Gene editing system including an RNA guide targeting stathmin 2 (STMN2) and uses thereof

Abstract

A system for gene editing of the Stathmin 2 (STMN2) gene, comprising: (i) a Cas12i2 polypeptide or a first nucleic acid encoding the Cas12i2 polypeptide; and (ii) an RNA guide or a second nucleic acid encoding the RNA guide, wherein the RNA guide comprises a spacer sequence specific for a target sequence in the STMN2 gene. Also provided herein are methods for editing the STMN2 gene and / or for treating a disease associated with the STMN2 gene using the gene editing system disclosed herein.

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 / 231,784, filed August 11, 2021, and U.S. Provisional Application No. 63 / 322,002, filed March 21, 2022, the contents of each of which are incorporated by reference herein in their entirety. [Background technology]

[0002] 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

[0003] The present disclosure is based, at least in part, on the development of a system for gene editing of stathmin 2 (STMN2) gene. The system includes a Cas12i polypeptide, such as a Cas12i2 polypeptide, and an RNA guide that mediates the cleavage at a gene site in the STMN2 gene by a CRISPR nuclease polypeptide. As reported herein, the gene editing system disclosed herein successfully edits the STMN2 gene with high editing efficiency and precision.

[0004] Without being bound by theory, the gene editing system disclosed herein may exhibit one or more of the following advantageous features: Compared to SpCas9 and Cas12a, Cas12i effectors are smaller (1033-1093aa), which, together with their short mature crRNA (40-43nt), is favorable in terms of delivery and synthesis costs. Cas12i cleavage results in larger deletions compared to the small deletions and +1 insertions induced by Cas9 cleavage. Also, the Cas12i PAM sequence is different from that of Cas9. Thus, compared to Cas9, a larger and different portion of the gene site of interest can be disrupted with the Cas12i polypeptide and RNA guide. Using the unbiased approach of tagmentation-based tag insertion site sequencing (TTISS), more potential off-target sites with a higher number of unique insertion events were identified for SpCas9 compared to Cas12i2. See WO / 2021 / 202800. Thus, Cas12i, e.g., Cas12i2, can be more specific than Cas9.

[0005] Thus, provided herein is a gene editing system for editing the STMN2 gene, a pharmaceutical composition or kit comprising such a gene editing system, a method of using the gene editing system to produce a genetically modified cell, and the resulting cell thus produced.Also provided herein is the use of the gene editing system disclosed herein, the pharmaceutical composition and kit comprising such a gene editing system, and / or the genetically modified cell thus produced for treating a neurodegenerative disease in a subject (e.g., amyotrophic lateral sclerosis (ALS) or frontotemporal dementia (FTD)).

[0006] In some embodiments, the disclosure features a system for gene editing of the Stathmin 2 (STMN2) gene, the system including (i) a Cas12i polypeptide or a first nucleic acid encoding a Cas12i polypeptide, and (ii) an RNA guide or a second nucleic acid encoding an RNA guide, the RNA guide including a spacer sequence specific to a target sequence in the STMN2 gene, the target sequence being adjacent to a protospacer adjacent motif (PAM), the PAM including a 5'-TTN-3' motif, the 5'-TTN-3' motif being located 5' to the target sequence.

[0007] In some embodiments, the Cas12i is a Cas12i2 polypeptide. In other embodiments, the Cas12i is a Cas12i4 polypeptide.

[0008] In some embodiments, the Cas12i polypeptide is a Cas12i2 polypeptide comprising an amino acid sequence at least 95% identical to SEQ ID NO: 448. In some cases, the Cas12i2 polypeptide may comprise one or more mutations relative to SEQ ID NO: 448. In some examples, the one or more mutations in the Cas12i2 polypeptide are at positions D581, G624, F626, P868, I926, V1030, E1035, and / or S1046 of SEQ ID NO: 448. In some examples, the one or more mutations are amino acid substitutions, optionally, D581R, G624R, F626R, P868T, I926R, V1030G, E1035R, S1046G, or a combination thereof.

[0009] 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).

[0010] An exemplary Cas12i2 polypeptide for use in any of the gene editing systems disclosed herein may comprise the amino acid sequence of any one of SEQ ID NOs: 449-453. In one example, an exemplary Cas12i2 polypeptide for use in any of the gene editing systems disclosed herein comprises the amino acid sequence of SEQ ID NO: 450. In another example, an exemplary Cas12i2 polypeptide for use in any of the gene editing systems disclosed herein comprises the amino acid sequence of SEQ ID NO: 453.

[0011] In some embodiments, the gene editing system may include a first nucleic acid encoding a Cas12i polypeptide (e.g., a Cas12i2 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 some cases, the first nucleic acid is a messenger RNA (mRNA). In some cases, the coding sequence of the Cas12i polypeptide is codon-optimized.

[0012] In some embodiments, the target sequence can be within exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, or an intron of the STMN2 gene.

[0013] In some embodiments, the RNA guide comprises the sequence of any one of SEQ ID NOs: 4508, 4512, 4559, and 4561, or the second nucleic acid encodes an RNA guide comprising any one of SEQ ID NOs: 4508, 4512, 4559, and 4561.

[0014] In some embodiments, the RNA guide comprises the sequence of any one of SEQ ID NOs: 4505, 4506, 4507, 4508, 4509, 4510, 4511, 4512, 4513, 4514, 4515, 4554, 4555, 4556, 4557, 4558, 4559, 4560, 4561, and 4562, or the second nucleic acid encodes an RNA guide comprising any one of SEQ ID NOs: 4505, 4506, 4507, 4508, 4509, 4510, 4511, 4512, 4513, 4514, 4515, 4554, 4555, 4556, 4557, 4558, 4559, 4560, 4561, and 4562.

[0015] In some embodiments, the spacer sequence may be 20 to 30 nucleotides in length. In some examples, the spacer sequence is 20 nucleotides in length.

[0016] In some embodiments, the RNA guide comprises a spacer and a direct repeat sequence. In some examples, the direct repeat sequence is 23-36 nucleotides in length. In one example, the direct repeat sequence is at least 90% identical to any one of SEQ ID NOs: 1-10, or a fragment thereof that is at least 23 nucleotides in length. In some specific examples, the direct repeat sequence is any one of SEQ ID NOs: 1-10, or a fragment thereof that is at least 23 nucleotides in length. As a non-limiting example, the direct repeat sequence is 5'-AGAAAUCCGUCUUUCAUUGACGG-3' (SEQ ID NO: 10).

[0017] In some embodiments, the system may include a second nucleic acid encoding an RNA guide. In some examples, the nucleic acid encoding the RNA guide may be located in a viral vector. In some examples, the viral vector includes both a first nucleic acid encoding a Cas12i polypeptide (e.g., a Cas12i2 polypeptide) and a second nucleic acid encoding an RNA guide.

[0018] In some embodiments, any of the systems described herein may include a first nucleic acid encoding a Cas12i polypeptide (e.g., a Cas12i2 polypeptide) located in a first vector and a second nucleic acid encoding an RNA guide located on a second vector. In some examples, the first vector and / or the second vector are viral vectors. In some specific examples, the first vector and the second vector are the same vector.

[0019] In some embodiments, any of the systems described herein may include one or more lipid nanoparticles (LNPs) that include a Cas12i polypeptide (e.g., a Cas12i2 polypeptide) or a first nucleic acid encoding a Cas12i polypeptide, an RNA guide or a second nucleic acid encoding an RNA guide, or both.

[0020] In some embodiments, the systems described herein may include a LNP that includes a Cas12i polypeptide (e.g., a Cas12i2 polypeptide) or a viral vector that includes a first nucleic acid encoding a Cas12i polypeptide and a second nucleic acid encoding an RNA guide. In some examples, the viral vector is an AAV vector. In other embodiments, the systems described herein may include a LNP that includes an RNA guide or a second nucleic acid encoding an RNA guide and a viral vector that includes a first nucleic acid encoding a Cas12i polypeptide. In some examples, the viral vector is an AAV vector.

[0021] In some embodiments, the present disclosure also provides pharmaceutical compositions comprising any of the gene editing systems disclosed herein, and kits comprising components of the gene editing systems.

[0022] In another aspect, the present disclosure also features a method for editing the stathmin 2 (STMN2) gene in a cell, the method comprising contacting the host cell with any of the systems disclosed herein to genetically edit the STMN2 gene in the host cell. In some examples, the host cell is cultured in vitro. In other examples, the contacting step is carried out by administering the system for editing the STMN2 gene to a subject comprising the host cell.

[0023] Also within the scope of the present disclosure are cells that contain a disrupted stathmin 2 (STMN2) gene, which can be produced by contacting a host cell with a system disclosed herein to genetically edit the STMN2 gene in the host cell.

[0024] Additionally, in other aspects, the present disclosure provides methods for treating a neurodegenerative disease in a subject (e.g., amyotrophic lateral sclerosis (ALS) or frontotemporal dementia (FTD)). The method may include administering to a subject in need thereof any of the systems for editing the stathmin 2 (STMN2) gene disclosed herein, or any of the cells disclosed herein.

[0025] Also provided herein is an RNA guide comprising: (i) a spacer sequence as disclosed herein that is specific for a target sequence in the stathmin 2 (STMN2) gene, wherein the target sequence is adjacent to a protospacer adjacent motif (PAM), the PAM comprising a 5'-TTN-3' motif, and the 5'-TTN-3' motif is located 5' to the target sequence; and (ii) a direct repeat sequence.

[0026] In some embodiments, the spacer can be 20 to 30 nucleotides in length. In some examples, the spacer is 20 nucleotides in length.

[0027] In some embodiments, the direct repeat sequence may be 23 to 36 nucleotides in length. In some examples, the direct repeat sequence is 23 nucleotides in length.

[0028] In some embodiments, the target sequence is within exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, or an intron of the STMN2 gene.

[0029] In some embodiments, the RNA guide comprises the sequence of any one of SEQ ID NOs: 4508, 4512, 4559, and 4561, or the second nucleic acid encodes an RNA guide comprising any one of SEQ ID NOs: 4508, 4512, 4559, and 4561.

[0030] In some embodiments, the RNA guide comprises the sequence of any one of SEQ ID NOs: 4505, 4506, 4507, 4508, 4509, 4510, 4511, 4512, 4513, 4514, 4515, 4554, 4555, 4556, 4557, 4558, 4559, 4560, 4561, and 4562, or the second nucleic acid encodes an RNA guide comprising any one of SEQ ID NOs: 4505, 4506, 4507, 4508, 4509, 4510, 4511, 4512, 4513, 4514, 4515, 4554, 4555, 4556, 4557, 4558, 4559, 4560, 4561, and 4562.

[0031] In some embodiments, the direct repeat sequence may be at least 90% identical to any one of SEQ ID NOs: 1-10, or a fragment thereof that is at least 23 nucleotides in length. In some examples, the direct repeat sequence is any one of SEQ ID NOs: 1-10, or a fragment thereof that is at least 23 nucleotides in length. As a non-limiting example, the direct repeat sequence is 5'-AGAAAUCCGUCUUUCAUUGACGG-3' (SEQ ID NO: 10).

[0032] Also provided herein are any of the gene editing systems disclosed herein, pharmaceutical compositions or kits comprising such gene editing systems, or genetically modified cells generated by the gene editing systems, for use in treating a neurodegenerative disease in a subject (e.g., amyotrophic lateral sclerosis (ALS) or frontotemporal dementia (FTD)), and use of the gene editing systems disclosed herein, pharmaceutical compositions or kits comprising such gene editing systems, or genetically modified cells generated by the gene editing systems, for the manufacture of a medicament for the treatment of a neurodegenerative disease in a subject (e.g., amyotrophic lateral sclerosis (ALS) or frontotemporal dementia (FTD)).

[0033] The details of one or more embodiments of the invention are set forth in the description below. Other features or advantages of the invention will become apparent from the following drawings and detailed description of several embodiments, and from the appended claims. [Brief description of the drawings]

[0034] 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.

[0035] [Figure 1] 1 shows the percentage editing of STMN2 intron target sequence with the indicated guides, as described in Example 1. [Figure 2A] Showing the disruption of more than 15% of cryptic splice sites in STMN2 intron 1 by guides 4, 8, 55, and 57. [Figure 2B] 1 shows greater than 15% disruption of at least one of the three TDP-43 binding motifs in STMN2 intron 1 by guides 12, 46, 47, 48, and 49. [Figure 2C] Showing greater than 15% disruption of the premature polyadenylation signal in STMN2 intron 1 by guides 17 and 18. [Diagram 3] FIG. 1 is a schematic showing where each of the indicated RNA guides binds in intron 1 of STMN2, relative to the locations of cryptic splice sites, TDP-43 binding motifs, and early polyadenylation signals. [Figure 4] 1 shows the indel activity of tested RNA guides in SH-SY5Y cells. [Figure 5A] 1 is a plot comparing the indel activity (% indels) demonstrated in HEK293T cells and SH-SY5Y cells from Example 1 and Example 2, respectively. [Figure 5B]1 is a plot comparing the disruption of splice site motifs demonstrated in HEK293T and SH-SY5Y cells from Examples 1 and 2, respectively. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0036] The present disclosure relates to a system for gene editing of the Stathmin 2 (STMN2) gene, comprising: (i) a Cas12i polypeptide or a first nucleic acid encoding a Cas12i2 polypeptide; and (ii) an RNA guide or a second nucleic acid encoding an RNA guide, wherein the RNA guide comprises a spacer sequence specific to a target sequence in the STMN2 gene, the target sequence is adjacent to a protospacer adjacent motif (PAM), the PAM comprises a 5'-TTN-3' motif, and the 5'-TTN-3' motif is located 5' to the target sequence. Also provided in the present disclosure are pharmaceutical compositions or kits comprising such systems, and uses thereof. Further disclosed herein are methods for editing the STMN2 gene in a cell, cells so produced containing a disrupted STMN2 gene, methods for treating a degenerative disease in a subject, and RNA guides comprising (i) a spacer sequence specific for a target sequence in the STMN2 gene, wherein the target sequence is adjacent to a protospacer adjacent motif (PAM), the PAM comprising a 5'-TTN-3' motif, and the 5'-TTN-3' motif is located 5' to the target sequence, and (ii) a direct repeat sequence, and uses thereof.

[0037] A Cas12i polypeptide for use in the gene editing system disclosed herein can be a Cas12i2 polypeptide, such as a wild-type Cas12i polypeptide or a variant thereof, such as those disclosed herein. In some examples, the Cas12i2 polypeptide comprises an amino acid sequence at least 95% identical to SEQ ID NO: 448, and comprises one or more mutations relative to SEQ ID NO: 448. In other examples, the Cas12i polypeptide can be a Cas12i4 polypeptide, which is also disclosed herein.

[0038] 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.

[0039] As used herein, the term "activity" refers to biological activity. In some embodiments, the activity includes enzymatic activity, such as the catalytic ability of the Cas12i polypeptide. For example, the activity can include nuclease activity.

[0040] As used herein, the term "STMN2" refers to "stathmin-2". STMN2 is a neuron-specific member of the stathmin family of proteins and plays a role in regulating microtubule stability and signal transduction. SEQ ID NO: 454 described herein provides an example of an STMN2 gene sequence. This sequence is also referenced in Gene ID: 11075 (www.ncbi.nlm.nih.gov / gene / 11075).

[0041] 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: 4503, 448, 4504, and 482 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).

[0042] 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.

[0043] 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). For example, the term "complex" can refer to a grouping of an RNA guide and a polypeptide (e.g., a Cas12i polypeptide). Alternatively, the term "complex" can refer to a grouping of an RNA guide, a polypeptide, and a complementary region of a target sequence. As used herein, the term "complex" can refer to a grouping of an STMN2-targeted RNA guide and a Cas12i polypeptide.

[0044] As used herein, the term "protospacer adjacent motif" or "PAM" refers to a DNA sequence adjacent to a target sequence (e.g., an STMN2 target sequence). 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 a target sequence disclosed herein.

[0045] In some embodiments, the PAM strand is a coding (e.g., sense) strand. In other embodiments, the PAM strand is a non-coding (e.g., antisense) strand. Because the RNA guide binds to the non-PAM strand via 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.

[0046] 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). In some embodiments, the target sequence is a sequence within the STMN2 gene sequence, including but not limited to the sequence set forth in SEQ ID NO: 454.

[0047] As used herein, the term "spacer" or "spacer sequence" refers to a portion within an RNA guide that is the RNA equivalent of a target sequence (DNA sequence). A spacer contains a sequence that is capable of binding to a 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.

