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Gene editing systems comprising a crispr nuclease and uses thereof — Arbor Biotechnologies, Inc. (US20240102007A1)

Arbor Biotechnologies, Inc. · Google Patents

Google Patents · Patents · License: Open Access

patent, google patents, intellectual property, US20240102007A1, Arbor Biotechnologies, Inc., David A. Scott, en, 2024

ABSTRACT

Abstract

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

Description

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit under 35 U.S.C. Â§ 119(e) of U.S. Provisional Application No. 63/195,621, filed Jun. 1, 2021, U.S. Provisional Application No. 63/236,047, filed Aug. 23, 2021, U.S. Provisional Application No. 63/272,937, filed Oct. 28, 2021, and U.S. Provisional Application No. 63/299,695, filed Jan. 14, 2022, the contents of each of which are incorporated by reference herein in their entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has been filed electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Jun. 1, 2022, is named 116928-0042-0001WO00_SEQ.txt and is 388,313 bytes in size.

BACKGROUND

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated (Cas) genes, collectively known as CRISPR-Cas or CRISPR/Cas systems, are adaptive immune systems in archaea and bacteria that defend particular species against foreign genetic elements.

SUMMARY OF THE INVENTION

The present disclosure is based, at least in part, on the development of a gene editing system involving a Type V CRISPR nuclease polypeptide (e.g., a Cas12i2 polypeptide) and a reverse transcriptase, as well as a guide RNA (gRNA) mediating cleavage at a genetic site of interest by the CRISPR nuclease polypeptide and a reverse transcription donor RNA mediating synthesis of desired sequences to be incorporated into the genomic site of interest. As reported herein, the gene editing system disclosed herein has achieved successful gene editing at various genomic sites with high editing efficiency and accuracy. Without being bound by theory, the gene editing system disclosed herein show at least one of the following advantageous features:

1. Many of the editing template RNAs described herein, such as those specific to a Cas12i polypeptide, do not require a trans-activating CRISPR RNA (tracrRNA) component and are thus smaller than prime editing guide RNAs (pegRNAs). Additionally, 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 preferable in terms of delivery and cost of synthesis. 2. Editing template RNAs described herein can be designed to have a longer primer binding site (PBS) than the PBS of pegRNAs. This feature could increase efficiency of edit incorporation into a target nucleic acid. 3. Gene editing systems comprising an editing template RNA designed to bind the non-PAM strand only (i.e., the complementary strand of the strand on which the PAM motif resides; also described herein as the target strand), as described herein, are capable of incorporating edits over a broader window compared to prime editing systems. In particular, Cas12i polypeptide-reverse transcriptase systems are capable of rewriting the full recognition sequence of the Cas12i polypeptide and an RNA guide. Therefore, these gene editing systems may be more efficient at evading retargeting of the target nucleic acid by the CRISPR nuclease-reverse transcriptase fusion and an editing template RNA.

Accordingly, provided herein are gene editing systems, pharmaceutical compositions or kits comprising such, methods of using the gene editing system to produce genetically modified cells, and the resultant cells thus produced.

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

In some embodiments, the Type V 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 instances, the Cas12i polypeptide is a Cas12i2 polypeptide, which comprises an amino acid sequence at least 95% identical to SEQ ID NO: 2.

In some instances, 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, which optionally is 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 still 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, the exemplary Cas12i2 polypeptide can comprise the amino acid sequence of SEQ ID NO: 4. In other examples, the exemplary Cas12i2 polypeptide can comprise the amino acid sequence of SEQ ID NO: 7.

In other instances, the Cas12i polypeptide has diminished crRNA processing activity, optionally wherein the Cas12i polypeptide comprises mutations at position H485 and/or position H486 of SEQ ID NO: 2.

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

In any of the gene editing systems disclosed herein, the RT polypeptide may be Moloney Murine Leukemia Virus (MMLV)-RT, mouse mammary tumor virus (MMTV)-RT, Marathon-RT, or RTx-RT (e.g., the MMLV RT, which may comprise the amino acid sequence of SEQ ID NO: 29). In some instances, the gene editing system comprises the RT polypeptide. Alternatively, the system comprises the second nucleic acid encoding the RT polypeptide. In some instances, the second nucleic acid is located in a second vector (e.g., a viral vector such as an adeno-associated viral vector or AAV vector). In one example, the gene editing system comprises a vector (e.g., a viral vector) that comprises both the first nucleic acid encoding the Type V CRISPR polypeptide and the second nucleic acid encoding the RT polypeptide. In other examples, the second nucleic acid encoding the RT is a second mRNA. In one example, the gene editing system comprises a single RNA molecule comprising both the first mRNA encoding the Type V CRISPR polypeptide and the second mRNA encoding the RT.

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

In any of the gene editing systems disclosed herein, the spacer sequence can be 20-30-nucleotide in length. In some examples, the spacer sequence is 20-nucleotide in length.

In some embodiments, the PAM comprises the motif of 5â²-TTN-3.â² In some instances (e.g., in association with a Cas12i2 polypeptide), the PAM may be located 5â² to the target sequence.

In some embodiments, the one or more CRISPR nuclease binding sites are direct repeat sequence(s). In some instances, each direct repeat sequence is 23-36-nucleotide in length. In one example, the direct repeat sequence is 23-nucleotide 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-nucleotide in length. In specific examples, 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-nucleotide in length.

In some embodiments, the gene editing system disclosed herein comprises the gRNA. Alternatively, the gene editing system comprises the third nucleic acid encoding the gRNA. In some examples, the third nucleic acid is located in a third vector, which optionally is a viral vector. In some examples, the gene editing system may comprise a vector such as a viral vector that comprises the third nucleic acid encoding the gRNA and the first and/or second nucleic acids encoding the Type V CRISPR nuclease polypeptide and/or the RT polypeptide.

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

In some embodiments, the template sequence in the RT donor RNA of any of the gene editing systems disclosed herein can be 5-100-nucleotide in length. For example, the template sequence may be 30-50-nucleotide in length. In some instances, the template sequence may be homologous to the genomic site of interest and comprises one or more nucleotide variations relative to the genomic site of interest. In some examples, at least one nucleotide variation is located within the target sequence. Alternatively or in addition, at least one nucleotide variation is located in the PAM.

In some embodiments, any of the gene editing system disclosed herein comprises the RT donor RNA. Alternatively, the gene editing system comprises the fourth nucleic acid encoding the RT donor RNA. In some examples, the fourth nucleic acid is located in a fourth vector, which optionally is a fourth viral vector. In some instances, the gene editing system comprises a vector such as a viral vector comprising the nucleic acid encoding the RT donor RNA, and one or more additional nucleic acids encoding the guide RNA, the Type V CRISPR nuclease polypeptide, and the RT polypeptide.

