or the Cas12i2 polypeptide includes an amino acid substitution of H485A, The gene editing system according to claim 2.

4. The gene editing system according to claim 1, wherein the Cas12i2 polypeptide includes an amino acid sequence of any one of SEQ ID NOs: 3 to 7.

5. The gene editing system according to claim 1, wherein the gene editing system includes the first nucleic acid encoding the type V CRISPR nuclease polypeptide, and the first nucleic acid is a DNA fragment located in messenger RNA (mRNA) or a viral vector.

6. The gene editing system according to claim 1, wherein the RT polypeptide is Moloney murine leukemia virus (MMLV)-RT, mouse mammary tumor virus (MMTV)-RT, Marathon-RT, or RTx-RT.

7. The gene editing system according to claim 1, wherein the system includes the second nucleic acid encoding the RT polypeptide, and the second nucleic acid is a DNA fragment located in mRNA or a viral vector.

8. The gene editing system according to any one of claims 1 to 7, wherein the gene editing system includes a fusion polypeptide or a nucleic acid encoding a fusion polypeptide, and the fusion polypeptide includes the Cas12i2 polypeptide and the RT polypeptide.

9. The gene editing system according to claim 8, wherein the gene editing system includes a nucleic acid encoding a fusion polypeptide, and the nucleic acid is a DNA fragment located in messenger RNA (mRNA) or a viral vector.

10. The gene editing system according to any one of claims 1 to 7, wherein the one or more CRISPR nuclease binding sites are direct repeat sequences, and the direct repeat sequences are at least 90% identical to any one of SEQ ID NOs: 15 to 17 and 241 to 247, or a fragment thereof having a length of at least 23 nucleotides.

11. The gene editing system according to claim 10, wherein the direct repeat sequence is any one of SEQ ID NOs: 15 to 17 and 241 to 247, or a fragment thereof having a length of at least 23 nucleotides.

12. The gene editing system according to any one of claims 1 to 7, wherein the PBS has a length of 5 to 100 nucleotides, the PBS binds to a PBS target site adjacent to a complementary region of the target sequence, and the PBS target site is upstream of the complementary region of the target sequence.

13. The PBS target site is 3 to 10 nucleotides upstream of the complementary region of the target sequence, the PBS target site overlaps with the complementary region of the target sequence, or the PBS target site is adjacent to or overlaps with the target sequence, The gene editing system according to claim 12.

14. The gene editing system according to any one of claims 1 to 7, wherein the template sequence has a length of 5 to 100 nucleotides, the template sequence is homologous to the target genomic site, and the target genomic site contains one or more nucleotide diversities.

15. The gene editing system according to claim 14, wherein at least one nucleotide diversity is located within the target sequence and / or at least one nucleotide diversity is located within the PAM.

16. The gene editing system according to any one of claims 1 to 7, wherein the system comprises the third nucleic acid encoding the gRNA and / or the fourth nucleic acid encoding the RT donor RNA.

17. The gene editing system according to any one of claims 1 to 7, wherein the gRNA and the RT donor RNA are located on a single RNA molecule, and the single RNA molecule comprises the CRISPR nuclease binding site, the gRNA comprising the spacer sequence, the RNA donor comprising the PBS, and the template sequence.

18. The single RNA molecule is, from 5' to 3', (i) the CRISPR nuclease binding site, the spacer sequence, the template sequence, and the PBS; (ii) the CRISPR nuclease binding site, the spacer sequence, the linker, the template sequence, and the PBS; (iii) the template sequence, the PBS, the CRISPR nuclease binding site, and the spacer sequence; or (iv)The gene editing system according to claim 17, comprising the template array, the PBS, the linker, the CRISPR nuclease binding site, and the spacer sequence.

19. The single RNA molecule further comprises a 5'-end protecting fragment, a 3'-end protecting fragment, or both, each of the 5'-end protecting fragment and the 3'-end protecting fragment forms a secondary structure, and the secondary structure is optionally a hairpin, pseudoknot, or triple-stranded structure, or The 5'-end protecting fragment and / or the 3'-end protecting fragment comprises one or more of the CRISPR nuclease binding sites and optionally comprises one or more segments that are not homologous to any human sequence. The gene editing system according to claim 18.

20. The gene editing system according to any one of claims 1 to 7, wherein the gRNA and the RT donor RNA are two separate RNA molecules.

21. The system according to any one of claims 1 to 7, wherein the system comprises one or more lipid nanoparticles (LNP), and the one or more LNP comprise element (a), (b), (c), (d), or any combination thereof.

22. The gene editing system according to any one of claims 1 to 7, wherein the gene editing system comprises a viral vector comprising a nucleotide sequence encoding a fusion polypeptide comprising the type V CRISPR nuclease and the RT polypeptide.

23. The gene editing system according to claim 22, wherein the viral vector further comprises a nucleotide sequence encoding a single RNA molecule comprising the gRNA and the RT donor RNA.

24. A pharmaceutical composition comprising the system according to any one of claims 1 to 7.

25. A kit comprising the elements (a) to (d) of the system according to any one of claims 1 to 7.

26. A method for gene editing of a cell, the method comprising contacting a host cell with the gene editing system according to any one of claims 1 to 7 or a pharmaceutical composition thereof to gene edit the host cell, wherein the host cell is cultured in vitro.

27. The gene editing system according to any one of claims 1 to 7 for gene editing of a host cell in a subject.

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