[0048] As used herein, the term "RNA guide" or "RNA guide sequence" refers to any RNA molecule or modified RNA molecule that facilitates targeting of a polypeptide described herein (e.g., a Cas12i polypeptide) to a target sequence (e.g., a sequence of the STMN2 gene). For example, an RNA guide can be a molecule designed to be complementary to a specific nucleic acid sequence (such as a target sequence in the STMN2 gene). An RNA guide can include a spacer sequence and a direct repeat (DR) sequence. In some cases, an 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, that binds to a sequence complementary to the target sequence. In some examples, the DNA binding sequence can contain a DNA sequence or a DNA / RNA hybrid sequence. The terms CRISPR RNA (crRNA), pre-crRNA, and mature crRNA are also used herein to refer to an RNA guide.

[0049] 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 allow an effector polypeptide complexed 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., having 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, ie, has 100% complementarity with the second polynucleotide.

[0050] 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 present disclosure. 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 present disclosure. 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.

[0051] As used herein, the term "editing" refers to one or more modifications being introduced into a target nucleic acid, for example, a target nucleic acid in the STMN2 gene. The editing can be one or more substitutions, one or more insertions, one or more deletions, or a combination thereof. 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. As used herein, the term "insertion" refers to the gain of one or more nucleotides in a nucleic acid sequence relative to a reference sequence. 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.

[0052] No particular process is suggested for how to make 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 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. The deletion can be a frameshift mutation or a non-frameshift mutation. The deletion described herein refers to a deletion of up to several kilobases.

[0053] As used herein, the terms "upstream" and "downstream" refer to relative positions within a single nucleic acid (e.g., DNA) sequence in a nucleic acid molecule. "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, a 5'-NTTN-3' or 5'-TTN-3' sequence is upstream of the indel described herein, and a Cas12i-induced indel is downstream of the 5'-NTTN-3' or 5'-TTN-3' sequence.

[0054] I. Gene editing systems In some embodiments, the present disclosure provides a gene editing system comprising the RNA guide that targets STMN2 gene.Such gene editing system can be used to edit STMN2 target gene, for example, can be used to disrupt STMN2 gene.

[0055] As used herein, the term "STMN2" refers to "stathmin-2". STMN2 is a neuron-specific member of the stathmin family of proteins and plays a role in regulating microtubule stability and signal transduction. SEQ ID NO: 454 described herein provides an example of an STMN2 gene sequence. This sequence is also referenced in Gene ID: 11075 (www.ncbi.nlm.nih.gov / gene / 11075).

[0056] In some embodiments, the RNA guide is composed of a direct repeat component and a spacer sequence. In some embodiments, the RNA guide is bound to a Cas12i polypeptide. In some embodiments, the spacer sequence is specific for the STMN2 target sequence, and the STMN2 target sequence is adjacent to a 5'-NTTN-3' or 5'-TTN-3' PAM sequence described herein. In the case of a double-stranded target, the RNA guide binds to the first strand of the target (i.e., the non-PAM strand) and the PAM sequence described herein is present in the second complementary strand (i.e., the PAM strand).

[0057] In some embodiments, the disclosure provides a composition comprising a complex, the complex comprising an RNA guide targeting STMN2. In some embodiments, the disclosure comprises a complex comprising an RNA guide and a Cas12i polypeptide. In some embodiments, the RNA guide and the Cas12i polypeptide bind to each other in a molar ratio of about 1:1. In some embodiments, the complex comprising an RNA guide and a Cas12i polypeptide binds to a complementary region of a target sequence in the STMN2 gene. In some embodiments, the complex comprising an RNA guide targeting STMN2 and a Cas12i polypeptide binds to a complementary region of a target sequence in the STMN2 gene in a molar ratio of about 1:1. In some embodiments, the complex comprises an enzymatic activity, e.g., a nuclease activity, capable of cleaving the STMN2 target sequence and / or the complementary sequence. The RNA guide, the Cas12i polypeptide, and the complementary region of the STMN2 target sequence do not naturally occur either alone or together. In some embodiments, the RNA guide in the complex comprises a direct repeat and / or a spacer sequence as described herein.

[0058] In some embodiments, the disclosure includes compositions comprising an RNA guide as described herein and / or an RNA encoding a Cas12i polypeptide as described herein. In some embodiments, the RNA guide and the RNA encoding a Cas12i polypeptide are included together in the same composition. In some embodiments, the RNA guide and the RNA encoding a Cas12i polypeptide are included in separate compositions. In some embodiments, the RNA guide comprises a direct repeat and / or a spacer sequence as described herein.

[0059] The use of the gene editing system disclosed herein has advantages over other known nuclease systems. Cas12i polypeptide is smaller than other nucleases. 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. Cas12i RNA guides, which do not require transactivating CRISPR RNA (tracrRNA), are also smaller than Cas9 RNA guides. Smaller Cas12i polypeptide and RNA guide sizes are beneficial for delivery. Compositions comprising Cas12i polypeptides also demonstrate reduced off-target activity compared to compositions comprising SpCas9 polypeptides. Please refer to WO / 2021 / 202800, the relevant disclosure of which is incorporated by reference for the subject matter and purposes referred to herein. Furthermore, the indels induced by compositions comprising Cas12i polypeptides are different from the indels induced by compositions comprising SpCas9 polypeptides. For example, SpCas9 polypeptides mainly induce insertions and deletions of 1 nucleotide length. However, Cas12i polypeptides induce larger deletions, which can be beneficial in disrupting larger portions of genes such as STMN2.

[0060] Also provided is a system for gene editing of the STMN2 gene, comprising: (i) a Cas12i polypeptide (e.g., a Cas12i2 polypeptide) or a first nucleic acid encoding a Cas12i polypeptide (e.g., a Cas12i2 polypeptide comprising an amino acid sequence at least 95% identical to SEQ ID NO: 448, which may include one or more mutations relative to SEQ ID NO: 448); and (ii) an RNA guide or a second nucleic acid encoding an RNA guide, wherein the RNA guide is located within the STMN2 gene (e.g., ST and an RNA guide or second nucleic acid comprising a spacer sequence specific for a target sequence in exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, or within an intron of the MN2 gene, wherein the target sequence is adjacent to a protospacer adjacent motif (PAM), wherein the PAM comprises a 5'-TTN-3' (5'-NTTN-3') motif, wherein the 5'-TTN-3' (5'-NTTN-3') motif is located 5' to the target sequence.

[0061] A. RNA guide In some embodiments, the gene editing systems described herein include RNA guides that target the STMN2 gene, e.g., target exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, or an intron of the STMN2 gene. In some embodiments, the gene editing systems described herein can include two or more (e.g., 2, 3, 4, 5, 6, 7, 8, or more) RNA guides that target STMN2.

[0062] The RNA guide can direct the Cas12i polypeptide contained in the gene editing system described herein to the STMN2 target sequence. Two or more RNA guides can direct two or more separate Cas12i polypeptides described herein (e.g., Cas12i polypeptides having the same or different sequences) to two or more (e.g., 2, 3, 4, 5, 6, 7, 8, or 9 or more) STMN2 target sequences. Those skilled in the art reading the following examples of specific types of RNA guides will understand that in some embodiments, the RNA guide is STMN2 target specific. That is, in some embodiments, the RNA guide specifically binds to one or more STMN2 target sequences (e.g., in a cell) and does not bind to non-target sequences (e.g., non-specific DNA or random sequences in the same cell).

[0063] In some embodiments, the RNA guide comprises a spacer sequence followed by a direct repeat sequence, which refers to the sequence in the 5' to 3' direction. In some embodiments, the RNA guide comprises a first direct repeat sequence followed by a spacer sequence and a second direct repeat sequence, which refers to the sequence in the 5' to 3' direction. In some embodiments, the first direct repeat and the second direct repeat of such an RNA guide are identical. In some embodiments, the first direct repeat and the second direct repeat of such an RNA guide are different.

[0064] In some embodiments, the spacer sequence and the direct repeat sequence of the RNA guide are present in the same RNA molecule. In some embodiments, the spacer and the direct repeat sequence are directly linked to each other. In some embodiments, a short linker is present between the spacer and the direct repeat sequence, for example, a 1, 2, or 3 nucleotide long RNA linker. In some embodiments, the spacer sequence and the direct repeat sequence of the RNA guide are present in separate molecules, and the separate molecules are linked to each other by base pairing interactions.

[0065] Additional information regarding exemplary direct repeat and spacer components of RNA guides is provided below.

[0066] (i) Direct Repeat In some embodiments, the RNA guide comprises a direct repeat sequence. In some embodiments, the direct repeat sequence of the RNA guide has a length of 12-100, 13-75, 14-50, or 15-40 nucleotides (e.g., 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 nucleotides).

[0067] In some embodiments, the direct repeat sequence is a sequence in Table 1 or a portion of a sequence in Table 1. The direct repeat sequence can include nucleotides 1 to 36 of any one of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, or 8. The direct repeat sequence can include nucleotides 2 to 36 of any one of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, or 8. The direct repeat sequence can include nucleotides 3 to 36 of any one of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, or 8. The direct repeat sequence can include nucleotides 4 to 36 of any one of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, or 8. The direct repeat sequence can include nucleotides 5 to 36 of any one of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, or 8. The direct repeat sequence can include nucleotides 6 to 36 of any one of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, or 8. The direct repeat sequence may comprise nucleotides 7 to 36 of any one of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, or 8. The direct repeat sequence may comprise nucleotides 8 to 36 of any one of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, or 8. The direct repeat sequence may comprise nucleotides 9 to 36 of any one of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, or 8. The direct repeat sequence may comprise nucleotides 10 to 36 of any one of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, or 8. The direct repeat sequence may comprise nucleotides 11 to 36 of any one of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, or 8. The direct repeat sequence may comprise nucleotides 12 to 36 of any one of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, or 8. The direct repeat sequence can include nucleotides 13 to 36 of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, or 8.The direct repeat sequence may comprise nucleotides 14 to 36 of any one of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, or 8. The direct repeat sequence may comprise nucleotides 1 to 34 of SEQ ID NO: 9. The direct repeat sequence may comprise nucleotides 2 to 34 of SEQ ID NO: 9. The direct repeat sequence may comprise nucleotides 3 to 34 of SEQ ID NO: 9. The direct repeat sequence may comprise nucleotides 4 to 34 of SEQ ID NO: 9. The direct repeat sequence may comprise nucleotides 5 to 34 of SEQ ID NO: 9. The direct repeat sequence may comprise nucleotides 6 to 34 of SEQ ID NO: 9. The direct repeat sequence may comprise nucleotides 7 to 34 of SEQ ID NO: 9. The direct repeat sequence may comprise nucleotides 8 to 34 of SEQ ID NO: 9. The direct repeat sequence may comprise nucleotides 9 to 34 of SEQ ID NO: 9. The direct repeat sequence may comprise nucleotides 10 to 34 of SEQ ID NO: 9. The direct repeat sequence can comprise nucleotides 11 to 34 of SEQ ID NO: 9. The direct repeat sequence can comprise nucleotides 12 to 34 of SEQ ID NO: 9. In some embodiments, the direct repeat sequence is set forth in SEQ ID NO: 10. In some embodiments, the direct repeat sequence comprises a portion of the sequence set forth in SEQ ID NO: 10.

[0068] In some embodiments, the direct repeat sequence has at least 90% identity (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) to a sequence in Table 1 or a portion of a sequence in Table 1. The direct repeat sequence can have at least 90% identity to a sequence that includes nucleotides 1 to 36 of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, or 8. The direct repeat sequence can have at least 90% identity to a sequence that includes nucleotides 2 to 36 of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, or 8. The direct repeat sequence can have at least 90% identity to a sequence that includes nucleotides 3 to 36 of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, or 8. The direct repeat sequence may have at least 90% identity to a sequence comprising 4 to 36 nucleotides of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, or 8. The direct repeat sequence may have at least 90% identity to a sequence comprising 5 to 36 nucleotides of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, or 8. The direct repeat sequence may have at least 90% identity to a sequence comprising 6 to 36 nucleotides of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, or 8. The direct repeat sequence may have at least 90% identity to a sequence comprising 7 to 36 nucleotides of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, or 8. The direct repeat sequence may have at least 90% identity to a sequence comprising 8 to 36 nucleotides of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, or 8. The direct repeat sequence can have at least 90% identity to a sequence comprising 9 to 36 nucleotides of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, or 8. The direct repeat sequence can have at least 90% identity to a sequence comprising 10 to 36 nucleotides of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, or 8.The direct repeat sequence may have at least 90% identity to a sequence comprising 11 to 36 nucleotides of any one of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, or 8. The direct repeat sequence may have at least 90% identity to a sequence comprising 12 to 36 nucleotides of any one of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, or 8. The direct repeat sequence may have at least 90% identity to a sequence comprising 13 to 36 nucleotides of any one of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, or 8. The direct repeat sequence may have at least 90% identity to a sequence comprising 14 to 36 nucleotides of any one of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, or 8. The direct repeat sequence may have at least 90% identity to a sequence comprising 14 to 36 nucleotides of any one of SEQ ID NO: 9. The direct repeat sequence may have at least 90% identity to a sequence comprising nucleotides 2 to 34 of SEQ ID NO:9. The direct repeat sequence may have at least 90% identity to a sequence comprising nucleotides 3 to 34 of SEQ ID NO:9. The direct repeat sequence may have at least 90% identity to a sequence comprising nucleotides 4 to 34 of SEQ ID NO:9. The direct repeat sequence may have at least 90% identity to a sequence comprising nucleotides 5 to 34 of SEQ ID NO:9. The direct repeat sequence may have at least 90% identity to a sequence comprising nucleotides 6 to 34 of SEQ ID NO:9. The direct repeat sequence may have at least 90% identity to a sequence comprising nucleotides 7 to 34 of SEQ ID NO:9. The direct repeat sequence may have at least 90% identity to a sequence comprising nucleotides 8 to 34 of SEQ ID NO:9. The direct repeat sequence may have at least 90% identity to a sequence comprising nucleotides 9 to 34 of SEQ ID NO:9. The direct repeat sequence can have at least 90% identity to a sequence comprising nucleotides 10 to 34 of SEQ ID NO:9. The direct repeat sequence can have at least 90% identity to a sequence comprising nucleotides 11 to 34 of SEQ ID NO:9.The direct repeat sequence can have at least 90% identity to a sequence comprising nucleotides 12 to 34 of SEQ ID NO: 9. In some embodiments, the direct repeat sequence has at least 90% identity (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) to SEQ ID NO: 10. In some embodiments, the direct repeat sequence has at least 90% identity to a portion of the sequence set forth in SEQ ID NO: 10.

[0069] In some embodiments, a composition comprising a Cas12i2 polypeptide and an RNA guide comprising a direct repeat of SEQ ID NO: 10 and a spacer length of 20 nucleotides is capable of introducing an indel into an STMN2 target sequence.

[0070] In some embodiments, the direct repeat sequence is at least 90% identical to the reverse complement of any one of SEQ ID NOs: 1-10 (see Table 1). In some embodiments, the direct repeat sequence is the reverse complement of any one of SEQ ID NOs: 1-10.

[0071] [Table 1]

[0072] In some embodiments, the direct repeat sequence is a sequence in Table 2 or a portion of a sequence in Table 2. The direct repeat sequence can include nucleotides 1 to 36 of any one of SEQ ID NOs: 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, or 479. The direct repeat sequence can include nucleotides 2 to 36 of any one of SEQ ID NOs: 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, or 479. The direct repeat sequence can include nucleotides 3 to 36 of any one of SEQ ID NOs: 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, or 479. The direct repeat sequence can include nucleotides 4 to 36 of any one of SEQ ID NOs: 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, or 479. The direct repeat sequence can include nucleotides 5 to 36 of any one of SEQ ID NOs: 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, or 479. The direct repeat sequence can include nucleotides 6 to 36 of any one of SEQ ID NOs: 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, or 479. The direct repeat sequence can include nucleotides 7 to 36 of any one of SEQ ID NOs: 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, or 479.The direct repeat sequence can include nucleotides 8 to 36 of any one of SEQ ID NOs: 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, or 479. The direct repeat sequence can include nucleotides 9 to 36 of any one of SEQ ID NOs: 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, or 479. The direct repeat sequence can include nucleotides 10 to 36 of any one of SEQ ID NOs: 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, or 479. The direct repeat sequence can include nucleotides 11 to 36 of any one of SEQ ID NOs: 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, or 479. The direct repeat sequence can include nucleotides 12 to 36 of any one of SEQ ID NOs: 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, or 479. The direct repeat sequence can include nucleotides 13 to 36 of any one of SEQ ID NOs: 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, or 479. The direct repeat sequence can include nucleotides 14 to 36 of any one of SEQ ID NOs: 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, or 479.