In some embodiments, the gene editing system disclosed herein comprises a single RNA molecule comprising the gRNA and the RT donor RNA. Such a single RNA comprises the CRISPR nuclease binding site, the spacer sequence, the PBS, and the 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 5â² to 3â²: the CRISPR nuclease binding site, the spacer sequence, the template sequence, and the PBS. In another example, the single RNA molecule comprises, from 5â² to 3â²: the CRISPR nuclease binding site, the spacer sequence, the linker, the template sequence, and the PBS. In yet another example, the single RNA molecule comprises, from 5â² to 3â²: the template sequence, the PBS, the CRISPR nuclease binding site, and the spacer sequence. In yet another example, the single RNA molecule comprises, from 5â² to 3â²: the template sequence, the PBS, the linker, the CRISPR nuclease binding site, and the spacer sequence.

In some instances, any of the single RNA molecule disclosed herein may further comprise a 5â² end protection fragment, a 3â² end protection fragment, or both. Each of the 5â² end protection fragment and the 3â² end protection fragment may form a secondary structure, for example, a hairpin, a pseudoknot, or a triplex structure. In some examples, the 5â² end protection fragment and/or the 3â² end protection fragment is an exoribonuclease-resistant RNA (xrRNA), a transfer RNA (tRNA), or a truncated tRNA. In specific examples, the 5â² end protection fragment, the 3â² end protection fragment, or both may comprise one or more of the CRISPR nuclease binding site. The 5â² end protection fragment, the 3â² end protection fragment, or both may further comprise one or more segments that are not homologous to any human sequence (cannot bind to any human sequences via base pairing).

In some embodiments, the gene editing system disclosed herein comprises 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 comprise a 5â² end protection fragment and/or a 3â² end protection fragment. Each of the protection fragment may form a secondary structure, for example, a hairpin, a pseudoknot, or a triplex structure. In some examples, the 5â² end protection fragment and/or the 3â² end protection fragment is an exoribonuclease-resistant RNA (xrRNA), a transfer RNA (tRNA), or a truncated tRNA. In other examples, the 5â² end protection fragment and/or the 3â² end protection fragment comprises one or more of the CRISPR nuclease binding site, and optionally one or more segments that are not homologous to any human sequence.

Any of the gene editing systems disclosed herein may comprise one or more lipid nanoparticles (LNPs), which encompass the Type V CRISPR nuclease polypeptide or the encoding nucleic acid, the RT polypeptide or the encoding nucleic acid, the guide RNA or the encoding nucleic acid, the RT donor RNA or the encoding nucleic acid, or any combination thereof. Alternatively, the gene editing system may comprise (i) one or more lipid nanoparticles (LNPs), which collectively encompass up to three components selected from of the Type V CRISPR nuclease polypeptide or the encoding nucleic acid, the RT polypeptide or the encoding nucleic acid, the guide RNA or the encoding nucleic acid, the RT donor RNA or the encoding nucleic acid; and (ii) one or more vectors encoding the remaining components in the gene editing system. In some instances, the one or more vectors can be one or more viral vectors, for example, one or more adeno-associated viral (AAV) vectors.

<div id="p-0025" num="0027" class="description-

CROSS REFERENCE TO RELATED APPLICATIONS

SEQUENCE LISTING

BACKGROUND

SUMMARY OF THE INVENTION

In any of the gene editing systems disclosed herein, the spacer sequence can be 20-30-nucleotide in length. In some examples, the spacer sequence is 20-nucleotide in length.

In some embodiments, the PAM comprises the motif of 5â²-TTN-3.â² In some instances (e.g., in association with a Cas12i2 polypeptide), the PAM may be located 5â² to the target sequence.

In some examples, the gene editing system disclosed herein comprises the Type V CRISPR nuclease polypeptide, the RT polypeptide, the gRNA, and the RT donor RNA. In some instances, the Type V CRISPR nuclease polypeptide and/or the RT polypeptide forms a complex (e.g., a ribonucleoprotein (RNP) complex) with the gRNA and/or the RT donor RNA.

In some aspects, the present disclosure also provides a pharmaceutical composition comprising any of the gene editing systems disclosed herein and a pharmaceutically acceptable carrier, and a kit comprising the components of the gene editing system.

In other aspects, the present disclosure also features a method for genetically editing a cell, the method comprising contacting a host cell any of the gene editing systems disclosed herein or the pharmaceutical composition comprising such to genetically 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 comprising the host cell.

Also within the scope of the present disclosure is a population of genetically modified cells, which can be produced by the gene editing system disclosed herein. In some examples, the genetically modified cells may comprise cells not editable by the gene editing system, for example, comprise one or more modifications in the PAM, in the target sequence, or in both.

In yet other aspects, the present disclosure features a gene editing RNA molecule, comprising: (i) one or more binding sites recognizable by a Type V CRISPR nuclease (CRISPR nuclease binding sites); (ii) a spacer sequence specific to a target sequence within a genetic 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.

In some examples, the RNA molecule comprises, from 5â² to 3â²: the CRISPR nuclease binding site, the spacer sequence, the template sequence, and the PBS. In other examples, the RNA molecule comprises, from 5â² to 3â²: the CRISPR nuclease binding site, the spacer sequence, the linker, the template sequence, and the PBS. In yet other examples, the RNA molecule comprises, from 5â² to 3â²: the template sequence, the PBS, the CRISPR nuclease binding site, and the spacer sequence. In still other examples, the RNA molecule comprises, from 5â² to 3â²: the template sequence, the PBS, the linker, the CRISPR nuclease binding site, and the spacer sequence.

Any of the gene editing RNA molecules disclosed herein may further comprise a 5â² end protection fragment, a 3â² end protection fragment, or both. Each of the protection fragment may form a secondary structure, for example, a hairpin, a pseudoknot, or a triplex structure. In some examples, the 5â² end protection fragment and/or the 3â² end protection fragment is an exoribonuclease-resistant RNA (xrRNA), a transfer RNA (tRNA), or a truncated tRNA. In other examples, the 5â² end protection fragment and/or the 3â² end protection fragment comprises one or more of the CRISPR nuclease binding site, and optionally one or more segments that are not homologous to any human sequence.