[0073] In some embodiments, the direct repeat sequence has at least 95% identity (e.g., at least 95%, 96%, 97%, 98%, or 99% identity) to a sequence in Table 2 or a portion of a sequence in Table 2. The direct repeat sequence can have at least 95% identity to a sequence comprising nucleotides 1 to 36 of any one of SEQ ID NOs: 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, or 479. The direct repeat sequence can have at least 95% identity to a sequence comprising 2 to 36 nucleotides of any one of SEQ ID NOs: 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, or 479. The direct repeat sequence can have at least 95% identity to a sequence comprising 3 to 36 nucleotides of any one of SEQ ID NOs: 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, or 479. The direct repeat sequence can have at least 95% identity to a sequence comprising 4 to 36 nucleotides of any one of SEQ ID NOs: 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, or 479. The direct repeat sequence can have at least 95% identity to a sequence comprising 5 to 36 nucleotides of any one of SEQ ID NOs: 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, or 479. The direct repeat sequence can have at least 95% identity to a sequence comprising 6 to 36 nucleotides of any one of SEQ ID NOs: 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, or 479.The direct repeat sequence can have at least 95% identity to a sequence comprising 7 to 36 nucleotides of any one of SEQ ID NOs: 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, or 479. The direct repeat sequence can have at least 95% identity to a sequence comprising 8 to 36 nucleotides of any one of SEQ ID NOs: 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, or 479. The direct repeat sequence can have at least 95% identity to a sequence comprising 9 to 36 nucleotides of any one of SEQ ID NOs: 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, or 479. The direct repeat sequence can have at least 95% identity to a sequence comprising 10 to 36 nucleotides of any one of SEQ ID NOs: 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, or 479. The direct repeat sequence can have at least 95% identity to a sequence comprising 11 to 36 nucleotides of any one of SEQ ID NOs: 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, or 479. The direct repeat sequence can have at least 95% identity to a sequence comprising 12 to 36 nucleotides of any one of SEQ ID NOs: 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, or 479. The direct repeat sequence can have at least 95% identity to a sequence comprising 13 to 36 nucleotides of any one of SEQ ID NOs: 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, or 479.

[0074] In some embodiments, the direct repeat sequence has at least 90% identity (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) to a sequence in Table 2 or a portion of a sequence in Table 2. The direct repeat sequence can have at least 90% identity to a sequence comprising nucleotides 1 to 36 of any one of SEQ ID NOs: 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, or 479. The direct repeat sequence can have at least 90% identity to a sequence comprising 2 to 36 nucleotides of any one of SEQ ID NOs: 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, or 479. The direct repeat sequence can have at least 90% identity to a sequence comprising 3 to 36 nucleotides of any one of SEQ ID NOs: 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, or 479. The direct repeat sequence can have at least 90% identity to a sequence comprising 4 to 36 nucleotides of any one of SEQ ID NOs: 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, or 479. The direct repeat sequence can have at least 90% identity to a sequence comprising 5 to 36 nucleotides of any one of SEQ ID NOs: 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, or 479. The direct repeat sequence can have at least 90% identity to a sequence comprising 6 to 36 nucleotides of any one of SEQ ID NOs: 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, or 479.The direct repeat sequence can have at least 90% identity to a sequence comprising 7 to 36 nucleotides of any one of SEQ ID NOs: 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, or 479. The direct repeat sequence can have at least 90% identity to a sequence comprising 8 to 36 nucleotides of any one of SEQ ID NOs: 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, or 479. The direct repeat sequence can have at least 90% identity to a sequence comprising 9 to 36 nucleotides of any one of SEQ ID NOs: 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, or 479. The direct repeat sequence can have at least 90% identity to a sequence comprising 10 to 36 nucleotides of any one of SEQ ID NOs: 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, or 479. The direct repeat sequence can have at least 90% identity to a sequence comprising 11 to 36 nucleotides of any one of SEQ ID NOs: 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, or 479. The direct repeat sequence can have at least 90% identity to a sequence comprising 12 to 36 nucleotides of any one of SEQ ID NOs: 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, or 479. The direct repeat sequence can have at least 90% identity to a sequence comprising 13 to 36 nucleotides of any one of SEQ ID NOs: 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, or 479.

[0075] In some embodiments, the direct repeat sequence is at least 90% identical to the reverse complement of any one of SEQ ID NOs: 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, or 479. In some embodiments, the direct repeat sequence is at least 95% identical to the reverse complement of any one of SEQ ID NOs: 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, or 479. In some embodiments, the direct repeat sequence is the reverse complement of any one of SEQ ID NOs: 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, or 479.

[0076] In some embodiments, the direct repeat sequence is at least 90% identical to SEQ ID NO: 480 or a portion of SEQ ID NO: 480. In some embodiments, the direct repeat sequence is at least 95% identical to SEQ ID NO: 480 or a portion of SEQ ID NO: 480. In some embodiments, the direct repeat sequence is at least 100% identical to SEQ ID NO: 480 or a portion of SEQ ID NO: 480.

[0077] [Table 2]

[0078] In some embodiments, the direct repeat sequence is a sequence in Table 3 or a portion of a sequence in Table 3. In some embodiments, the direct repeat sequence has at least 95% identity (e.g., at least 95%, 96%, 97%, 98%, or 99% identity) with a sequence in Table 3 or a portion of a sequence in Table 3. In some embodiments, the direct repeat sequence has at least 90% identity (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) with a sequence in Table 3 or a portion of a sequence in Table 3. In some embodiments, the direct repeat sequence is at least 90% identical to the reverse complement of any one of SEQ ID NOs: 485-487. In some embodiments, the direct repeat sequence is at least 95% identical to the reverse complement of any one of SEQ ID NOs: 485-487. In some embodiments, the direct repeat sequence is the reverse complement of any one of SEQ ID NOs: 485-487.

[0079] [Table 3]

[0080] In some embodiments, the direct repeat sequence is a sequence in Table 4 or a portion of a sequence in Table 4. In some embodiments, the direct repeat sequence has at least 95% identity (e.g., at least 95%, 96%, 97%, 98%, or 99% identity) to a sequence in Table 4 or a portion of a sequence in Table 4. In some embodiments, the direct repeat sequence has at least 90% identity (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) to a sequence in Table 4 or a portion of a sequence in Table 4. In some embodiments, the direct repeat sequence is at least 90% identical to the reverse complement of any one of SEQ ID NOs: 488-490. In some embodiments, the direct repeat sequence is at least 95% identical to the reverse complement of any one of SEQ ID NOs: 488-490. In some embodiments, the direct repeat sequence is the reverse complement of any one of SEQ ID NOs: 488-490.

[0081] [Table 4]

[0082] In some embodiments, the direct repeat sequences described herein include uracil (U). In some embodiments, the direct repeat sequences described herein include thymine (T). In some embodiments, the direct repeat sequences according to Tables 1-4 include sequences that include a thymine at one or more positions shown as uracil in Tables 1-4.

[0083] (ii) Spacer sequence In some embodiments, the RNA guide comprises a DNA target or spacer sequence. In some embodiments, the spacer sequence of the RNA guide has a length of 12-100, 13-75, 14-50, or 15-30 nucleotides (e.g., 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides) and is complementary to a non-PAM strand sequence. In some embodiments, the spacer sequence is designed to be complementary to a specific DNA strand, e.g., a specific DNA strand of a genomic locus.

[0084] In some embodiments, the RNA guide spacer sequence is substantially identical to the complementary strand of the target sequence. In some embodiments, the RNA guide comprises 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 with a reference nucleic acid sequence, e.g., the complementary strand of the target sequence. 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 software programs or algorithms (e.g., BLAST, ALIGN, CLUSTAL).

[0085] In some embodiments, the RNA guide comprises a spacer sequence, the spacer sequence having a length of 12-100, 13-75, 14-50, or 15-30 nucleotides (e.g., 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides) and is at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% complementary to a region on the non-PAM strand that is complementary to the target sequence. In some embodiments, the RNA guide comprises a sequence that is at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% complementary to a target DNA sequence. In some embodiments, the RNA guide comprises a sequence that is at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% complementary to the target genome sequence. In some embodiments, the RNA guide comprises a sequence, e.g., an RNA sequence, where the RNA sequence is up to 50 in length and is at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% complementary to the region on the non-PAM strand that is complementary to the target sequence. In some embodiments, the RNA guide comprises a sequence that is at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% complementary to the target DNA sequence. In some embodiments, the RNA guide comprises a sequence that is at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% complementary to the target genome sequence.

[0086] In some embodiments, the spacer sequence is a sequence in Table 5A or 5B or a portion of a sequence in Table 5A or 5B. The references SEQ ID NOs: 229-446 or 2497-4502 should be considered equivalent to the list of SEQ ID NOs: 229-446 or 2497-4502, with each of the intervening numbers present in the list, i.e., 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440,441, 442, 443, 444, 445, and 446, or 2497, 2498, 2499, 2500, 2501, 2502, 2503, 2504, 2505, 2506, 2507, 2508, 2509, 2510, 2511, 2512, 2513, 2514, 2515, 2 516, 2517, 2518, 2519, 2520, 2521, 2522, 2523, 2524, 2525, 2526, 2527, 2528, 2529, 2530, 2531, 2532, 2533, 2534, 2535, 2536, 2537, 2538, 2539, 2540, 2 541, 2542, 2543, 2544, 2545, 2546, 2547, 2548, 2549, 2550, 2551, 2552, 2553, 2554, 2555, 2556, 2557, 2558, 2559, 2560, 2561, 2562, 2563, 2564, 2565, 2 566, 2567, 2568, 2569, 2570, 2571, 2572, 2573, 2574, 2575, 2576, 2577, 2578, 2579, 2580, 2581, 2582, 2583, 2584, 2585, 2586, 2587, 2588, 2589, 2590, 2 591, 2592, 2593, 2594, 2595, 2596, 2597, 2598, 2599, 2600, 2601, 2602, 2603, 2604, 2605, 2606, 2607, 2608, 2609, 2610, 2611, 2612, 2613, 2614, 2615, 2616, 2617, 2618, 2619, 2620, 2621, 2622, 2623, 2624, 2625, 2626, 2627, 2628, 2629, 2630, 2631, 2632, 2633, 2634, 2635, 2636, 2637, 2638, 2639, 2640, 2641, 2642, 2643, 2644, 2645, 2646, 2647, 2648, 2649, 2650, 2651, 2652, 2653, 2654, 2655, 2656, 2657, 2658, 2659, 2660, 2661, 2662, 2663, 2664, 2665, 2666, 2667, 2668, 2669, 2670, 2671, 2672, 2673, 2674, 2675, 2676, 2677, 2678, 2679, 2680, 2681, 2682, 2683, 2684, 2685, 2686, 2687, 2688, 2689, 2690,2691、2692、2693、2694、2695、2696、2697、2698、2699、2700、2701、2702、2703、2704、2705、2706、2707、2708、2709、2710、2711、2712、2713、2714、2715、2716、2717、2718、2719、2720、2721、2722、2723、2724、2725、2726、2727、2728、2729、2730、2731、2732、2733、2734、2735、2736、2737、2738、2739、2740、2741、2742、2743、2744、2745、2746、2747、2748、2749、2750、2751、2752、2753、2754、2755、2756、2757、2758、2759、2760、2761、2762、2763、2764、2765、2766、2767、2768、2769、2770、2771、2772、2773、2774、2775、2776、2777、2778、2779、2780、2781、2782、2783、2784、2785、2786、2787、2788、2789、2790、2791、2792、2793、2794、2795、2796、2797、2798、2799、2800、2801、2802、2803、2804、2805、2806、2807、2808、2809、2810、2811、2812、2813、2814、2815、2816、2817、2818、2819、2820、2821、2822、2823、2824、2825、2826、2827、2828、2829、2830、2831、2832、2833、2834、2835、2836、2837、2838、2839、2840、2841、2842、2843、2844、2845、2846、2847、2848、2849、2850、2851、2852、2853、2854、2855、2856、2857、2858、2859、2860、2861、2862、2863、2864、2865、2866、2867、2868、2869、2870、2871、2872、2873、2874、2875、2876、2877、2878、2879、2880、2881、2882、2883、2884、2885、2886、2887、2888、2889、2890、2891、2892、2893、2894、2895、2896、2897、2898、2899、2900、2901、2902、2903、2904、2905、2906、2907、2908、2909、2910、2911、2912、2913、2914、2915、2916、2917、2918、2919、2920、2921、2922、2923、2924、2925、2926、2927、2928、2929、2930、2931、2932、2933、2934、2935、2936、2937、2938、2939、2940、2941、2942、2943、2944、2945、2946、2947、2948、2949、2950、2951、2952、2953、2954、2955、2956、2957、2958、2959、2960、2961、2962、2963、2964、2965、2966、2967、2968、2969、2970、2971、2972、2973、2974、2975、2976、2977、2978、2979、2980、2981、2982、2983、2984、2985、2986、2987、2988、2989、2990、2991、2992、2993、2994、2995、2996、2997、2998、2999、3000、3001、3002、 3003、3004、3005、3006、3007、3008、3009、3010、3011、3012、3013、3014、3015、3016、3017、3018、3019、3020、3021、3022、3023、3024、3025、3026、3027、3028、3029、3030、3031、3032、3033、3034、3035、3036、3037、3038、3039、3040、3041、3042、3043、3044、3045、3046、3047、3048、3049、3050、3051、3052、3053、3054、3055、3056、3057、3058、3059、3060、3061、3062、3063、3064、3065、3066、3067、3068、3069、3070、3071、3072、3073、3074、3075、3076、3077、3078、3079、3080、3081、3082、3083、3084、3085、3086、3087、3088、3089、3090、3091、3092、3093、3094、3095、3096、3097、3098、3099、3100、3101、3102、3103、3104、3105、3106、3107、3108、3109、3110、3111、3112、3113、3114、3115、3116、3117、3118、3119、3120、3121、3122、3123、3124、3125、3126、3127、3128、3129、3130、3131、3132、3133、3134、3135、3136、3137、3138、3139、3140、3141、3142、3143、3144、3145、3146、3147、3148、3149、3150、3151、3152、3153、3154、3155、3156、3157、3158、3159、3160、3161、3162、3163、3164、3165、3166、3167、3168、3169、3170、3171、3172、3173、3174、3175、3176、3177、3178、3179、3180、3181、3182、3183、3184、3185、3186、3187、3188、3189、3190、3191、3192、3193、3194、3195、3196、3197、3198、3199、3200、3201、3202、3203、3204、3205、3206、3207、3208、3209、3210、3211、3212、3213、3214、3215、3216、3217、3218、3219、3220、3221、3222、3223、3224、3225、3226、3227、3228、3229、3230、3231、3232、3233、3234、3235、3236、3237、3238、3239、3240、3241、3242、3243、3244、3245、3246、3247、3248、3249、3250、3251、3252、3253、3254、3255、3256、3257、3258、3259、3260、3261、3262、3263、3264、3265、3266、3267、3268、3269、3270、3271、3272、3273、3274、3275、3276、3277、3278、3279、3280、3281、3282、3283、3284、3285、3286、3287、3288、3289、3290、3291、3292、3293、3294、3295、3296、3297、3298、3299、3300、3301、3302、3303、3304、3305、3306、3307、3308、3309、3310、3311、3312、3313、3314、3315、3316、3317、3318、3319、3320、3321、3322、3323、3324、3325、3326、3327、3328、3329、3330、3331、3332、3333、3334、3335、3336、3337、3338、3339、3340、3341、3342、3343、3344、3345、3346、3347、3348、3349、3350、3351、3352、3353、3354、3355、3356、3357、3358、3359、3360、3361、3362、3363、3364、3365、3366、3367、3368、3369、3370、3371、3372、3373、3374、3375、3376、3377、3378、3379、3380、3381、3382、3383、3384、3385、3386、3387、3388、3389、3390、3391、3392、3393、3394、3395、3396、3397、3398、3399、3400、3401、3402、3403、3404、3405、3406、3407、3408、3409、3410、3411、3412、3413、3414、3415、3416、3417、3418、3419、3420、3421、3422、3423、3424、3425、3426、3427、3428、3429、3430、3431、3432、3433、3434、3435、3436、3437、3438、3439、3440、3441、3442、3443、3444、3445、3446、3447、3448、3449、3450、3451、3452、3453、3454、3455、3456、3457、3458、3459、3460、3461、3462、3463、3464、3465、3466、3467、3468、3469、3470、3471、3472、3473、3474、3475、3476、3477、3478、3479、3480、3481、3482、3483、3484、3485、3486、3487、3488、3489、3490、3491、3492、3493、3494、3495、3496、3497、3498、3499、3500、3501、3502、3503、3504、3505、3506、3507、3508、3509、3510、3511、3512、3513、3514、3515、3516、3517、3518、3519、3520、3521、3522、3523、3524、3525、3526、3527、3528、3529、3530、3531、3532、3533、3534、3535、3536、3537、3538、3539、3540、3541、3542、3543、3544、3545、3546、3547、3548、3549、3550、3551、3552、3553、3554、3555、3556、3557、3558、3559、3560、3561、3562、3563、3564、3565、3566、3567、3568、3569、3570、3571、3572、3573、3574、3575、3576、3577、3578、3579、3580、3581、3582、3583、3584、3585、3586、3587、3588、3589、3590、3591、3592、3593、3594、3595、3596、3597、3598、3599、3600、3601、3602、3603、3604、3605、3606、3607、3608、3609、3610、3611、3612、3613、3614、3615、3616、3617、3618、3619、3620、3621、3622、3623、3624、3625、3626、3627、3628、3629、3630、3631、3632、3633、3634、3635、3636、3637、3638、3639、3640、3641、3642、3643、3644、3645、3646、3647、3648、3649、3650、3651、3652、3653、3654、3655、3656、3657、3658、3659、3660、3661、3662、3663、3664、3665、3666、3667、3668、3669、3670、3671、3672、3673、3674、3675、3676、3677、3678、3679、3680、3681、3682、3683、3684、3685、3686、3687、3688、3689、3690、3691、3692、3693、3694、3695、3696、3697、3698、3699、3700、3701、3702、3703、3704、3705、3706、3707、3708、3709、3710、3711、3712、3713、3714、3715、3716、3717、3718、3719、3720、3721、3722、3723、3724、3725、3726、3727、3728、3729、3730、3731、3732、3733、3734、3735、3736、3737、3738、3739、3740、3741、3742、3743、3744、3745、3746、3747、3748、3749、3750、3751、3752、3753、3754、3755、3756、3757、3758、3759、3760、3761、3762、3763、3764、3765、3766、3767、3768、3769、3770、3771、3772、3773、3774、3775、3776、3777、3778、3779、3780、3781、3782、3783、3784、3785、3786、3787、3788、3789、3790、3791、3792、3793、3794、3795、3796、3797、3798、3799、3800、3801、3802、3803、3804、3805、3806、3807、3808、3809、3810、3811、3812、3813、3814、3815、3816、3817、3818、3819、3820、3821、3822、3823、3824、3825、3826、3827、3828、3829、3830、3831、3832、3833、3834、3835、3836、3837、3838、3839、3840、3841、3842、3843、3844、3845、3846、3847、3848、3849、3850、3851、3852、3853、3854、3855、3856、3857、3858、3859、3860、3861、3862、3863、3864、3865、3866、3867、3868、3869、3870、3871、3872、3873、3874、3875、3876、3877、3878、3879、3880、3881、3882、3883、3884、3885、3886、3887、3888、3889、3890、3891、3892、3893、3894、3895、3896、3897、3898、3899、3900、3901、3902、3903、3904、3905、3906、3907、3908、3909、3910、3911、3912、3913、3914、3915、3916、3917、3918、3919、3920、3921、3922、3923、3924、3925、3926、3927、3928、3929、3930、3931、3932、3933、3934、3935、3936、3937、3938、3939、3940、3941、3942、3943、3944、3945、3946、3947、3948、3949、3950、3951、3952、3953、3954、3955、3956、3957、3958、3959、3960、3961、3962、3963、3964、3965、3966、3967、3968、3969、3970、3971、3972、3973、3974、3975、3976、3977、3978、3979、3980、3981、3982、3983、3984、3985、3986、3987、3988、3989、3990、3991、3992、3993、3994、3995、3996、3997、3998、3999、4000、4001、4002、4003、4004、4005、4006、4007、4008、4009、4010、4011、4012、4013、4014、4015、4016、4017、4018、4019、4020、4021、4022、4023、4024、4025、4026、4027、4028、4029、4030、4031、4032、4033、4034、4035、4036、4037、4038、4039、4040、4041、4042、4043、4044、4045、4046、4047、4048、4049、4050、4051、4052、4053、4054、4055、4056、4057、4058、4059、4060、4061、4062、4063、4064、4065、4066、4067、4068、4069、4070、4071、4072、4073、4074、4075、4076、4077、4078、4079、4080、4081、4082、4083、4084、4085、4086、4087、4088、4089、4090、4091、4092、4093、4094、4095、4096、4097、4098、4099、4100、4101、4102、4103、4104、4105、4106、4107、4108、4109、4110、4111、4112、4113、4114、4115、4116、4117、4118、4119、4120、4121、4122、4123、4124、4125、4126、4127、4128、4129、4130、4131、4132、4133、4134、4135、4136、4137、4138、4139、4140、4141、4142、4143、4144、4145、4146、4147、4148、4149、4150、4151、4152、4153、4154、4155、4156、4157、4158、4159、4160、4161、4162、4163、4164、4165、4166、4167、4168、4169、4170、4171、4172、4173、4174、4175、4176、4177、4178、4179、4180、4181、4182、4183、4184、4185、4186、4187、4188、4189、4190、4191、4192、4193、4194、4195、4196、4197、4198、4199、4200、4201、4202、4203、4204、4205、4206、4207、4208、4209、4210、4211、4212、4213、4214、4215、4216、4217、4218、4219、4220、4221、4222、4223、4224、4225、4226、4227、4228、4229、4230、4231、4232、4233、4234、4235、4236、4237、4238、4239、4240、4241、4242、4243、4244、4245、4246、4247、4248、4249、4250、4251、4252、4253、4254、4255、4256、4257、4258、4259、4260、4261、4262、4263、4264、4265、4266、4267、4268、4269、4270、4271、4272、4273、4274、4275、4276、4277、4278、4279、4280、4281、4282、4283、4284、4285、4286、4287、4288、4289、4290、4291、4292、4293、4294、4295、4296、4297、4298、4299、4300、4301、4302、4303、4304、4305、4306、4307、4308、4309、4310、4311、4312、4313、4314、4315、4316、4317、4318、4319、4320、4321、4322、4323、4324、4325、4326、4327、4328、4329、4330、4331、4332、4333、4334、4335、4336、4337、4338、4339、4340、4341、4342、4343、4344、4345、4346、4347、4348、4349、4350、4351、4352、4353、4354、4355、4356、4357、4358、4359、4360、4361、4362、4363、4364、4365、4366、4367、4368、4369、4370、4371、4372、4373、4374、4375、4376、4377、4378、4379、4380、4381、4382、4383、4384、4385、4386、4387、4388、4389、4390、4391、4392、4393、4394、4395、4396、4397、4398、4399、4400、4401、4402、4403, 4404, 4405, 4406, 4407, 4408, 4409, 4410, 4411, 4412, 4413, 4414, 4415, 4416, 4417, 4418, 4419, 4420, 4421, 4422, 4423, 4424, 4425, 4426, 4427, 44 28, 4429, 4430, 4431, 4432, 4433, 4434, 4435, 4436, 4437, 4438, 4439, 4440, 4441, 4442, 4443, 4444, 4445, 4446, 4447, 4448, 4449, 4450, 4451, 4452, 4453 , 4454, 4455, 4456, 4457, 4458, 4459, 4460, 4461, 4462, 4463, 4464, 4465, 4466, 4467, 4468, 4469, 4470, 4471, 4472, 4473, 4474, 4475, 4476, 4477, 4478, 4 479, 4480, 4481, 4482, 4483, 4484, 4485, 4486, 4487, 4488, 4489, 4490, 4491, 4492, 4493, 4494, 4495, 4496, 4497, 4498, 4499, 4500, 4501, or 4502.

[0087] The spacer sequence may include nucleotides 1 to 16 of any one of SEQ ID NOs: 229 to 446 or 2497 to 4502. The spacer sequence may include nucleotides 1 to 17 of any one of SEQ ID NOs: 229 to 446 or 2497 to 4502. The spacer sequence may include nucleotides 1 to 18 of any one of SEQ ID NOs: 229 to 446 or 2497 to 4502. The spacer sequence may include nucleotides 1 to 19 of any one of SEQ ID NOs: 229 to 446 or 2497 to 4502. The spacer sequence may include nucleotides 1 to 20 of any one of SEQ ID NOs: 229 to 446 or 2497 to 4502. The spacer sequence may include nucleotides 1 to 21 of any one of SEQ ID NOs: 229 to 446 or 2497 to 4502. The spacer sequence may include nucleotides 1 to 22 of any one of SEQ ID NOs: 229 to 446 or 2497 to 4502. The spacer sequence may include nucleotides 1 to 23 of any one of SEQ ID NOs: 229 to 446 or 2497 to 4502. The spacer sequence may include nucleotides 1 to 24 of any one of SEQ ID NOs: 229 to 446 or 2497 to 4502. The spacer sequence may include nucleotides 1 to 25 of any one of SEQ ID NOs: 229 to 446 or 2497 to 4502. The spacer sequence may include nucleotides 1 to 26 of any one of SEQ ID NOs: 229 to 446 or 2497 to 4502. The spacer sequence may include nucleotides 1 to 27 of any one of SEQ ID NOs: 229 to 446 or 2497 to 4502. The spacer sequence can include nucleotides 1 to 28 of any one of SEQ ID NOs: 229 to 446 or 2497 to 4502. The spacer sequence can include nucleotides 1 to 29 of any one of SEQ ID NOs: 229 to 446 or 2497 to 4502.The spacer sequence can include nucleotides 1 to 30 of any one of SEQ ID NOs: 229-446 or 2497-4502.

[0088] In some embodiments, the spacer sequence has at least 90% identity (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) to a sequence of Table 5A or 5B or a portion of a sequence of Table 5A or 5B. The spacer sequence can have at least 90% identity to a sequence including nucleotides 1 to 16 of any one of SEQ ID NOs: 229-446 or 2497-4502. The spacer sequence can have at least 90% identity to a sequence including nucleotides 1 to 17 of any one of SEQ ID NOs: 229-446 or 2497-4502. The spacer sequence can have at least 90% identity to a sequence including nucleotides 1 to 18 of any one of SEQ ID NOs: 229-446 or 2497-4502. The spacer sequence may have at least 90% identity to a sequence including nucleotides 1 to 19 of any one of SEQ ID NOs: 229 to 446 or 2497 to 4502. The spacer sequence may have at least 90% identity to a sequence including nucleotides 1 to 20 of any one of SEQ ID NOs: 229 to 446 or 2497 to 4502. The spacer sequence may have at least 90% identity to a sequence including nucleotides 1 to 21 of any one of SEQ ID NOs: 229 to 446 or 2497 to 4502. The spacer sequence may have at least 90% identity to a sequence including nucleotides 1 to 22 of any one of SEQ ID NOs: 229 to 446 or 2497 to 4502. The spacer sequence may have at least 90% identity to a sequence including nucleotides 1 to 23 of any one of SEQ ID NOs: 229 to 446 or 2497 to 4502. The spacer sequence can have at least 90% identity to a sequence including nucleotides 1 to 24 of any one of SEQ ID NOs: 229 to 446 or 2497 to 4502. The spacer sequence can have at least 90% identity to a sequence including nucleotides 1 to 25 of any one of SEQ ID NOs: 229 to 446 or 2497 to 4502.The spacer sequence may have at least 90% identity to a sequence including nucleotides 1 to 26 of any one of SEQ ID NOs: 229 to 446 or 2497 to 4502. The spacer sequence may have at least 90% identity to a sequence including nucleotides 1 to 27 of any one of SEQ ID NOs: 229 to 446 or 2497 to 4502. The spacer sequence may have at least 90% identity to a sequence including nucleotides 1 to 28 of any one of SEQ ID NOs: 229 to 446 or 2497 to 4502. The spacer sequence may have at least 90% identity to a sequence including nucleotides 1 to 29 of any one of SEQ ID NOs: 229 to 446 or 2497 to 4502. The spacer sequence may have at least 90% identity to a sequence including nucleotides 1 to 30 of any one of SEQ ID NOs: 229 to 446 or 2497 to 4502.

[0089] [Table 5-1]

[0090] [Table 5-2]

[0091] [Table 5-3]

[0092] [Table 5-4]

[0093] [Table 5-5]

[0094] [Table 5-6]

[0095]

Table 5-7

[0096]

Table 5-8

[0097]

Table 5-9

[0098]

Table 5-10

[0099]

Table 6-1

[0100]

Table 6-2

[0101]

Table 6-3

[0102]

Table 6-4

[0103]

Table 6-5

[0104]

Table 6-6

[0105]

Table 6-7

[0106]

Table 6-8

[0107]

Table 6-9

[0108]

Table 6-10

[0109]

Table 6-11

[0110]

Table 6-12

[0111]

Table 6-13

[0112]

Table 6-14

[0113]

Table 6-15

[0114]

Table 6-16

[0115]

Table 6-17

[0116]

Table 6-18

[0117]

Table 6-19

[0118]

Table 6-20

[0119]

Table 6-21

[0120]

Table 6-22

[0121]

Table 6-23

[0122]

Table 6-24

[0123]

Table 6-25

[0124]

Table 6-26

[0125]

Table 6-27

[0126]

Table 6-28

[0127]

Table 6-29

[0128]

Table 6-30

[0129]

Table 6-31

[0130]

Table 6-32

[0131]

Table 6-33

[0132]

Table 6-34

[0133]

Table 6-35

[0134]

Table 6-36

[0135]

Table 6-37

[0136]

Table 6-38

[0137]

Table 6-39

[0138]

Table 6-40

[0139]

Table 6-41

[0140]

Table 6-42

[0141]

Table 6-43

[0142]

Table 6-44

[0143]

Table 6-45

[0144]

Table 6-46

[0145]

Table 6-47

[0146]

Table 6-48

[0147]

Table 6-49

[0148]

Table 6-50

[0149]

Table 6-51

[0150]

Table 6-52

[0151]

Table 6-53

[0152]

Table 6-54

[0153]

Table 6-55

[0154]

Table 6-56

[0155]

Table 6-57

[0156]

Table 6-58

[0157]

Table 6-59

[0158]

Table 6-60

[0159]

Table 6-61

[0160]

Table 6-62

[0161]

Table 6-63

[0162]

Table 6-64

[0163]

Table 6-65

[0164]

Table 6-66

[0165]

Table 6-67

[0166]

Table 6-68

[0167]

Table 6-69

[0168]

Table 6-70

[0169]

Table 6-71

[0170]

Table 6-72

[0171]

Table 6-73

[0172]

Table 6-74

[0173]

Table 6-75

[0174]

Table 6-76

[0175]

Table 6-77

[0176]

Table 6-78

[0177]

Table 6-79

[0178]

Table 6-80

[0179]

Table 6-81

[0180]

Table 6-82

[0181]

Table 6-83

[0182]

Table 6-84

[0183]

Table 6-85

[0184]

Table 6-86

[0185]

Table 6-87

[0186]

Table 6-88

[0187]

Table 6-89

[0188]

Table 6-90

[0189]

Table 6-91

[0190]

Table 6-92

[0191]

Table 6-93

[0192]

Table 6-94

[0193]

Table 6-95

[0194]

Table 6-96

[0195]

Table 6-97

[0196]

Table 6-98

[0197]

Table 6-99

[0198]

Table 6-100

[0199]

Table 6-101

[0200]

Table 6-102

[0201]

Table 6-103

[0202]

Table 6-104

[0203]

Table 6-105

[0204]

Table 6-106

[0205]

Table 6-107

[0206]

Table 6-108

[0207]

Table 6-109

[0208]

Table 6-110

[0209]

Table 6-111

[0210]

Table 6-112

[0211]

Table 6-113

[0212]

Table 6-114

[0213]

Table 6-115

[0214]

Table 6-116

[0215]

Table 6-117

[0216]

Table 6-118

[0217] [Table 6-119]

[0218] The present disclosure includes all combinations of the direct repeat sequences and spacer sequences listed above that are consistent with the present disclosure herein.