In addition, the present disclosure features a set of gene editing RNA molecules (two separate RNA molecules), comprising: (i) a guide RNA comprising one or more binding sites recognizable by the Type V CRISPR nuclease (CRISPR nuclease binding sites), and a spacer sequence specific to a target sequence within a genetic 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, wherein the RT donor RNA comprises a primer binding site (PBS) and a template sequence. In some examples, the gRNA, the RT donor RNA, or both further comprise a 5â² end protection fragment and/or a 3â² end protection fragment. Each of the protection fragment may form a secondary structure, for example, a hairpin, a pseudoknot, or a triplex structure. In some examples, the 5â² end protection fragment and/or the 3â² end protection fragment is an exoribonuclease-resistant RNA (xrRNA), a transfer RNA (tRNA), or a truncated tRNA. In other examples, the 5â² end protection fragment and/or the 3â² end protection fragment comprises one or more of the CRISPR nuclease binding site, and optionally one or more segments that are not homologous to any human sequence.

Also provided herein is a DNA molecule or a set of DNA molecules, which encode the gene editing RNA molecule or the set of gene editing RNA molecules as disclosed herein. In some examples, the DNA molecule or the set of DNA molecules of claim 76 , which is included in a vector or a set of vectors, optionally wherein the vector or set of vectors are viral vectors.

In addition, provided herein is a fusion polypeptide comprising a CRISPR nuclease and a reverse transcriptase. 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 Type V CRISPR nuclease, for example, a Cas12i polypeptide. In some examples, the Cas12i polypeptide is a Cas12i2 polypeptide, e.g., those disclosed herein. In specific examples, the fusion polypeptide may comprise the amino acid sequence of 25-26 and 219-223.

In some embodiments, the Cas12i polypeptide is a Cas12i4 polypeptide. In some examples, the Cas12i4 polypeptide may be fused with a reverse transcriptase, such as an MMLV RT. Such a fusion Cas12i4-RT fusion polypeptide may comprise the amino acid sequence of SEQ ID NO: 53.

Any of the nucleic acids encoding any of the CRISPR nuclease-RT fusion polypeptides, including vectors such as expression vectors (e.g., viral vectors), is also within the scope of the present disclosure.

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

BRIEF DESCRIPTION OF THE DRAWINGS

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 drawing in combination with the detailed description of specific embodiments presented herein.

FIGS. 1 A- 1 B include schematics showing exemplary gene editing systems disclosed herein. FIG. 1 A is a schematic showing a gene editing system comprising a CRISPR nuclease (e.g., a Cas12i polypeptide) fused to a reverse transcriptase polypeptide and an RNA guide fused to an RT donor RNA at the 3â² end of the RNA guide. The RT donor RNA comprises a reverse transcription template sequence and a PBS. The PBS comprises substantial complementarity to the PAM-strand (a.k.a., the non-target strand) of a target nucleic acid. FIG. 1 B shows a Cas9 nickase fused to a reverse transcriptase (left) and a Cas12i nickase fused to a reverse transcriptase (right). Using an RT donor RNA fused to the 3â² end of an RNA guide, an edit is incorporated into the PAM strand of a target nucleic acid.

FIG. 2 is a schematic showing an exemplary gene editing system comprising a CRISPR nuclease (e.g., a Cas12i polypeptide) fused to a reverse transcriptase polypeptide and an RNA guide fused to an RT donor RNA at the 5â² end of the RNA guide. The RT donor RNA comprises a PBS and a reverse transcription template sequence. The PBS comprises complementarity to the PAM strand of a target nucleic acid.

FIG. 3 is a schematic showing a CRISPR nuclease (e.g., a Cas12i polypeptide), a reverse transcriptase polypeptide, an RNA guide, and an RT donor RNA. The RT donor RNA comprises a reverse transcription template sequence and a PBS. An edit is incorporated into the genome following cleavage by the CRISPR nuclease.

FIG. 4 is a schematic showing a CRISPR nuclease (e.g., a 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. An edit is incorporated into the genome in the presence of the CRISPR nuclease.

FIG. 5 is a schematic showing an exemplary gene editing system comprising a CRISPR nuclease (e.g., a Cas12i polypeptide) fused to a reverse transcriptase polypeptide and an RNA guide containing mismatches to the target nucleic acid, fused to an RT donor RNA at the 3â² end of the RNA guide. The RT donor RNA comprises a PBS. The PBS comprises complementarity to the non-PAM strand (a.k.a., target strand or TS) of a target nucleic acid.

FIGS. 6 A- 6 B include schematics showing exemplary gene editing systems disclosed herein. FIG. 6 A is a schematic showing an exemplary gene editing system comprising a CRISPR nuclease (e.g., a Cas12i polypeptide) fused to a reverse transcriptase polypeptide and an RNA guide fused to an RT donor RNA at the 3â² end of the RNA guide. The RT donor RNA comprises a reverse transcription template sequence and a PBS. When the spacer sequence of the RNA guide and the PBS are bound to the target nucleic acid, the reverse transcription template sequence forms a loop of unpaired nucleotides. The PBS comprises complementarity to the non-PAM strand of a target nucleic acid. The variant Cas12i2 cleavage sites in the PAM strand and non-PAM strand are indicated by the triangles. Using an RT donor RNA fused to the 3â² end of an RNA guide, an edit is incorporated into the non-PAM strand of a target nucleic acid. FIG. 6 B shows the positioning of an edit, reverse transcription template sequence, and PBS, wherein the length of the reverse transcription template sequence and PBS can be varied.

FIG. 7 is a schematic showing an exemplary gene editing system comprising a CRISPR nuclease (e.g., a Cas12i polypeptide) fused to a reverse transcriptase polypeptide and an RNA guide fused to an RT donor RNA at the 5â² end of the RNA guide. The RT donor RNA comprises a PBS and a reverse transcription template sequence. The PBS comprises complementarity to the non-PAM strand of a target nucleic acid.

FIGS. 8 A- 8 C include schematics showing exemplary Cas12i2 RNA guide-RT donor RNA fusions. FIG. 8 A is a schematic of a variant Cas12i2 RNA guide fused to an RT donor RNA, which was tested in Example 1. The spacer of the RNA guide binds to the non-PAM strand adjacent to a 5â²-TIT-3â² PAM. The RT donor RNA comprises a reverse transcription template sequence and a PBS. When the spacer sequence and the PBS are bound to the target nucleic acid, the reverse transcription template sequence forms a loop of unpaired nucleotides. The PBS comprises complementarity to the non-PAM strand of a target nucleic acid. In this schematic, the PBS is 13 nucleotides in length and the reverse transcription template sequence is 34 nucleotides in length. The PBS is designed such that complementarity to non-PAM strand begins at a cleavage site (triangle). FIG. 8 B shows exemplary RNA guide-RT donor RNA fusions targeting an AAVS1_T7 genomic site, as tested in Example 1. Various PBS lengths were tested (13, 30, and 60 nucleotides). The RNA guide-RT donor RNA fusions were designed to introduce substitutions (S), an insertion (I), a deletion (D), or a hairpin (H) into the target sequence. FIG. 8 C shows encoded edits (substitutions, insertions, and deletions) introduced into an AAVS1_T7 genomic site (top panel), an EMX1_T6 genomic site (middle panel), and a VEGFA_T5 genomic site (bottom panel) as described in Example 1. Sequences in FIG. 8 A , from top to bottom, are SEQ ID NOs: 65-67. Sequences in FIG. 8 B , from top to bottom, are SEQ ID NOs: 74-80, and 87-89. Sequences in FIG. 8 C , from top to bottom, are SEQ ID NOs: 248-259.