[0219] In some embodiments, a spacer sequence described herein comprises uracil (U). In some embodiments, a spacer sequence described herein comprises thymine (T). In some embodiments, a spacer sequence according to Table 5A or 5B includes a sequence that includes a thymine at one or more (e.g., all) of the locations shown as uracil in Table 5A or 5B.

[0220] The present disclosure includes RNA guides that include any combination of direct repeats and spacers described herein (e.g., described in Tables 5A or 5B above).

[0221] In some embodiments, the RNA guide has at least 90% identity (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) to any one of SEQ ID NOs: 4505-4562. In some embodiments, the RNA guide has at least 95% identity (e.g., at least 95%, 96%, 97%, 98%, or 99% identity) to any one of SEQ ID NOs: 4505-4562. In some embodiments, the RNA guide has a sequence set forth in any one of SEQ ID NOs: 4505-4562.

[0222] In some embodiments, the RNA guide comprises the sequence of any one of SEQ ID NOs: 4508, 4512, 4559, and 4561, or the second nucleic acid encodes an RNA guide comprising any one of SEQ ID NOs: 4508, 4512, 4559, and 4561.

[0223] In some embodiments, the RNA guide comprises the sequence of any one of SEQ ID NOs: 4505, 4506, 4507, 4508, 4509, 4510, 4511, 4512, 4513, 4514, 4515, 4554, 4555, 4556, 4557, 4558, 4559, 4560, 4561, and 4562, or the second nucleic acid encodes an RNA guide comprising any one of SEQ ID NOs: 4505, 4506, 4507, 4508, 4509, 4510, 4511, 4512, 4513, 4514, 4515, 4554, 4555, 4556, 4557, 4558, 4559, 4560, 4561, and 4562.

[0224] B. Nucleic acid modification An RNA guide may include one or more covalent modifications to a reference sequence, particularly a parent polyribonucleotide, and these covalent modifications are included within the scope of the present disclosure.

[0225] 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.

[0226] The RNA guide may include any useful modification, such as a modification 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 some 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.

[0227] 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.

[0228] 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 nucleotide analogs or other modifications can be placed at any position in 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.

[0229] 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.

[0230] 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.

[0231] 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).

[0232] 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.

[0233] 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).

[0234] Other internucleoside linkages that can be used in accordance with the present disclosure are described herein, including internucleoside linkages that do not contain a phosphorus atom.

[0235] 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).

[0236] 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.

[0237] 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.

[0238] In some embodiments, one or more of the nucleotides of the RNA guide comprise a 2'-O-methyl phosphorothioate modification. In some embodiments, each of the first three nucleotides of the RNA guide comprise a 2'-O-methyl phosphorothioate modification. In some embodiments, each of the last four nucleotides of the RNA guide comprise a 2'-O-methyl phosphorothioate modification. In some embodiments, each of the first to last, second to last, and third to last nucleotides of the RNA guide comprise a 2'-O-methyl phosphorothioate modification, and the last nucleotide of the RNA guide is unmodified. In some embodiments, each of the first three nucleotides of the RNA guide comprise a 2'-O-methyl phosphorothioate modification, and each of the first to last, second to last, and third to last nucleotides of the RNA guide comprise a 2'-O-methyl phosphorothioate modification.

[0239] When the gene editing system disclosed herein comprises a nucleic acid, e.g., an mRNA molecule, encoding a Cas12i polypeptide disclosed herein, such a nucleic acid molecule may contain any of the modifications disclosed herein, if applicable.

[0240] C. Cas12i Polypeptides In some embodiments, a composition or system of the present disclosure comprises a Cas12i polypeptide as described in WO / 2019 / 178427, the relevant disclosure of which is incorporated by reference for the subject matter and purposes referenced herein.

[0241] In some embodiments, the gene editing system of the disclosure comprises a Cas12i2 polypeptide described herein (e.g., a polypeptide comprising SEQ ID NO:448 and / or encoded by SEQ ID NO:447 (or a version thereof in which T is replaced with U)). In some embodiments, the Cas12i2 polypeptide comprises at least one RuvC domain. In some embodiments, the gene editing system of the disclosure comprises a nucleic acid molecule (e.g., a DNA molecule or a polyribonucleotide molecule) encoding a Cas12i polypeptide.

[0242] Nucleic acid sequences encoding the Cas12i2 polypeptides described herein can be substantially identical to a reference nucleic acid sequence, such as SEQ ID NO: 447 (or a version thereof in which T is replaced with U). In some embodiments, the Cas12i2 polypeptide 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, such as SEQ ID NO: 447 (or a version thereof in which T is replaced with U). 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 software programs or algorithms (e.g., BLAST, ALIGN, CLUSTAL) using standard parameters. One indication that two nucleic acid sequences are substantially identical is that the nucleic acid molecules hybridize to the complementary sequence of the other under stringent conditions of temperature and ionic strength (e.g., within a range of moderate to high stringency). See, for example, Tijssen, "Hybridization with Nucleic Acid Probes. Part I. Theory and Nucleic Acid Preparation" (Laboratory Techniques in Biochemistry and Molecular Biology, Vol 24).

[0243] In some embodiments, the Cas12i2 polypeptide is encoded by a nucleic acid sequence that 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%, or at least about 99% or more sequence identity to a reference nucleic acid sequence, e.g., SEQ ID NO:447 (or a version thereof in which T is replaced with U), but does not have 100% sequence identity.

[0244] In some embodiments, a Cas12i2 polypeptide of the disclosure comprises a polypeptide 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:448.

[0245] In some embodiments, the disclosure describes Cas12i2 polypeptides that have a certain degree of amino acid sequence identity with one or more reference 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%, or even at least 99%, but not 100%, sequence identity with the amino acid sequence of SEQ ID NO: 448. Homology or identity can be determined by amino acid sequence alignment, for example, using programs such as BLAST, ALIGN, or CLUSTAL, as described herein.

[0246] Also provided is a Cas12i2 polypeptide of the disclosure that has enzymatic activity, e.g., nuclease or endonuclease activity, and comprises an amino acid sequence that differs from the amino acid sequence of SEQ ID NO: 448 by 50, 40, 35, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, or 0 amino acid residues when aligned using any of the alignment methods described above.

[0247] In some embodiments, the Cas12i2 polypeptide can contain one or more mutations, e.g., at positions D581, G624, F626, P868, I926, V1030, E1035, S1046, or any combination thereof, relative to SEQ ID NO: 448. In some cases, the one or more mutations are amino acid substitutions, e.g., D581R, G624R, F626R, P868T, I926R, V1030G, E1035R, S1046G, or any combination thereof.

[0248] In some embodiments, the Cas12i2 polypeptide comprises a polypeptide having a sequence of SEQ ID NO:449, SEQ ID NO:450, SEQ ID NO:451, SEQ ID NO:452, or SEQ ID NO:453. In some examples, the Cas12i2 polypeptide contains mutations at positions D581, D911, I926, and V1030. Such a Cas12i2 polypeptide may contain amino acid substitutions of D581R, D911R, I926R, and V1030G (e.g., SEQ ID NO:449). In some examples, the Cas12i2 polypeptide contains mutations at positions D581, I926, and V1030. Such a Cas12i2 polypeptide may contain amino acid substitutions of D581R, I926R, and V1030G (e.g., SEQ ID NO:450). In some examples, the Cas12i2 polypeptide may contain mutations at positions D581, I926, V1030, and S1046. Such Cas12i2 polypeptides may contain amino acid substitutions of D581R, I926R, V1030G, and S1046G (e.g., SEQ ID NO: 451). In some examples, the Cas12i2 polypeptide may contain mutations at positions D581, G624, F626, I926, V1030, E1035, and S1046. Such Cas12i2 polypeptides may contain amino acid substitutions of D581R, G624R, F626R, I926R, V1030G, E1035R, and S1046G (e.g., SEQ ID NO: 452). In some instances, the Cas12i2 polypeptide may contain mutations at positions D581, G624, F626, P868, I926, V1030, E1035, and S1046. Such Cas12i2 polypeptides may contain amino acid substitutions of D581R, G624R, F626R, P868T, I926R, V1030G, E1035R, and S1046G (e.g., SEQ ID NO: 453).

[0249] In some embodiments, a Cas12i2 polypeptide of the disclosure comprises a polypeptide 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:449, SEQ ID NO:450, SEQ ID NO:451, SEQ ID NO:452, or SEQ ID NO:453. In some embodiments, a Cas12i2 polypeptide 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:449, SEQ ID NO:450, SEQ ID NO:451, SEQ ID NO:452, or SEQ ID NO:453 maintains the amino acid changes (or at least one, two, three, etc. of these changes) that distinguish the polypeptide from its respective parent / reference sequence.

[0250] In some embodiments, the disclosure describes Cas12i2 polypeptides that have a certain degree of amino acid sequence identity with one or more reference 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%, or even at least 99%, but not 100%, sequence identity with the amino acid sequence of SEQ ID NO: 449, SEQ ID NO: 450, SEQ ID NO: 451, SEQ ID NO: 452, or SEQ ID NO: 453. Homology or identity can be determined by amino acid sequence alignment, for example, using programs such as BLAST, ALIGN, or CLUSTAL, as described herein.

[0251] Also provided is a Cas12i2 polypeptide of the present disclosure having enzymatic activity, e.g., nuclease or endonuclease activity, comprising an amino acid sequence that differs from the amino acid sequence of SEQ ID NO:449, SEQ ID NO:450, SEQ ID NO:451, SEQ ID NO:452, or SEQ ID NO:453 by 50, 40, 35, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, or 0 amino acid residues when aligned using any of the alignment methods described above.

[0252] In some embodiments, a composition of the disclosure comprises a Cas12i4 polypeptide described herein (e.g., a polypeptide comprising SEQ ID NO:482 and / or encoded by SEQ ID NO:481 (or a version thereof in which T is replaced with U)). In some embodiments, the Cas12i4 polypeptide comprises at least one RuvC domain.

[0253] Nucleic acid sequences encoding the Cas12i4 polypeptides described herein can be substantially identical to a reference nucleic acid sequence, such as SEQ ID NO: 481 (or a version thereof in which T is replaced with U). In some embodiments, the Cas12i4 polypeptide 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, such as SEQ ID NO: 481 (or a version thereof in which T is replaced with U). 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 software programs or algorithms (e.g., BLAST, ALIGN, CLUSTAL) using standard parameters. One indication that two nucleic acid sequences are substantially identical is that the nucleic acid molecules hybridize to the complementary sequence of the other under stringent conditions of temperature and ionic strength (e.g., within a range of moderate to high stringency).

[0254] In some embodiments, the Cas12i4 polypeptide is encoded by a nucleic acid sequence that 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%, or at least about 99% or more sequence identity to a reference nucleic acid sequence, e.g., SEQ ID NO:481 (or a version thereof in which T is replaced with U), but does not have 100% sequence identity.

[0255] In some embodiments, a Cas12i4 polypeptide of the disclosure comprises a polypeptide 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:482.

[0256] In some embodiments, the disclosure describes Cas12i4 polypeptides that have a certain degree of amino acid sequence identity with one or more reference 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%, or even at least 99%, but not 100%, sequence identity with the amino acid sequence of SEQ ID NO: 482. Homology or identity can be determined by amino acid sequence alignment, for example, using programs such as BLAST, ALIGN, or CLUSTAL, as described herein.

[0257] Also provided is a Cas12i4 polypeptide of the present disclosure having enzymatic activity, e.g., nuclease or endonuclease activity, comprising an amino acid sequence that differs from the amino acid sequence of SEQ ID NO: 482 by 50, 40, 35, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, or 0 amino acid residues when aligned using any of the alignment methods described above.

[0258] In some embodiments, the Cas12i4 polypeptide comprises a polypeptide comprising the sequence of SEQ ID NO:483 or SEQ ID NO:484.

[0259] In some embodiments, a Cas12i4 polypeptide of the disclosure comprises a polypeptide 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: 483 or SEQ ID NO: 484. In some embodiments, a Cas12i4 polypeptide 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: 483 or SEQ ID NO: 484 maintains the amino acid changes (or at least one, two, three, etc. of these changes) that distinguish it from its respective parent / reference sequence.

[0260] In some embodiments, the disclosure describes Cas12i4 polypeptides that have a certain degree of amino acid sequence identity with one or more reference 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%, or even at least 99%, but not 100%, sequence identity with the amino acid sequence of SEQ ID NO: 483 or SEQ ID NO: 484. Homology or identity can be determined by amino acid sequence alignment, for example, using programs such as BLAST, ALIGN, or CLUSTAL, as described herein.

[0261] Also provided is a Cas12i4 polypeptide of the present disclosure having enzymatic activity, e.g., nuclease or endonuclease activity, comprising an amino acid sequence that differs from the amino acid sequence of SEQ ID NO: 483 or SEQ ID NO: 484 by 50, 40, 35, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, or 0 amino acid residues when aligned using any of the alignment methods described above.

[0262] In some embodiments, a composition of the disclosure comprises a Cas12i1 polypeptide described herein (e.g., a polypeptide comprising SEQ ID NO: 4503). In some embodiments, the Cas12i1 polypeptide comprises at least one RuvC domain.

[0263] In some embodiments, a Cas12i1 polypeptide of the disclosure comprises a polypeptide 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:4503.

[0264] In some embodiments, the disclosure describes Cas12i1 polypeptides that have a certain degree of amino acid sequence identity with one or more reference 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%, or even at least 99%, but not 100%, sequence identity with the amino acid sequence of SEQ ID NO: 4503. Homology or identity can be determined by amino acid sequence alignment, for example, using programs such as BLAST, ALIGN, or CLUSTAL, as described herein.

[0265] Also provided is a Cas12i1 polypeptide of the present disclosure having enzymatic activity, e.g., nuclease or endonuclease activity, comprising an amino acid sequence that differs from the amino acid sequence of SEQ ID NO: 4503 by 50, 40, 35, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, or 0 amino acid residues when aligned using any of the alignment methods described above.

[0266] In some embodiments, a composition of the disclosure comprises a Cas12i3 polypeptide described herein (e.g., a polypeptide comprising SEQ ID NO: 4504). In some embodiments, the Cas12i3 polypeptide comprises at least one RuvC domain.

[0267] In some embodiments, a Cas12i3 polypeptide of the disclosure comprises a polypeptide 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:4504.

[0268] In some embodiments, the disclosure describes Cas12i3 polypeptides that have a certain degree of amino acid sequence identity with one or more reference 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%, or even at least 99%, but not 100%, sequence identity with the amino acid sequence of SEQ ID NO: 4504. Homology or identity can be determined by amino acid sequence alignment, for example, using programs such as BLAST, ALIGN, or CLUSTAL, as described herein.

[0269] Also provided is a Cas12i3 polypeptide of the present disclosure having enzymatic activity, e.g., nuclease or endonuclease activity, comprising an amino acid sequence that differs from the amino acid sequence of SEQ ID NO: 4504 by 50, 40, 35, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, or 0 amino acid residues when aligned using any of the alignment methods described above.

[0270] The changes described herein may be one or more amino acid changes, but the changes to the Cas12i polypeptide may also be of a substantial nature, for example, fusion of a polypeptide as an amino and / or carboxyl terminal extension. For example, the Cas12i polypeptide may contain an additional peptide, for example, one or more peptides. Examples of additional peptides may include epitope peptides for labeling, for example, polyhistidine tags (His tags), Myc, and FLAG. In some embodiments, the Cas12i polypeptides described herein may be fused to a detectable moiety, for example, a fluorescent protein (e.g., green fluorescent protein (GFP) or yellow fluorescent protein (YFP)).

[0271] In some embodiments, the Cas12i polypeptide comprises at least one (e.g., 2, 3, 4, 5, or more) nuclear localization signal (NLS). In some embodiments, the Cas12i polypeptide comprises at least one (e.g., 2, 3, 4, 5, or more) nuclear export signal (NES). In some embodiments, the Cas12i polypeptide comprises at least one (e.g., 2, 3, 4, 5, or more) NLS and at least one (e.g., 2, 3, 4, 5, or more) NES.

[0272] In some embodiments, the Cas12i polypeptides described herein can be self-inactivating. See Epstein et al., "Engineering a Self-Inactivating CRISPR System for AAV Vectors," Mol. Ther., 24(2016):S50, which is incorporated by reference in its entirety.

[0273] In some embodiments, the nucleotide sequence encoding the Cas12i polypeptide described herein can be codon-optimized for use in a particular host cell or organism. For example, the nucleic acid can 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, at the "Codon Usage Database" available at the World Wide Web at kazusa.orjp / codon / , and these tables can 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, for example, Gene Forge (Aptagen, Jacobus, PA). In some examples, the nucleic acid encoding the Cas12i polypeptide, such as the Cas12i2 polypeptide disclosed herein, can be an mRNA molecule that can be codon-optimized.

[0274] Exemplary Cas12i polypeptide sequences and corresponding nucleotide sequences are listed in Table 7.

[0275] [Table 7-1]

[0276] [Table 7-2]

[0277]

Table 7-3

[0278]

Table 7-4

[0279]

Table 7-5

[0280]

Table 7-6

[0281]

Table 7-7

[0282]

Table 7-8

[0283]

Table 7-9

[0284]

Table 7-10

[0285]

Table 7-11

[0286]

Table 7-12

[0287]

Table 7-13

[0288]

Table 7-14

[0289]

Table 7-15

[0290]

Table 7-16

[0291]

Table 7-17

[0292]

Table 7-18

[0293]

Table 7-19

[0294]

Table 7-20

[0295]

Table 7-21

[0296]

Table 7-22

[0297] In some embodiments, the gene editing system disclosed herein may include a Cas12i polypeptide disclosed herein. In other embodiments, the gene editing system may include a nucleic acid encoding a Cas12i polypeptide. For example, the gene editing system may include a vector (e.g., a viral vector, e.g., an AAV vector, e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, and AAV12) encoding a Cas12i polypeptide. Alternatively, the gene editing system may include an mRNA molecule encoding a Cas12i polypeptide. In some cases, the mRNA molecule may be codon-optimized.

[0298] II. Preparation of gene editing system components The present disclosure provides components of the gene editing system disclosed herein, such as methods for producing RNA guides, methods for producing Cas12i polypeptides, and methods for forming complexes between RNA guides and Cas12i polypeptides.

[0299] A. RNA guide In some embodiments, the RNA guide is produced by in vitro transcription of a DNA molecule. Thus, for example, in some embodiments, the RNA guide is produced by in vitro transcription of a DNA molecule encoding the RNA guide using an upstream promoter sequence (e.g., a T7 polymerase promoter sequence).

[0300] In some embodiments, the DNA molecule encodes multiple RNA guides, or the in vitro transcription reaction includes multiple different DNA molecules, each encoding a different RNA guide. In some embodiments, the RNA guide is produced using chemical synthesis methods. In some embodiments, the RNA guide is produced by expressing an RNA guide sequence in a cell transfected with a plasmid that includes a sequence encoding the RNA guide. In some embodiments, the plasmid encodes multiple different RNA guides. In some embodiments, multiple different plasmids, each encoding a different RNA guide, are transfected into the cell. In some embodiments, the RNA guide is expressed from a plasmid that encodes the RNA guide and also encodes a Cas12i polypeptide. In some embodiments, the RNA guide is expressed from a plasmid that expresses the RNA guide but does not express a Cas12i polypeptide. In some embodiments, the RNA guide is purchased from a commercial vendor. In some embodiments, the RNA guide is synthesized using one or more modified nucleotides, such as those described above.

[0301] B. Cas12i Polypeptides In some embodiments, the Cas12i polypeptide of the present disclosure can be prepared by (a) culturing bacteria that produce the Cas12i polypeptide of the present disclosure, isolating the Cas12i polypeptide, optionally purifying the Cas12i polypeptide, and forming a complex between the Cas12i polypeptide and an RNA guide. The Cas12i polypeptide can also be prepared by (b) known genetic engineering techniques, specifically by isolating the gene encoding the Cas12i polypeptide of the present disclosure from bacteria, constructing a recombinant expression vector, and then introducing the vector into a suitable host cell, where the host cell expresses the RNA guide for expression of the recombinant protein, and the recombinant protein forms a complex with the RNA guide in the host cell. Alternatively, the Cas12i polypeptide can be prepared by (c) an in vitro coupled transcription-translation system, followed by complex formation with the RNA guide.

[0302] In some embodiments, a host cell is used to express Cas12i polypeptide. 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 a 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.

[0303] After the host is transformed with the expression vector, the host cell may be cultured, cultivated, or propagated for the production of the Cas12i polypeptide. After expression of the Cas12i polypeptide, the host cell may be harvested and the Cas12i polypeptide may be purified from the culture or the like by conventional methods (e.g., filtration, centrifugation, cell disruption, gel filtration chromatography, ion exchange chromatography, etc.).

[0304] In some embodiments, the methods for Cas12i polypeptide 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 Cas12i polypeptide. 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 a Cas12i polypeptide.

[0305] Various methods can be used to determine the level of production of Cas12i polypeptide in a host cell. Such methods include, but are not limited to, methods using polyclonal or monoclonal antibodies specific for Cas12i polypeptide 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] ).

[0306] The present disclosure provides a method for in vivo expression of a Cas12i polypeptide in a cell, the method comprising providing a host cell with a polyribonucleotide encoding a Cas12i polypeptide, wherein the polyribonucleotide encodes the Cas12i polypeptide, expressing the Cas12i polypeptide in the cell, and obtaining the Cas12i polypeptide from the cell.

[0307] The present disclosure further provides a method of in vivo expression of a Cas12i polypeptide in a cell, comprising providing a host cell with a polyribonucleotide encoding a Cas12i polypeptide, the polyribonucleotide encoding the Cas12i polypeptide, and expressing the Cas12i polypeptide in the cell. In some embodiments, the polyribonucleotide encoding the Cas12i polypeptide is delivered to the cell with an RNA guide, and upon expression in the cell, the Cas12i polypeptide and the RNA guide form a complex. In some embodiments, the polyribonucleotide encoding the Cas12i polypeptide and the RNA guide are delivered to the cell in a single composition. In some embodiments, the polyribonucleotide encoding the Cas12i polypeptide and the RNA guide are contained in separate compositions. In some embodiments, the host cell is present in a subject, e.g., a human patient.

[0308] C. complex In some embodiments, the RNA guide targeting STMN2 is complexed with a Cas12i polypeptide to form a ribonucleoprotein (RNP). In some embodiments, the complexation of the RNA guide with the Cas12i polypeptide occurs at a temperature lower than about any one of 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, 41°C, 42°C, 43°C, 44°C, 45°C, 50°C, or 55°C. In some embodiments, the RNA guide does not dissociate from the Cas12i polypeptide over an incubation period of at least about any one of 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 35 minutes, 40 minutes, 45 minutes, 50 minutes, 55 minutes, 1 hour, 2 hours, 3 hours, or 4 hours or more at about 37°C.

[0309] In some embodiments, the RNA guide and the Cas12i polypeptide are complexed in a complexation buffer. In some embodiments, the Cas12i polypeptide is stored in a buffer, and the buffer is replaced with a complexation buffer, and the Cas12i polypeptide is complexed with the RNA guide. In some embodiments, the Cas12i polypeptide is stored in a complexation buffer.

[0310] In some embodiments, the complex formation buffer has a pH in the range of about 7.3 to 8.6. In one embodiment, the pH of the complex formation buffer is about 7.3. In one embodiment, the pH of the complex formation buffer is about 7.4. In one embodiment, the pH of the complex formation buffer is about 7.5. In one embodiment, the pH of the complex formation buffer is about 7.6. In one embodiment, the pH of the complex formation buffer is about 7.7. In one embodiment, the pH of the complex formation buffer is about 7.8. In one embodiment, the pH of the complex formation buffer is about 7.9. In one embodiment, the pH of the complex formation buffer is about 8.0. In one embodiment, the pH of the complex formation buffer is about 8.1. In one embodiment, the pH of the complex formation buffer is about 8.2. In one embodiment, the pH of the complex formation buffer is about 8.3. In one embodiment, the pH of the complex formation buffer is about 8.4. In one embodiment, the pH of the complex formation buffer is about 8.5. In one embodiment, the pH of the complex formation buffer is about 8.6.

[0311] In some embodiments, the Cas12i polypeptide may be overexpressed in a host cell and may form a complex with an RNA guide before purification as described herein. In some embodiments, an mRNA or DNA encoding the Cas12i polypeptide is introduced into a cell such that the Cas12i polypeptide is expressed in the cell. In some embodiments, the RNA guide is also introduced into the cell simultaneously, separately, or sequentially with a single mRNA or DNA construct such that an RNP complex is formed in the cell.

[0312] III. Gene Editing Methods The present disclosure also provides a method of modifying a target site in the STMN2 gene. In some embodiments, the method includes introducing an STMN2-targeting RNA guide and a Cas12i polypeptide into a cell. The STMN2-targeting RNA guide and a Cas12i polypeptide may be introduced into a cell as a ribonucleoprotein complex. The STMN2-targeting RNA guide and a Cas12i polypeptide may be introduced on a nucleic acid vector. The Cas12i polypeptide may be introduced as an mRNA. The RNA guide and template DNA may be directly introduced into a cell. In some embodiments, the compositions described herein are delivered to a cell / tissue / person to reduce STMN2 in the cell / tissue / person. In some embodiments, the compositions described herein are delivered to a cell / tissue / person to reduce STMN2 production in the cell / tissue / person. In some embodiments, the compositions described herein are delivered to a cell / tissue / person to treat a neurodegenerative disease (e.g., amyotrophic lateral sclerosis (ALS) or frontotemporal dementia (FTD)) in the cell / tissue / person. In some embodiments, the compositions described herein are delivered to a person with a neurodegenerative disease (e.g., amyotrophic lateral sclerosis (ALS) or frontotemporal dementia (FTD)).

[0313] Any of the gene editing systems disclosed herein can be used to engineer STMN2 gene. The gene editing system can include a guide RNA, a Cas12i2 polypeptide, and a template DNA. The guide RNA includes a spacer sequence specific to a target sequence in the STMN2 gene, for example, specific to exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, or a region within an intron of the STMN2 gene.

[0314] A. Target Sequence In some embodiments, the RNA guides disclosed herein are designed to be complementary to a target sequence adjacent to a 5'-TTN-3'PAM sequence or a 5'-NTTN-3'PAM sequence.

[0315] In some embodiments, the target sequence is within the STMN2 gene or a locus of the STMN2 gene (e.g., exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, or an intron) to which the RNA guide can bind via base pairing. In some embodiments, the cell has only one copy of the target sequence. In some embodiments, the cell has two or more copies of the target sequence, such as at least about any one of 2, 3, 4, 5, 10, or 100 or more copies.

[0316] In some embodiments, the STMN2 gene is a mammalian gene. In some embodiments, the STMN2 gene is a human gene. For example, in some embodiments, the target sequence is within the sequence of SEQ ID NO: 454, or a reverse complement thereof. In some embodiments, the target sequence is within an exon of the STMN2 gene set forth in SEQ ID NO: 454, or a reverse complement thereof, for example, within any one of the sequences of SEQ ID NOs: 455-461 (or a reverse complement of any of them). Target sequences within the exon regions of the STMN2 gene of SEQ ID NO: 454 are listed in Table 6. Exon sequences are listed in Table 7. In some embodiments, the target sequence is within an intron of the STMN2 gene set forth in SEQ ID NO: 454, or a reverse complement thereof. In some embodiments, the target sequence is within a variant (e.g., a polymorphic variant) of the STMN2 gene sequence set forth in SEQ ID NO: 454, or a reverse complement thereof. In some embodiments, the STMN2 gene sequence is a homolog of the sequence set forth in SEQ ID NO: 454, or a reverse complement thereof. In some embodiments, the STMN2 gene sequence is a non-human STMN2 sequence.

[0317] In some embodiments, the target sequence is adjacent to a 5'-NTTN-3' PAM sequence, where N is any nucleotide. The 5'-NTTN-3' sequence may be directly 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. In some embodiments, the 5'-NTTN-3' sequence is 5'-NTTY-3', 5'-NTTC-3', 5'-NTTT-3', 5'-NTTA-3', 5'-NTTB-3', 5'-NTTG-3', 5'-CTTY-3', 5'-DTTR'3', 5'-CTTR-3', 5'-DTTT-3', 5'-ATTN-3', or 5'-GTTN-3', where Y is C or T, B is any nucleotide except A, D is any nucleotide except C, and R is A or G. In some embodiments, the 5'-NTTN-3' sequence is 5'-ATTA-3', 5'-ATTT-3', 5'-ATTG-3', 5'-ATTC-3', 5'-TTTA-3', 5'-TTTT-3', 5'-TTTG-3', 5'-TTTC-3', 5'-GTTA-3', 5'-GTTT-3', 5'-GTTG-3', 5'-GTTC-3', 5'-CTTA-3', 5'-CTTT-3', 5'-CTTG-3', or 5'-CTTC-3'. The PAM sequence can be 5' to the target sequence.

[0318] 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, such as methylated nucleotides, damaged nucleotides, or nucleotide analogs. In some embodiments, the target sequence is unmodified. In some embodiments, the RNA guide binds to a first strand (e.g., a target strand or a spacer complementary strand) of a double-stranded target sequence, and the 5'-NTTN-3' PAM sequence is present in a second complementary strand (e.g., a non-target strand or a non-spacer complementary strand). In some embodiments, the RNA guide binds adjacent to the 5'-NAAN-3' sequence on the target strand (e.g., a spacer complementary strand).

[0319] The 5'-NTTN-3' sequence may be immediately adjacent to the target sequence or may be within, for example, a small number (e.g., 1, 2, 3, 4, or 5) nucleotides of the target sequence. In some embodiments, the 5'-NTTN-3' sequence is 5'-NTTY-3', 5'-NTTC-3', 5'-NTTT-3', 5'-NTTA-3', 5'-NTTB-3', 5'-NTTG-3', 5'-CTTY-3', 5'-DTTR-3', 5'-CTTR-3', 5'-DTTT-3', 5'-ATTN-3', or 5'-GTTN-3', where Y is C or T, B is any nucleotide except A, D is any nucleotide except C, and R is A or G. In some embodiments, the 5'-NTTN-3' sequence is 5'-ATTA-3', 5'-ATTT-3', 5'-ATTG-3', 5'-ATTC-3', 5'-TTTA-3', 5'-TTTT-3', 5'-TTTG-3', 5'-TTTC-3', 5'-GTTA-3', 5'-GTTT-3', 5'-GTTG-3', 5'-GTTC-3', 5'-CTTA-3', 5'-CTTT-3', 5'-CTTG-3', or 5'-CTTC-3'. In some embodiments, the RNA guide is designed to bind to a first strand (i.e., the non-PAM strand) of a double-stranded target nucleic acid, and the 5'-NTTN-3' PAM sequence is present in the second, complementary strand (i.e., the PAM strand). In some embodiments, the RNA guide binds to a region on the non-PAM strand that is complementary to a target sequence on the PAM strand adjacent to the 5'-NAAN-3' sequence.

[0320] In some embodiments, the target sequence is present within a cell. In some embodiments, the target sequence is present in the nucleus of a cell. In some embodiments, the target sequence is endogenous to the cell. In some embodiments, the target sequence is genomic DNA. In some embodiments, the target sequence is chromosomal DNA. In some embodiments, the target sequence is a protein-coding gene or a functional region thereof, such as a coding region, or a regulatory element, such as a promoter, enhancer, 5' or 3' untranslated region, etc.

[0321] In some embodiments, the target sequence is within an easily accessible region of the target sequence. In some embodiments, the target sequence is within an exon of the target gene. In some embodiments, the target sequence spans an exon-intron junction of the target gene. In some embodiments, the target sequence is within a non-coding region of the gene, such as a regulatory region.

[0322] B. Gene Editing In some embodiments, the Cas12i polypeptide has enzymatic activity (e.g., nuclease activity). In some embodiments, the Cas12i polypeptide induces one or more DNA double-strand breaks in a cell. In some embodiments, the Cas12i polypeptide induces one or more DNA single-strand breaks in a cell. In some embodiments, the Cas12i polypeptide induces one or more DNA nicks in a cell. In some embodiments, the DNA breaks and / or nicks result in the formation of one or more indels (e.g., one or more deletions).