FIGS. 9 A- 9 J include diagrams showing gene editing efficiencies resulting from exemplary gene editing systems disclosed herein. FIG. 9 A shows percentage of NGS reads analyzed with indels and encoded edits induced by variant Cas12i2 of SEQ ID NO: 4 and C-terminal and N-terminal Cas12i2-MMLV RT fusions of SEQ ID NO: 25 and SEQ ID NO: 26 with an RNA guide targeting an AAVS1_T6 genomic site. FIG. 9 B shows the percentage of NGS reads analyzed 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 an AAVS1_T6 genomic site. The RNA guide-RT donor RNA fusions had a PBS length of 13, 30, or 60 nucleotides and were designed to introduce substitutions (S), an insertion (I), a deletion (D), or a hairpin (H) into the AAVS1_T6 genomic site. FIG. 9 C shows the percentage of NGS reads analyzed with indels and encoded edits induced by variant Cas12i2 of SEQ ID NO: 4 and C-terminal and N-terminal Cas12i2-MMLV RT fusions of SEQ ID NO: 25 and SEQ ID NO: 26 with an RNA guide targeting an AAVS1_T7 genomic site. FIG. 9 D shows the percentage of NGS reads analyzed 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 an AAVS1_T7 genomic site. The RNA guide-RT donor RNA fusions had a PBS length of 13, 30, or 60 nucleotides and were designed to introduce substitutions (S), an insertion (I), a deletion (D), or a hairpin (H) into the AAVS1_T7 genomic site. FIG. 9 E shows the percentage of NGS reads analyzed with indels and encoded edits induced by variant Cas12i2 of SEQ ID NO: 4 and C-terminal and N-terminal Cas12i2-MMLV RT fusions of SEQ ID NO: 25 and SEQ ID NO: 26 with an RNA guide targeting an EMX1_T6 genomic site. FIG. 9 F shows the percentage of NGS reads analyzed with indels and 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 an EMX1_T6 genomic site. The RNA guide-RT donor RNA fusions had a PBS length of 13, 30, or 60 nucleotides and were designed to introduce substitutions (S), an insertion (I), a deletion (D), or a hairpin (H) into the EMX1_T6 genomic site. FIG. 9 G shows the percentage of NGS reads analyzed with indels and encoded edits induced by variant Cas12i2 of SEQ ID NO: 4 and C-terminal and N-terminal Cas12i2-MMLV RT fusions of SEQ ID NO: 25 and SEQ ID NO: 26 with an RNA guide targeting a VEGFA_T2 genomic site. FIG. 9 H shows the percentage of NGS reads analyzed 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 a VEGFA_T2 genomic site. The RNA guide-RT donor RNA fusions had a PBS length of 13, 30, or 60 nucleotides and were designed to introduce substitutions (S), an insertion (I), a deletion (D), or a hairpin (H) into the VEGFA_T2 genomic site. FIG. 9 I shows the percentage of NGS reads analyzed with indels and encoded edits induced by variant Cas12i2 of SEQ ID NO: 4 and C-terminal and N-terminal Cas12i2-MMLV RT fusions of SEQ ID NO: 25 and SEQ ID NO: 26 with an RNA guide targeting a VEGFA_T5 genomic site. FIG. 9 J shows the percentage of NGS reads analyzed 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 a VEGFA_T5 genomic site. The RNA guide-RT donor RNA fusions had a PBS length of 13, 30, or 60 nucleotides and were designed to introduce substitutions (S), an insertion (I), a deletion (D), or a hairpin (H) into the VEGFA_T5 genomic site.

FIG. 10 is a schematic showing a Cas12i polypeptide (e.g., a Cas12i2 nickase) fused to a reverse transcriptase. Using an RT donor RNA fused to the 5â² end or the 3â² end of an RNA guide, an encoded edit is incorporated into the PAM strand of a 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 between about 3-100 nucleotides and comprise substantial complementarity to the PAM strand. Structured RNA such as hairpins can be introduced between the spacer and the reverse transcription template sequence.

FIG. 11 is a schematic showing an RNA guide-RT donor RNA further fused to a second direct repeat (DR)-spacer sequence. The additional DR-spacer inhibits exonuclease activity.

FIGS. 12 A- 12 B include schematics showing exemplary designs of editing template RNAs (gene editing RNAs). FIG. 12 A is a schematic depicting editing template RNAs (5â²-nuclease binding sequenceâDNA-binding sequenceâreverse transcription templateâPBS-3â²) further comprising 3â² end protection. The 3â² end protection can be a chemical end protection (top portion of the figure) or a hairpin (bottom portion of the figure). The hairpin can be a nuclease binding sequence such as a direct repeat sequence. FIG. 12 B is a schematic depicting editing template RNAs (5â²-reverse transcription templateâPBS nuclease binding sequenceâDNA-binding sequence-3â²) with and without 5â² end protection. The 5â² end protection can be a hairpin (e.g., a nuclease binding sequence such as a direct repeat sequence), as shown in the bottom portion of the figure.