[0323] In some embodiments, the RNA guide disclosed herein forms a complex with a Cas12i polypeptide and directs the Cas12i polypeptide to a target sequence adjacent to a 5'-NTTN-3' sequence. In some embodiments, the complex induces a deletion (e.g., a nucleotide deletion or a DNA deletion) adjacent to a 5'-NTTN-3' sequence. In some embodiments, the complex induces a deletion adjacent to a 5'-ATTA-3', 5'-ATTT-3', 5'-ATTG-3', 5'-ATTC-3', 5'-TTTA-3', 5'-TTTT-3', 5'-TTTG-3', 5'-TTTC-3', 5'-GTTA-3', 5'-GTTT-3', 5'-GTTG-3', 5'-GTTC-3', 5'-CTTA-3', 5'-CTTT-3', 5'-CTTG-3', or 5'-CTTC-3' sequence. In some embodiments, the complex induces a deletion adjacent to a T / C rich sequence.

[0324] In some embodiments, the deletion is downstream of the 5'-NTTN-3' sequence. In some embodiments, the deletion is downstream of the 5'-ATTA-3', 5'-ATTT-3', 5'-ATTG-3', 5'-ATTC-3', 5'-TTTA-3', 5'-TTTT-3', 5'-TTTG-3', 5'-TTTC-3', 5'-GTTA-3', 5'-GTTT-3', 5'-GTTG-3', 5'-GTTC-3', 5'-CTTA-3', 5'-CTTT-3', 5'-CTTG-3', or 5'-CTTC-3' sequence. In some embodiments, the deletion is downstream of the T / C rich sequence.

[0325] In some embodiments, the deletion alters expression of the STMN2 gene. In some embodiments, the deletion alters function of the STMN2 gene. In some embodiments, the deletion inactivates the STMN2 gene. In some embodiments, the deletion is a frameshift deletion. In some embodiments, the deletion is a non-frameshift deletion. In some embodiments, the deletion results in cytotoxicity or cell death (e.g., apoptosis).

[0326] In some embodiments, the deletion begins within about 5 to about 15 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) of the 5'-NTTN-3' sequence. In some embodiments, deletions begin within about 5 to about 15 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) of the 5'-ATTA-3', 5'-ATTT-3', 5'-ATTG-3', 5'-ATTC-3', 5'-TTTA-3', 5'-TTTT-3', 5'-TTTG-3', 5'-TTTC-3', 5'-GTTA-3', 5'-GTTT-3', 5'-GTTG-3', 5'-GTTC-3', 5'-CTTA-3', 5'-CTTT-3', 5'-CTTG-3', or 5'-CTTC-3' sequence. In some embodiments, the deletion begins within about 5 to about 15 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) of the T / C rich sequence.

[0327] In some embodiments, the deletion begins within about 5 to about 15 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) downstream of the 5'-NTTN-3' sequence. In some embodiments, the deletion begins within about 5 to about 15 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) downstream of the 5'-ATTA-3', 5'-ATTT-3', 5'-ATTG-3', 5'-ATTC-3', 5'-TTTA-3', 5'-TTTT-3', 5'-TTTG-3', 5'-TTTC-3', 5'-GTTA-3', 5'-GTTT-3', 5'-GTTG-3', 5'-GTTC-3', 5'-CTTA-3', 5'-CTTT-3', 5'-CTTG-3', or 5'-CTTC-3' sequence. In some embodiments, the deletion begins within about 5 to about 15 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) downstream of the T / C rich sequence.

[0328] In some embodiments, the deletion begins within about 5 to about 10 nucleotides (eg, about 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides) of the 5'-NTTN-3' sequence. In some embodiments, the deletion begins within about 5 to about 10 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides) of the 5'-ATTA-3', 5'-ATTT-3', 5'-ATTG-3', 5'-ATTC-3', 5'-TTTA-3', 5'-TTTT-3', 5'-TTTG-3', 5'-TTTC-3', 5'-GTTA-3', 5'-GTTT-3', 5'-GTTG-3', 5'-GTTC-3', 5'-CTTA-3', 5'-CTTT-3', 5'-CTTG-3', or 5'-CTTC-3' sequence. In some embodiments, the deletion begins within about 5 to about 10 nucleotides (eg, about 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides) of the T / C rich sequence.

[0329] In some embodiments, the deletion begins within about 5 to about 10 nucleotides (eg, about 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides) downstream of the 5'-NTTN-3' sequence. In some embodiments, the deletion begins within about 5 to about 10 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides) downstream of the 5'-ATTA-3', 5'-ATTT-3', 5'-ATTG-3', 5'-ATTC-3', 5'-TTTA-3', 5'-TTTT-3', 5'-TTTG-3', 5'-TTTC-3', 5'-GTTA-3', 5'-GTTT-3', 5'-GTTG-3', 5'-GTTC-3', 5'-CTTA-3', 5'-CTTT-3', 5'-CTTG-3', or 5'-CTTC-3' sequence. In some embodiments, the deletion begins within about 5 to about 10 nucleotides (eg, about 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides) downstream of the T / C rich sequence.

[0330] In some embodiments, the deletion begins within about 10 to about 15 nucleotides (eg, about 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) of the 5'-NTTN-3' sequence. In some embodiments, the deletion begins within about 10 to about 15 nucleotides (e.g., about 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) of the 5'-ATTA-3', 5'-ATTT-3', 5'-ATTG-3', 5'-ATTC-3', 5'-TTTA-3', 5'-TTTT-3', 5'-TTTG-3', 5'-TTTC-3', 5'-GTTA-3', 5'-GTTT-3', 5'-GTTG-3', 5'-GTTC-3', 5'-CTTA-3', 5'-CTTT-3', 5'-CTTG-3', or 5'-CTTC-3' sequence. In some embodiments, the deletion begins within about 10 to about 15 nucleotides (eg, about 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) of the T / C rich sequence.

[0331] In some embodiments, the deletion begins within about 10 to about 15 nucleotides (eg, about 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) downstream of the 5'-NTTN-3' sequence. In some embodiments, the deletion begins within about 10 to about 15 nucleotides (e.g., about 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) downstream of the 5'-ATTA-3', 5'-ATTT-3', 5'-ATTG-3', 5'-ATTC-3', 5'-TTTA-3', 5'-TTTT-3', 5'-TTTG-3', 5'-TTTC-3', 5'-GTTA-3', 5'-GTTT-3', 5'-GTTG-3', 5'-GTTC-3', 5'-CTTA-3', 5'-CTTT-3', 5'-CTTG-3', or 5'-CTTC-3' sequence. In some embodiments, the deletion begins within about 10 to about 15 nucleotides (eg, about 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) downstream of the T / C rich sequence.

[0332] In some embodiments, the deletion ends within about 20 to about 30 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) of the 5'-NTTN-3' sequence. In some embodiments, the deletion ends within about 20 to about 30 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) of the 5'-ATTA-3', 5'-ATTT-3', 5'-ATTG-3', 5'-ATTC-3', 5'-TTTA-3', 5'-TTTT-3', 5'-TTTG-3', 5'-TTTC-3', 5'-GTTA-3', 5'-GTTT-3', 5'-GTTG-3', 5'-GTTC-3', 5'-CTTA-3', 5'-CTTT-3', 5'-CTTG-3', or 5'-CTTC-3' sequence. In some embodiments, the deletion ends within about 20 to about 30 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) of the T / C rich sequence.

[0333] In some embodiments, the deletion ends within about 20 to about 30 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) downstream of the 5'-NTTN-3' sequence. In some embodiments, the deletion ends within about 20 to about 30 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) downstream of the 5'-ATTA-3', 5'-ATTT-3', 5'-ATTG-3', 5'-ATTC-3', 5'-TTTA-3', 5'-TTTT-3', 5'-TTTG-3', 5'-TTTC-3', 5'-GTTA-3', 5'-GTTT-3', 5'-GTTG-3', 5'-GTTC-3', 5'-CTTA-3', 5'-CTTT-3', 5'-CTTG-3', or 5'-CTTC-3' sequence. In some embodiments, the deletion ends within about 20 to about 30 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) downstream of the T / C rich sequence.

[0334] In some embodiments, the deletion ends within about 20 to about 25 nucleotides (eg, about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 nucleotides) of the 5'-NTTN-3' sequence. In some embodiments, the deletion ends within about 20 to about 25 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 nucleotides) of the 5'-ATTA-3', 5'-ATTT-3', 5'-ATTG-3', 5'-ATTC-3', 5'-TTTA-3', 5'-TTTT-3', 5'-TTTG-3', 5'-TTTC-3', 5'-GTTA-3', 5'-GTTT-3', 5'-GTTG-3', 5'-GTTC-3', 5'-CTTA-3', 5'-CTTT-3', 5'-CTTG-3', or 5'-CTTC-3' sequence. In some embodiments, the deletion ends within about 20 to about 25 nucleotides (eg, about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 nucleotides) of the T / C rich sequence.

[0335] In some embodiments, the deletion ends within about 20 to about 25 nucleotides (eg, about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 nucleotides) downstream of the 5'-NTTN-3' sequence. In some embodiments, the deletion ends within about 20 to about 25 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 nucleotides) downstream of the 5'-ATTA-3', 5'-ATTT-3', 5'-ATTG-3', 5'-ATTC-3', 5'-TTTA-3', 5'-TTTT-3', 5'-TTTG-3', 5'-TTTC-3', 5'-GTTA-3', 5'-GTTT-3', 5'-GTTG-3', 5'-GTTC-3', 5'-CTTA-3', 5'-CTTT-3', 5'-CTTG-3', or 5'-CTTC-3' sequence. In some embodiments, the deletion ends within about 20 to about 25 nucleotides (eg, about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 nucleotides) downstream of the T / C rich sequence.

[0336] In some embodiments, the deletion ends within about 25 to about 30 nucleotides (eg, about 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) of the 5'-NTTN-3' sequence. In some embodiments, the deletion ends within about 25 to about 30 nucleotides (e.g., about 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) of the 5'-ATTA-3', 5'-ATTT-3', 5'-ATTG-3', 5'-ATTC-3', 5'-TTTA-3', 5'-TTTT-3', 5'-TTTG-3', 5'-TTTC-3', 5'-GTTA-3', 5'-GTTT-3', 5'-GTTG-3', 5'-GTTC-3', 5'-CTTA-3', 5'-CTTT-3', 5'-CTTG-3', or 5'-CTTC-3' sequence. In some embodiments, the deletion ends within about 25 to about 30 nucleotides (eg, about 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) of the T / C rich sequence.

[0337] In some embodiments, the deletion ends within about 25 to about 30 nucleotides (eg, about 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) downstream of the 5'-NTTN-3' sequence. In some embodiments, the deletion ends within about 25 to about 30 nucleotides (e.g., about 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) downstream of the 5'-ATTA-3', 5'-ATTT-3', 5'-ATTG-3', 5'-ATTC-3', 5'-TTTA-3', 5'-TTTT-3', 5'-TTTG-3', 5'-TTTC-3', 5'-GTTA-3', 5'-GTTT-3', 5'-GTTG-3', 5'-GTTC-3', 5'-CTTA-3', 5'-CTTT-3', 5'-CTTG-3', or 5'-CTTC-3' sequence. In some embodiments, the deletion ends within about 25 to about 30 nucleotides (e.g., about 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) downstream of the T / C rich sequence.

[0338] In some embodiments, the deletion begins within about 5 to about 15 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) and ends within about 20 to about 30 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) of the 5'-NTTN-3' sequence. In some embodiments, the deletion is 5'-ATTA-3', 5'-ATTT-3', 5'-ATTG-3', 5'-ATTC-3', 5'-TTTA-3', 5'-TTTT-3', 5'-TTTG-3', 5'-TTTC-3', 5'-GTTA-3', 5'-GTTT-3', 5'-GTTG-3', 5'-GTTC-3', 5'-CTTA-3', 5'-CTTT-3', 5'-CTTG- It starts within about 5 to about 15 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) of the 3' or 5'-CTTC-3' sequence and ends within about 20 to about 30 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides). In some embodiments, the deletion begins within about 5 to about 15 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) and ends within about 20 to about 30 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) of the T / C-rich sequence.

[0339] In some embodiments, the deletion begins within about 5 to about 15 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) downstream of the 5'-NTTN-3' sequence and ends within about 20 to about 30 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) downstream of the 5'-NTTN-3' sequence. In some embodiments, the deletion begins within about 5 to about 15 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) downstream of the 5'-ATTA-3', 5'-ATTT-3', 5'-ATTG-3', 5'-ATTC-3', 5'-TTTA-3', 5'-TTTT-3', 5'-TTTG-3', 5'-TTTC-3', 5'-GTTA-3', 5'-GTTT-3', 5'-GTTG-3', 5'-GTTC-3', 5'-CTTA-3', 5'-CTTT-3', 5'-CTTG-3', or 5'-CTTC-3' sequence. and terminates within about 20 to about 30 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) downstream of the 5'-ATTA-3', 5'-ATTT-3', 5'-ATTG-3', 5'-ATTC-3', 5'-TTTA-3', 5'-TTTT-3', 5'-TTTG-3', 5'-TTTC-3', 5'-GTTA-3', 5'-GTTT-3', 5'-GTTG-3', 5'-GTTC-3', 5'-CTTA-3', 5'-CTTT-3', 5'-CTTG-3', or 5'-CTTC-3' sequence. In some embodiments, the deletion begins within about 5 to about 15 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) downstream of the T / C-rich sequence and ends within about 20 to about 30 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) downstream of the T / C-rich sequence.

[0340] In some embodiments, the deletion begins within about 5 to about 15 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) and ends within about 20 to about 25 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 nucleotides) of the 5'-NTTN-3' sequence. In some embodiments, the deletions are 5'-ATTA-3', 5'-ATTT-3', 5'-ATTG-3', 5'-ATTC-3', 5'-TTTA-3', 5'-TTTT-3', 5'-TTTG-3', 5'-TTTC-3', 5'-GTTA-3', 5'-GTTT-3', 5'-GTTG-3', 5'-GTTC-3', 5'-CTTA-3', 5'-CTTT ... It starts within about 5 to about 15 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) of the 5'-CTTG-3' or 5'-CTTC-3' sequence and ends within about 20 to about 25 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 nucleotides). In some embodiments, the deletion begins within about 5 to about 15 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) and ends within about 20 to about 25 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 nucleotides) of the T / C-rich sequence.

[0341] In some embodiments, the deletion begins within about 5 to about 15 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) downstream of the 5'-NTTN-3' sequence and ends within about 20 to about 25 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 nucleotides) downstream of the 5'-NTTN-3' sequence. In some embodiments, the deletion is about 5 to about 15 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) downstream of the 5'-ATTA-3', 5'-ATTT-3', 5'-ATTG-3', 5'-ATTC-3', 5'-TTTA-3', 5'-TTTT-3', 5'-TTTG-3', 5'-TTTC-3', 5'-GTTA-3', 5'-GTTT-3', 5'-GTTG-3', 5'-GTTC-3', 5'-CTTA-3', 5'-CTTT-3', 5'-CTTG-3', or 5'-CTTC-3' sequence. and ends within about 20 to about 25 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 nucleotides) downstream of the 5'-ATTA-3', 5'-ATTT-3', 5'-ATTG-3', 5'-ATTC-3', 5'-TTTA-3', 5'-TTTT-3', 5'-TTTG-3', 5'-TTTC-3', 5'-GTTA-3', 5'-GTTT-3', 5'-GTTG-3', 5'-GTTC-3', 5'-CTTA-3', 5'-CTTT-3', 5'-CTTG-3', or 5'-CTTC-3' sequence. In some embodiments, the deletion begins within about 5 to about 15 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) downstream of the T / C rich sequence and ends within about 20 to about 25 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 nucleotides) downstream of the T / C rich sequence.

[0342] In some embodiments, the deletion begins within about 5 to about 15 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) and ends within about 25 to about 30 nucleotides (e.g., about 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) of the 5'-NTTN-3' sequence. In some embodiments, the deletions are 5'-ATTA-3', 5'-ATTT-3', 5'-ATTG-3', 5'-ATTC-3', 5'-TTTA-3', 5'-TTTT-3', 5'-TTTG-3', 5'-TTTC-3', 5'-GTTA-3', 5'-GTTT-3', 5'-GTTG-3', 5'-GTTC-3', 5'-CTTA-3', 5'-CTTT ... It starts within about 5 to about 15 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) of the 5'-CTTG-3' or 5'-CTTC-3' sequence and ends within about 25 to about 30 nucleotides (e.g., about 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides). In some embodiments, the deletion begins within about 5 to about 15 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) and ends within about 25 to about 30 nucleotides (e.g., about 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) of the T / C-rich sequence.