FIGS. 13 A- 13 D include diagrams showing gene editing efficiencies resulting from exemplary gene editing systems disclosed herein. FIG. 13 A shows activity of Cas12i2 (SEQ ID NO: 4) and Cas12i2-RT (SEQ ID NO: 25) with the RNA guide of SEQ ID NO: 112 or the editing template RNAs of SEQ ID NOs: 123-137 at an AAVS1_T7 genomic site (SEQ ID NO: 30). % NGS reads analyzed as having an indel are shown in the white bars for Cas12i2 and grey bars for Cas12i2-RT. % NGS reads analyzed as having the encoded edit are shown in the checkered bars for Cas12i2 and black bars for Cas12i2-RT. FIG. 13 B shows activity of Cas12i2 (SEQ ID NO: 4) and Cas12i2-RT (SEQ ID NO: 25) with the RNA guide of SEQ ID NO: 114 or the editing template RNAs of SEQ ID NOs: 138-152 at an EMX1_T6 genomic site (SEQ ID NO: 34). % NGS reads analyzed as having an indel are shown in the white bars for Cas12i2 and grey bars for Cas12i2-RT. % reads analyzed as having the encoded edit are shown in the checkered bars for Cas12i2 and black bars for Cas12i2-RT. FIG. 13 C shows activity of Cas12i2 (SEQ ID NO: 4) and Cas12i2-RT (SEQ ID NO: 25) with the RNA guide of SEQ ID NO: 116 or the editing template RNAs of SEQ ID NOs: 153-167 at VEGFA_T2 (SEQ ID NO: 36). % NGS reads analyzed as having an indel are shown in the white bars for Cas12i2 and grey bars for Cas12i2-RT. % NGS reads analyzed as having the encoded edit are shown in the checkered bars for Cas12i2 and black bars for Cas12i2-RT. FIG. 13 D shows activity of Cas12i2 (SEQ ID NO: 4) and Cas12i2-RT (SEQ ID NO: 25) with the RNA guide of SEQ ID NO: 118 or the editing template RNAs of SEQ ID NOs: 168-182 at a VEGFA_T5 genomic site (SEQ ID NO: 38). % NGS reads analyzed as having an indel are shown in the white bars for Cas12i2 and grey bars for Cas12i2-RT. % NGS reads analyzed as having the encoded edit are shown in the checkered bars for Cas12i2 and black bars for Cas12i2-RT.

FIG. 14 A- 14 C include schematics depicting the steps of an assay used to identify cleavage patterns of Cas12i2 with an RNA guide or an editing template RNA. FIG. 14 A shows an oligo configuration comprising a target sequence and a barcode. FIG. 14 B shows treatment of cleavage products to blunt 5â² and 3â² overhangs or end repair to fill in the 5â² overhangs. FIG. 14 C shows amplification of cleavage products.

FIGS. 15 A- 15 E include diagrams showing gene editing using exemplary gene editing systems disclosed herein. FIG. 15 A is a schematic depicting in vitro cleavage sites (triangles) induced by Cas12i2 of SEQ ID NO: 2 on the PAM strand and non-PAM strand of an AAVS1_T2 genomic site. FIG. 15 B is a histogram of read lengths obtained from amplification of 5â² cleavage products following fill-in treatment. FIG. 15 C is a histogram of read lengths obtained from amplification of 3â² cleavage products following fill-in treatment. FIG. 15 D is a histogram of read lengths obtained from amplification of 5â² cleavage products following blunting treatment. FIG. 15 E is a histogram of read lengths obtained from amplification of 3â² cleavage products following blunting treatment. Each read length histogram is mapped to the target sequence as shown on the x-axis of FIGS. 15 B- 15 E .

FIGS. 16 A- 16 B show in vitro cleavage sites (triangles) induced by Cas12i2 of SEQ ID NO: 2 or variant Cas12i2 of SEQ ID NO: 4 on the PAM strand or the non-PAM strand of an EMX1_T6 genomic site ( FIG. 16 A ) and a VEGFA_T5 genomic site ( FIG. 16 B ). The scale bar (right) represents the cleavage frequency as measured by the number of sequencing reads.

FIGS. 17 A- 17 B include diagrams showing gene editing results at exemplary genomic sizes. FIG. 17 A shows activity by editing template RNAs introducing 4-nucleotide insertions into an AAVS1_T7 genomic site (SEQ ID NO: 30), an EMX1_T6 genomic site (SEQ ID NO: 34), or a VEGFA_T5 genomic site (SEQ ID NO: 38). The editing template RNAs comprised a 34-nucleotide reverse transcription template sequence and a 3, 8, 13, 30, or 60-nucleotide PBS. Ratio of encoded edits to total edits is shown on the y-axis. FIG. 17 B shows activity by editing template RNAs in 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). The editing template RNAs comprised a 13-nucleotide PBS and a 14, 24, 34, 44, or 54-nucleotide reverse transcription template sequence. Ratio of encoded edits to total edits is shown on the y-axis. Sequences in FIG. 17 A , from top to bottom, are SEQ ID NOs: 90-92. Sequences in FIG. 17 B , from top to bottom, are SEQ ID NOs: 90-92.

FIG. 18 shows encoded edits incorporated into an AAVS1_T7 genomic site (SEQ ID NO: 32) and an EMX1_T6 genomic site (SEQ ID NO: 34) in U2OS cells.

FIGS. 19 A- 19 B include schematics illustrating gene editing procedures using exemplary gene editing systems disclosed herein. FIG. 19 A is a schematic depicting a Cas9 prime editor comprising a Cas9 fused to a reverse transcriptase and a pegRNA. A primer on the target DNA is generated following cleavage of the PAM strand by Cas9. Hybridization of the primer with the pegRNA initiates reverse transcription. FIG. 19 B is a schematic depicting a Type V CRISPR nuclease fused to a reverse transcriptase and an editing template RNA. A primer on the target DNA is generated following cleavage of the non-PAM strand by the Type V CRISPR nuclease. Hybridization of the primer with the editing template RNA initiates reverse transcription.

FIGS. 20 A- 20 C include diagrams showing edits at various genomic sites with Cas12i2-RT fusion polypeptides as indicated. FIG. 20 A is a plot showing % of NGS reads comprising an indel edit (white bars) or an encoded edit (grey bar) introduced by a variant Cas12i2-RT fusion of SEQ ID NOs: 219-223 at an AAVS1 genomic site. FIG. 20 B is a plot showing % of NGS reads comprising an indel edit (white bars) or an encoded edit (grey bar) introduced by a variant Cas12i2-RT fusion of SEQ ID NOs: 219-223 at an EMX1 genomic site. FIG. 20 C is a plot showing % of NGS reads comprising an indel edit (white bars) or an encoded edit (grey bar) introduced by a variant Cas12i2-RT fusion of SEQ ID NOs: 219-223 at a VEGFA genomic site.

FIG. 21 is a plot showing % of NGS reads comprising an indel edit or an encoded edit introduced by a variant Cas12i2 (SEQ ID NO: 4) or variant Cas12i2-RT fusion (SEQ ID NO: 219) and an RNA guide or an editing template RNA. The RNA guides and editing template RNAs were either unmodified or comprised terminal phosphorothioate backbone linkages and/or 2â²O-methyl nucleotides.

FIG. 22 is a plot showing % of NGS reads comprising an indel edit (white bars) or an encoded edit (grey bar) introduced by a variant Cas12i4-RT fusion at an AAVS1 genomic site.

FIG. 23 is a plot showing % of NGS reads comprising an indel edit (white bars) or an encoded edit (grey bar) introduced by a variant Cas12i2 or a variant Cas12i2-RT fusion, an RNA guide, and an RT donor RNA at an AAVS1, EMX1, or VEGFA genomic site.

DETAILED DESCRIPTION

The present disclosure relates to gene editing systems comprising a Type V nuclease or a nucleic acid encoding such, an RNA guide or a nucleic acid encoding such, a reverse transcriptase or a nucleic acid encoding such, and an RT donor RNA or a nucleic acid encoding such. Also provided herein are pharmaceutical compositions and kits comprising any of the gene editing systems disclosed herein, methods for genetically editing a cell using any of the gene editing systems disclosed herein, genetically engineered cells thus produced, and gene editing RNA molecules or a set of RNA molecules involved in the gene editing system, as well as DNA molecule(s) for producing such.

Definitions

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. Terms as set forth hereinafter are generally to be understood in their common sense unless indicated otherwise.

As used herein, the term âactivityâ refers to a biological activity. In some embodiments, the activity refers to effector activity. In some embodiments, activity includes enzymatic activity, e.g., 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.

As used herein, the term âadjacent toâ refers to a nucleotide or amino acid sequence in close proximity to another nucleotide or amino acid sequence. In some embodiments, a nucleotide sequence is adjacent to another nucleotide sequence if no nucleotides separate the two sequences (i.e., immediately adjacent). In some embodiments, a nucleotide sequence is adjacent to another nucleotide sequence if a small number of nucleotides separate the two sequences (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). 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 up to 2 nucleotides, up to 5 nucleotides, up to 8 nucleotides, up to 10 nucleotides, up to 12 nucleotides, or up to 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.

As used herein, the term âCRISPR nucleaseâ refers to an RNA-guided effector that is capable of binding a nucleic acid and introducing a single-stranded break or double-stranded break. In some embodiments, a CRISPR nuclease is a Type II CRISPR nuclease or a Type V CRISPR nuclease. In some embodiments, a CRISPR nuclease is an effector as described in Makarova et al. âClassification and Nomenclature of CRISPR-Cas Systems: Where from Here?â CRISPRJ. 1(5):325-36 (2018).

As used herein, the term âType IIâ and âType II nucleaseâ refers to a nuclease comprising a RuvC domain and an HNH domain. The Type II nuclease can be a Type II-A nuclease, a Type II-B nuclease, or a Type II-C nuclease. In some embodiments, the Type II nuclease requires a tracrRNA. In some embodiments, the Type II nuclease is a Cas9 polypeptide. The Cas9 polypeptide can cleave a double-stranded DNA target or be a nickase.

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

As used herein, the term âCas12i polypeptideâ (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, wherein the polypeptide has at least some amino acid sequence homology to 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 purpose referenced herein. In some embodiments, a 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: 8, 2, 11, and 9 of the present application. In some embodiments, a Cas12i polypeptide of the disclosure is a Cas12i2 polypeptide as described in WO/2021/202800, the relevant disclosures of which are incorporated by reference for the subject matter and purpose referenced herein. In some embodiments, the Cas12i polypeptide cleaves a target nucleic acid (e.g., as a nick or a double strand break).

The âpercent identityâ (a.k.a., sequence identity) of two nucleic acids or of 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, word length=3 to obtain amino acid sequences homologous to the protein molecules of the invention. Where gaps exist between two sequences, Gapped BLAST can be utilized as described in Altschul et al., Nucleic Acids Res. 25(17):3389-3402, 1997. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used.

As used herein, the term âcomplexâ refers to a grouping of two or more molecules. In some embodiments, the complex comprises a polypeptide and a nucleic acid molecule interacting with (e.g., binding to, coming into contact with, adhering to) one another. In some embodiments, the term âcomplexâ is used to refer to association of a CRISPR nuclease (e.g., a Type V nuclease such as a Cas12i polypeptide) and a reverse transcriptase polypeptide. For example, a complex of a CRISPR nuclease (e.g., a Cas12i2 polypeptide as disclosed herein) and a reverse transcriptase polypeptide may 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 âcomplexâ is used to refer to association of an RNA guide and an RT donor RNA. In some embodiments, the term âcomplexâ is used to refer to association of a CRISPR nuclease (e.g., a Type V nuclease such as a Cas12i polypeptide), a reverse transcriptase polypeptide, an RNA guide, and an RT donor RNA. In some embodiments, the term âcomplexâ is used to refer to association of a reverse transcriptase polypeptide and an RT donor RNA.

As used herein, the term âbinding site recognizable by a nucleaseâ or ânuclease binding sequenceâ refers to a sequence that is capable of binding to a 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, a nuclease binding sequence is capable of binding to a Type II CRISPR nuclease or a Type V CRISPR nuclease (e.g., binding site recognizable by a Type II CRISPR nuclease, or binding site recognizable by a Typ

CLAIMS

Claims ( 88 )

1 . A gene editing system comprising:

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

2 . The gene editing system of claim 1 , wherein the Type V CRISPR nuclease polypeptide is a Cas12 polypeptide.

3 . The gene editing system of claim 2 , wherein the Cas12 polypeptide is a Cas12i polypeptide, which optionally is a Cas12i2 polypeptide.

4 . The gene editing system of claim 3 , wherein the Cas12i polypeptide is a Cas12i2 polypeptide, which comprises an amino acid sequence at least 95% identical to SEQ ID NO: 2.

5 . The gene editing system of claim 4 , wherein 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.

6 . The gene editing system of claim 5 , wherein the one or more mutations are amino acid substitutions, which optionally is D581R, G624R, F626R, P868T, I926R, V1030G, E1035R, S1046G, or a combination thereof.

7 . The gene editing system of claim 5 , wherein the Cas12i2 polypeptide comprises:

(i) mutations at positions D581, D911, I926, and V1030, which optionally are amino acid substitutions of D581R, D911R, I926R, and V1030G; (ii) mutations at positions D581, I926, and V1030, which optionally are amino acid substitutions of D581R, I926R, and V1030G; (iii) mutations at positions D581, I926, V1030, and S1046, which optionally are amino acid substitutions of D581R, I926R, V1030G, and S1046G; (iv) mutations at positions D581, G624, F626, I926, V1030, E1035, and S1046, which optionally are amino acid substitutions of D581R, G624R, F626R, I926R, V1030G, E1035R, and S1046G; or (v) mutations at positions D581, G624, F626, P868, I926, V1030, E1035, and S1046, which optionally are amino acid substitutions of D581R, G624R, F626R, P868T, I926R, V1030G, E1035R, and S1046G.

8 . The gene editing system of claim 7 , wherein the Cas12i2 polypeptide comprises the amino acid sequence of any one of SEQ ID NO: 3-7, optionally SEQ ID NO:4 or SEQ ID NO: 7.

9 . The gene editing system of claim 4 , wherein the Cas12i polypeptide has diminished crRNA processing activity, optionally wherein the Cas12i polypeptide comprises mutations at position H485 and/or position H486 of SEQ ID NO: 2.

10 . The gene editing system of claim 1 , wherein the system comprises the Type V CRISPR nuclease polypeptide.

11 . The gene editing system of claim 1 , wherein the system comprises the first nucleic acid encoding the Type V CRISPR nuclease polypeptide.

12 . The gene editing system of claim 11 , wherein the first nucleic acid is located in a first vector, which optionally is a first viral vector.

13 . The gene editing system of claim 11 , wherein the first nucleic acid is a first messenger RNA (mRNA).

14 . The gene editing system of claim 1 , wherein the RT polypeptide is Moloney Murine Leukemia Virus (MMLV)-RT, mouse mammary tumor virus (MMTV)-RT, Marathon-RT, or RTx-RT.

15 . The gene editing system of claim 1 , wherein the system comprises the RT polypeptide.

16 . The gene editing system of claim 1 , wherein the system comprises the second nucleic acid encoding the RT polypeptide.

17 . The gene editing system of claim 16 , wherein the second nucleic acid is located in a second vector, which optionally is a second viral vector.

18 . The gene editing system of claim 17 , wherein the second vector is the same as the first vector.

19 . The gene editing system of claim 16 , wherein the second nucleic acid is a second mRNA.

20 . The gene editing system of claim 17 , wherein the first mRNA and the second mRNA are located on a single RNA molecule.

21 . The gene editing system of claim 1 , wherein the gene editing system comprises a fusion polypeptide, which comprises the Type V CRISPR nuclease polypeptide and the RT polypeptide.

22 . The gene editing system of claim 1 , wherein the Type V CRISPR nuclease polypeptide and the RT polypeptide are separate polypeptides.

23 . The gene editing system of claim 1 , wherein the spacer sequence is 20-30-nucleotide in length, optionally 20-nucleotide in length.

24 . The gene editing system of claim 3 , wherein the PAM comprises the motif of 5â²-TTN-3â², which optionally is located 5â² to the target sequence.

25 . The gene editing system of claim 3 , wherein the one or more CRISPR nuclease binding sites are direct repeat sequence(s).

26 . The gene editing system of claim 25 , wherein each direct repeat sequence is 23-36-nucleotide in length, optionally 23-nucleotide in length.

27 . The gene editing system of claim 26 , wherein the direct repeat sequence is at least 90% identical to any one of SEQ ID NOs: 15-17 and 241-247, or a fragment thereof that is at least 23-nucleotide in length.

28 . The gene editing system of claim 27 , wherein the direct repeat sequence is any one of SEQ ID NOs: 15-17 and 241-247, or a fragment thereof that is at least 23-nucleotide in length; optionally wherein the direct repeat sequence is SEQ ID NO: 17.

29 . The gene editing system of claim 1 , wherein the system comprises the gRNA.

30 . The gene editing system of claim 1 , wherein the system comprises the third nucleic acid encoding the gRNA.

31 . The gene editing system of claim 30 , wherein the third nucleic acid is located in a third vector, which optionally is a viral vector.

32 . The gene editing system of claim 31 , wherein the third vector is the same as the first vector and/or the second vector.

33 . The gene editing system of claim 1 , wherein the PBS is 5-100-nucleotide in length, optionally 10-60-nucleotide in length, preferably 10-30-nucleotide in length.

34 . The gene editing system of claim 1 , wherein the PBS binds a PBS-targeting site that is adjacent to the complementary region of the target sequence, and wherein the PBS-targeting site is upstream to the complementary region of the target sequence.

35 . The gene editing system of claim 34 , wherein the PBS-targeting site is 3-10-nucleotide upstream to the complementary region of the target sequence.

36 . The gene editing system of claim 1 , wherein the PBS-targeting site overlaps with the complementary region of the target sequence.

37 . The gene editing system of claim 1 , wherein the PBS-targeting site is adjacent to or overlap with the target sequence.

38 . The gene editing system of claim 1 , wherein the template sequence is 5-100-nucleotide in length, optionally 30-50-nucleotide in length.

39 . The gene editing system of claim 1 , wherein the template sequence is homologous to the genomic site of interest and comprises one or more nucleotide variations relative to the genomic site of interest.

40 . The gene editing system of claim 39 , wherein at least one nucleotide variation is located within the target sequence.

41 . The gene editing system of claim 39 , wherein at least one nucleotide variation is located in the PAM.

42 . The gene editing system of claim 1 , wherein the system comprises the RT donor RNA.

43 . The gene editing system of claim 1 , wherein the system comprises the fourth nucleic acid encoding the RT donor RNA.

44 . The gene editing system of claim 43 , wherein the fourth nucleic acid is located in a fourth vector, which optionally is a fourth viral vector.

45 . The gene editing system of claim 44 , wherein the four vector is the same as the first vector, the second vector, and/or the third vector.

46 . The gene editing system of claim 1 , wherein the gRNA and the RT donor RNA are located on a single RNA molecule, which comprises the CRISPR nuclease binding site, the spacer sequence, the PBS, and the template sequence.

47 . The gene editing system of claim 46 , wherein the single RNA molecule further comprises a linker between the gRNA and the RT donor RNA.

48 . The gene editing system of claim 47 , wherein the linker comprises a hairpin.

49 . The gene editing system of claim 46 , wherein the single RNA molecule comprises, from 5â² to 3â²:

(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 template sequence, the PBS, the linker, the CRISPR nuclease binding site, and the spacer sequence.

50 . The gene editing system of claim 46 , wherein the single RNA molecule further comprises a 5â² end protection fragment, a 3â² end protection fragment, or both, each of the 5â² end protection fragment and the 3â² end protection fragment forming a secondary structure, which optionally is a hairpin, a pseudoknot, or a triplex structure.

51 . The gene editing system of claim 50 , wherein the 5â² end protection fragment and/or the 3â² end protection fragment is an exoribonuclease-resistant RNA (xrRNA), a transfer RNA (tRNA), or a truncated tRNA.

52 . The gene editing system of claim 50 , wherein the 5â² end protection fragment and/or the 3â² end protection fragment comprises one or more of the CRISPR nuclease binding site, and optionally one or more segments that are not homologous to any human sequence.

53 . The gene editing system of claim 1 , wherein the gRNA and the RT donor RNA are two separate RNA molecules.

54 . The gene editing system of claim 53 , wherein the gRNA, the RT donor RNA, or both further comprise a 5â² end protection fragment and/or a 3â² end protection fragment.

55 . The gene editing system of claim 54 , wherein the 5â² end protection fragment and/or the 3â² end protection fragment forms a secondary structure, which optionally is a hairpin, a pseudoknot, or a triplex structure, or wherein the 5â² end protection fragment and/or the 3â² end protection fragment is an exoribonuclease-resistant RNA (xrRNA), a transfer RNA (tRNA), or a truncated tRNA.

56 . The gene editing system of claim 54 , wherein the 5â² end protection fragment and/or the 3â² end protection fragment comprises one or more of the CRISPR nuclease binding site, and optionally one or more segments that are not homologous to any human sequence.

57 . The gene editing system of claim 1 , wherein the system comprises one or more lipid nanoparticles (LNPs), which encompass element (a), (b), (c), (d), or any combination thereof.

58 . The gene editing system of claim 1 , wherein the system comprises (i) one or more lipid nanoparticles (LNPs), which collectively encompass up to three elements of (a)-(d), and (ii) one or more vectors.

59 . The gene editing system of claim 58 , wherein the one or more vectors are one or more viral vectors, which optionally are adeno-associated viral (AAV) vectors.

60 . The gene editing system of claim 56 , wherein the system comprises the Type V CRISPR nuclease polypeptide, the RT polypeptide, the gRNA, and the RT donor RNA.

61 . The gene editing system of claim 60 , wherein the Type V CRISPR nuclease polypeptide and/or the RT polypeptide forms a complex with the gRNA and/or the RT donor RNA.

62 . A pharmaceutical composition comprising the system of claim 1 .

63 . A kit comprising the elements of (a)-(d) of the system set forth in claim 1 .

64 . A method for genetically editing a cell, the method comprising contacting a host cell the gene editing system of claim 1 or the pharmaceutical composition comprising the gene editing system to genetically edit the host cell.

65 . The method of claim 64 , wherein the host cell is cultured in vitro.

66 . The method of claim 65 , wherein the contacting step is performed by administering the gene editing system to a subject comprising the host cell.

67 . A population of genetically modified cells, which is produced by the gene editing system of claim 1 .

68 . The population of genetically modified cells of claim 67 , which comprises genetically modified cells not editable by the gene editing system.

69 . The population of genetically modified cells of claim 68 , wherein the genetically modified cells comprise one or more modifications in the PAM, in the target sequence, or in both.

70 . A gene editing RNA molecule, comprising:

(i) one or more binding sites recognizable by a Type V CRISPR nuclease (CRISPR nuclease binding sites); (ii) a spacer sequence specific to a target sequence within a genetic site, the target sequence being adjacent to a protospacer adjacent motif (PAM); (iii) a primer binding site (PBS); and (iv) a template sequence.

71 . The gene editing RNA molecule of claim 70 , which further comprises one or more linkers.

72 . The gene editing RNA molecule of claim 70 , wherein the RNA molecule comprises, from 5â² to 3â²:

(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 template sequence, the PBS, the linker, the CRISPR nuclease binding site, and the spacer sequence.

73 . The gene editing RNA molecule of claim 70 , which further comprises a 5â² end protection fragment, a 3â² end protection fragment, or both.

74 . The gene editing RNA molecule of claim 73 , wherein the 5â² end protection fragment and/or the 3â² end protection fragment forms a secondary structure, which optionally is a hairpin, a pseudoknot, or a triplex structure, or wherein the 5â² end protection fragment and/or the 3â² end protection fragment is an exoribonuclease-resistant RNA (xrRNA), a transfer RNA (tRNA), or a truncated tRNA.

75 . The gene editing RNA molecule of claim 73 , wherein the 5â² end protection fragment and/or the 3â² end protection fragment comprises one or more of the CRISPR nuclease binding site, and optionally one or more segments that are not homologous to any human sequence.

76 . A set of gene editing RNA molecules, comprising:

(i) a guide RNA comprising one or more binding sites recognizable by the Type V CRISPR nuclease (CRISPR nuclease binding sites) and a spacer sequence specific to a target sequence within a genetic 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, wherein the RT donor RNA comprises a primer binding site (PBS) and a template sequence.

77 . The set of gene editing RNA molecules of claim 76 , wherein the gRNA, the RT donor RNA, or both further comprise a 5â² end protection fragment and/or a 3â² end protection fragment.

78 . The set of gene editing RNA molecules of claim 77 , wherein the 5â² end protection fragment and/or the 3â² end protection fragment forms a secondary structure, which optionally is a hairpin, a pseudoknot, or a triplex structure, or wherein the 5â² end protection fragment and/or the 3â² end protection fragment is an exoribonuclease-resistant RNA (xrRNA), a transfer RNA (tRNA), or a truncated tRNA.

79 . The set of gene editing RNA molecules of claim 77 , wherein the 5â² end protection fragment and/or the 3â² end protection fragment comprises one or more of the CRISPR nuclease binding site, and optionally one or more segments that are not homologous to any human sequence.

80 . The gene editing RNA molecule of claim 70 wherein:

(i) the Type V CRISPR nuclease binding site comprises one or more direct repeat sequences;

(ii) the spacer sequence is 20-30-nucleotide in length;

(iii) the PBS is 5-100-nucleotide in length; and/or

(iv) the template sequence is 5-100-nucleotide in length.

81 . A DNA molecule or a set of DNA molecules, which encode the gene editing RNA molecule or the set of gene editing RNA molecules set forth in claim 1 .

82 . The DNA molecule or the set of DNA molecules of claim 81 , which is included in a vector or a set of vectors, optionally wherein the vector or set of vectors are viral vectors.

83 . A fusion polypeptide comprising a CRISPR nuclease and a reverse transcriptase.

84 . The fusion polypeptide of claim 83 , wherein the CRISPR nuclease is a Type V CRISPR nuclease, which optionally is a Cas12i polypeptide.

85 . The fusion polypeptide of claim 84 , wherein the Cas12i polypeptide is a Cas12i2 polypeptide, which optionally is set forth in claim 1 .

86 . The fusion polypeptide of claim 85 , which comprises the amino acid sequence of any one of SEQ ID NOs: 25-26 and 219-223.

87 . A nucleic acid comprising a nucleotide sequence encoding a fusion polypeptide of claim 1 .

88 . The nucleic acid of claim 87 , which is a vector, optionally an expression vector.

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