[0343] In some embodiments, the deletion begins within about 5 to about 15 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) downstream of the 5'-NTTN-3' sequence and ends within about 25 to about 30 nucleotides (e.g., about 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) downstream of the 5'-NTTN-3' sequence. In some embodiments, the deletion is about 5 to about 15 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) downstream of the 5'-ATTA-3', 5'-ATTT-3', 5'-ATTG-3', 5'-ATTC-3', 5'-TTTA-3', 5'-TTTT-3', 5'-TTTG-3', 5'-TTTC-3', 5'-GTTA-3', 5'-GTTT-3', 5'-GTTG-3', 5'-GTTC-3', 5'-CTTA-3', 5'-CTTT-3', 5'-CTTG-3', or 5'-CTTC-3' sequence. and ends within about 25 to about 30 nucleotides (e.g., about 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) downstream of the 5'-ATTA-3', 5'-ATTT-3', 5'-ATTG-3', 5'-ATTC-3', 5'-TTTA-3', 5'-TTTT-3', 5'-TTTG-3', 5'-TTTC-3', 5'-GTTA-3', 5'-GTTT-3', 5'-GTTG-3', 5'-GTTC-3', 5'-CTTA-3', 5'-CTTT-3', 5'-CTTG-3', or 5'-CTTC-3' sequence. In some embodiments, the deletion begins within about 5 to about 15 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) downstream of the T / C rich sequence and ends within about 25 to about 30 nucleotides (e.g., about 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) downstream of the T / C rich sequence.

[0344] In some embodiments, the deletion begins within about 5 to about 10 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides) and ends within about 20 to about 30 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) of the 5'-NTTN-3' sequence. In some embodiments, the deletions are 5'-ATTA-3', 5'-ATTT-3', 5'-ATTG-3', 5'-ATTC-3', 5'-TTTA-3', 5'-TTTT-3', 5'-TTTG-3', 5'-TTTC-3', 5'-GTTA-3', 5'-GTTT-3', 5'-GTTG-3', 5'-GTTC-3', 5'-CTTA-3', 5'-CTTT ... It starts within about 5 to about 10 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides) of the 5'-CTTG-3' or 5'-CTTC-3' sequence and ends within about 20 to about 30 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides). In some embodiments, the deletion begins within about 5 to about 10 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides) and ends within about 20 to about 30 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) of the T / C-rich sequence.

[0345] In some embodiments, the deletion begins within about 5 to about 10 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides) downstream of the 5'-NTTN-3' sequence and ends within about 20 to about 30 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) downstream of the 5'-NTTN-3' sequence. In some embodiments, the deletion begins within about 5 to about 10 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides) downstream of the 5'-ATTA-3', 5'-ATTT-3', 5'-ATTG-3', 5'-ATTC-3', 5'-TTTA-3', 5'-TTTT-3', 5'-TTTG-3', 5'-TTTC-3', 5'-GTTA-3', 5'-GTTT-3', 5'-GTTG-3', 5'-GTTC-3', 5'-CTTA-3', 5'-CTTT-3', 5'-CTTG-3', or 5'-CTTC-3' sequence and terminates with the 5'-AT and terminates within about 20 to about 30 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) downstream of the 5'-ATTT-3', 5'-ATTG-3', 5'-ATTC-3', 5'-TTTA-3', 5'-TTTT-3', 5'-TTTG-3', 5'-TTTC-3', 5'-GTTA-3', 5'-GTTT-3', 5'-GTTG-3', 5'-GTTC-3', 5'-CTTA-3', 5'-CTTT-3', 5'-CTTG-3', or 5'-CTTC-3' sequence. In some embodiments, the deletion begins within about 5 to about 10 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides) downstream of the T / C rich sequence and ends within about 20 to about 30 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) downstream of the T / C rich sequence.

[0346] In some embodiments, the deletion begins within about 5 to about 10 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides) and ends within about 20 to about 25 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 nucleotides) of the 5'-NTTN-3' sequence. The deletion begins within about 5 to about 10 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides) and ends within about 20 to about 25 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 nucleotides) of the TTT-3', 5'-CTTG-3', or 5'-CTTC-3' sequence. In some embodiments, the deletion begins within about 5 to about 10 nucleotides and ends within about 20 to about 25 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 nucleotides) (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides) of the T / C rich sequence.

[0347] In some embodiments, the deletion begins within about 5 to about 10 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides) downstream of the 5'-NTTN-3' sequence and ends within about 20 to about 25 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 nucleotides) downstream of the 5'-NTTN-3' sequence. In some embodiments, the deletion begins within about 5 to about 10 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides) downstream of the 5'-ATTA-3', 5'-ATTT-3', 5'-ATTG-3', 5'-ATTC-3', 5'-TTTA-3', 5'-TTTT-3', 5'-TTTG-3', 5'-TTTC-3', 5'-GTTA-3', 5'-GTTT-3', 5'-GTTG-3', 5'-GTTC-3', 5'-CTTA-3', 5'-CTTT-3', 5'-CTTG-3', or 5'-CTTC-3' sequence. and terminates within about 20 to about 25 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 nucleotides) downstream of the 5'-ATTA-3', 5'-ATTT-3', 5'-ATTG-3', 5'-ATTC-3', 5'-TTTA-3', 5'-TTTT-3', 5'-TTTG-3', 5'-TTTC-3', 5'-GTTA-3', 5'-GTTT-3', 5'-GTTG-3', 5'-GTTC-3', 5'-CTTA-3', 5'-CTTT-3', 5'-CTTG-3', or 5'-CTTC-3' sequence. In some embodiments, the deletion begins within about 5 to about 10 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides) downstream of the T / C-rich sequence and ends within about 20 to about 25 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 nucleotides) downstream of the T / C-rich sequence.

[0348] In some embodiments, the deletion begins within about 5 to about 10 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides) and ends within about 25 to about 30 nucleotides (e.g., about 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) of the 5'-NTTN-3' sequence. In some embodiments, the deletion begins within about 5 to about 10 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides) and ends within about 25 to about 30 nucleotides (e.g., about 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) of the T / C rich sequence.

[0349] In some embodiments, the deletion begins within about 5 to about 10 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides) downstream of the 5'-NTTN-3' sequence and ends within about 25 to about 30 nucleotides (e.g., about 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) downstream of the 5'-NTTN-3' sequence. In some embodiments, the deletion begins within about 5 to about 10 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides) downstream of the 5'-ATTA-3', 5'-ATTT-3', 5'-ATTG-3', 5'-ATTC-3', 5'-TTTA-3', 5'-TTTT-3', 5'-TTTG-3', 5'-TTTC-3', 5'-GTTA-3', 5'-GTTT-3', 5'-GTTG-3', 5'-GTTC-3', 5'-CTTA-3', 5'-CTTT-3', 5'-CTTG-3', or 5'-CTTC-3' sequence. and terminates within about 25 to about 30 nucleotides (e.g., about 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) downstream of the 5'-ATTA-3', 5'-ATTT-3', 5'-ATTG-3', 5'-ATTC-3', 5'-TTTA-3', 5'-TTTT-3', 5'-TTTG-3', 5'-TTTC-3', 5'-GTTA-3', 5'-GTTT-3', 5'-GTTG-3', 5'-GTTC-3', 5'-CTTA-3', 5'-CTTT-3', 5'-CTTG-3', or 5'-CTTC-3' sequence. In some embodiments, the deletion begins within about 5 to about 10 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides) downstream of the T / C rich sequence and ends within about 25 to about 30 nucleotides (e.g., about 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) downstream of the T / C rich sequence.

[0350] In some embodiments, the deletion begins within about 10 to about 15 nucleotides (e.g., about 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) and ends within about 20 to about 30 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) of the 5'-NTTN-3' sequence. In some embodiments, the deletion is 5'-ATTA-3', 5'-ATTT-3', 5'-ATTG-3', 5'-ATTC-3', 5'-TTTA-3', 5'-TTTT-3', 5'-TTTG-3', 5'-TTTC-3', 5'-GTTA-3', 5'-GTTT-3', 5'-GTTG-3', 5'-GTTC-3', 5'-CTTA-3', 5'-CTTT-3', 5'-C It starts within about 10 to about 15 nucleotides (e.g., about 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) of the TTG-3' or 5'-CTTC-3' sequence and ends within about 20 to about 30 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides). In some embodiments, the deletion begins within about 10 to about 15 nucleotides (e.g., about 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) and ends within about 20 to about 30 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) of the T / C-rich sequence.

[0351] In some embodiments, the deletion begins within about 10 to about 15 nucleotides (e.g., about 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) downstream of the 5'-NTTN-3' sequence and ends within about 20 to about 30 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) downstream of the 5'-NTTN-3' sequence. In some embodiments, the deletion begins within about 10 to about 15 nucleotides (e.g., about 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) downstream of the 5'-ATTA-3', 5'-ATTT-3', 5'-ATTG-3', 5'-ATTC-3', 5'-TTTA-3', 5'-TTTT-3', 5'-TTTG-3', 5'-TTTC-3', 5'-GTTA-3', 5'-GTTT-3', 5'-GTTG-3', 5'-GTTC-3', 5'-CTTA-3', 5'-CTTT-3', 5'-CTTG-3', or 5'-CTTC-3' sequence and ends within 5'- It terminates within about 20 to about 30 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) downstream of the ATTA-3', 5'-ATTT-3', 5'-ATTG-3', 5'-ATTC-3', 5'-TTTA-3', 5'-TTTT-3', 5'-TTTG-3', 5'-TTTC-3', 5'-GTTA-3', 5'-GTTT-3', 5'-GTTG-3', 5'-GTTC-3', 5'-CTTA-3', 5'-CTTT-3', 5'-CTTG-3', or 5'-CTTC-3' sequence. In some embodiments, the deletion begins within about 10 to about 15 nucleotides (e.g., about 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) downstream of the T / C-rich sequence and ends within about 20 to about 30 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) downstream of the T / C-rich sequence.

[0352] In some embodiments, the deletion begins within about 10 to about 15 nucleotides (e.g., about 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) and ends within about 20 to about 25 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 nucleotides) of the 5'-NTTN-3' sequence. In some embodiments the deletion is 5'-ATTA-3', 5'-ATTT-3', 5'-ATTG-3', 5'-ATTC-3', 5'-TTTA-3', 5'-TTTT-3', 5'-TTTG-3', 5'-TTTC-3', 5'-GTTA-3', 5'-GTTT-3', 5'-GTTG-3', 5'-GTTC-3', 5'-CTTA-3', 5'-CTTT In some embodiments, the deletion begins within about 10 to about 15 nucleotides (e.g., about 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) of the 5'-CTTG-3', 5'-CTTC-3', or 5'-CTTG-3' sequence and ends within about 20 to about 25 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 nucleotides) of the T / C rich sequence.

[0353] In some embodiments, the deletion begins within about 10 to about 15 nucleotides (e.g., about 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) downstream of the 5'-NTTN-3' sequence and ends within about 20 to about 25 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 nucleotides) downstream of the 5'-NTTN-3' sequence. In some embodiments, the deletion is within about 10 to about 15 nucleotides (e.g., about 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) downstream of the 5'-ATTA-3', 5'-ATTT-3', 5'-ATTG-3', 5'-ATTC-3', 5'-TTTA-3', 5'-TTTT-3', 5'-TTTG-3', 5'-TTTC-3', 5'-GTTA-3', 5'-GTTT-3', 5'-GTTG-3', 5'-GTTC-3', 5'-CTTA-3', 5'-CTTT-3', 5'-CTTG-3', or 5'-CTTC-3' sequence. and ends within about 20 to about 25 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 nucleotides) downstream of the 5'-ATTA-3', 5'-ATTT-3', 5'-ATTG-3', 5'-ATTC-3', 5'-TTTA-3', 5'-TTTT-3', 5'-TTTG-3', 5'-TTTC-3', 5'-GTTA-3', 5'-GTTT-3', 5'-GTTG-3', 5'-GTTC-3', 5'-CTTA-3', 5'-CTTT-3', 5'-CTTG-3', or 5'-CTTC-3' sequence. In some embodiments, the deletion begins within about 10 to about 15 nucleotides (e.g., about 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) downstream of the T / C-rich sequence and ends within about 20 to about 25 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 nucleotides) downstream of the T / C-rich sequence.

[0354] In some embodiments, the deletion begins within about 10 to about 15 nucleotides (e.g., about 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) and ends within about 25 to about 30 nucleotides (e.g., about 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) of the 5'-NTTN-3' sequence. In some embodiments the deletion is 5'-ATTA-3', 5'-ATTT-3', 5'-ATTG-3', 5'-ATTC-3', 5'-TTTA-3', 5'-TTTT-3', 5'-TTTG-3', 5'-TTTC-3', 5'-GTTA-3', 5'-GTTT-3', 5'-GTTG-3', 5'-GTTC-3', 5'-CTTA-3', 5'-CTTT In some embodiments, the deletion begins within about 10 to about 15 nucleotides (e.g., about 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) and ends within about 25 to about 30 nucleotides (e.g., about 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) of the T / C-rich sequence.

[0355] In some embodiments, the deletion begins within about 10 to about 15 nucleotides (e.g., about 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) downstream of the 5'-NTTN-3' sequence and ends within about 25 to about 30 nucleotides (e.g., about 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) downstream of the 5'-NTTN-3' sequence. In some embodiments, the deletion is within about 10 to about 15 nucleotides (e.g., about 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) downstream of the 5'-ATTA-3', 5'-ATTT-3', 5'-ATTG-3', 5'-ATTC-3', 5'-TTTA-3', 5'-TTTT-3', 5'-TTTG-3', 5'-TTTC-3', 5'-GTTA-3', 5'-GTTT-3', 5'-GTTG-3', 5'-GTTC-3', 5'-CTTA-3', 5'-CTTT-3', 5'-CTTG-3', or 5'-CTTC-3' sequence. and ends within about 25 to about 30 nucleotides (e.g., about 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) downstream of the 5'-ATTA-3', 5'-ATTT-3', 5'-ATTG-3', 5'-ATTC-3', 5'-TTTA-3', 5'-TTTT-3', 5'-TTTG-3', 5'-TTTC-3', 5'-GTTA-3', 5'-GTTT-3', 5'-GTTG-3', 5'-GTTC-3', 5'-CTTA-3', 5'-CTTT-3', 5'-CTTG-3', or 5'-CTTC-3' sequence. In some embodiments, the deletion begins within about 10 to about 15 nucleotides (e.g., about 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) downstream of the T / C-rich sequence and ends within about 25 to about 30 nucleotides (e.g., about 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) downstream of the T / C-rich sequence.

[0356] In some embodiments, the deletion is up to about 50 nucleotides in length (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, or 55 nucleotides). In some embodiments, the deletion is up to about 40 nucleotides in length (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, or 45 nucleotides). In some embodiments, the deletion is about 4 nucleotides to about 40 nucleotides in length (e.g., about 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, or 45 nucleotides). In some embodiments, the deletion is about 4 nucleotides to about 25 nucleotides in length (e.g., about 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, or 28 nucleotides). In some embodiments, the deletion is about 10 to about 25 nucleotides in length (e.g., about 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 nucleotides). In some embodiments, the deletion is about 10 to about 15 nucleotides in length (e.g., about 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides).

[0357] In some embodiments, two or more RNA guides described herein are used to introduce a deletion having a length of more than 40 nucleotides. In some embodiments, two or more RNA guides described herein are used to introduce a deletion of at least about 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 16, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, or 400 nucleotides. In some embodiments, two or more RNA guides described herein are used to delete all or a portion of the STMN2 gene or SEQ ID NO:454.

[0358] In some embodiments, the methods described herein are used to engineer cells that contain a deletion described herein in the STMN2 gene. In some embodiments, the methods are performed using a complex that includes a Cas12i enzyme described herein and an RNA guide that includes a direct repeat sequence and a spacer sequence described herein.

[0359] In some embodiments, the RNA guide targeting STMN2 is encoded in a plasmid. In some embodiments, the RNA guide targeting STMN2 is synthetic or purified RNA. In some embodiments, the Cas12i polypeptide is encoded in a plasmid. In some embodiments, the Cas12i polypeptide is encoded by RNA, and the RNA is synthetic or purified.

[0360] C. Delivery Any of the components of the gene editing system disclosed herein can be formulated, for example, in a carrier, such as a carrier and / or a polymeric carrier, such as a liposome, and delivered to a cell (e.g., prokaryote, eukaryote, plant, mammal, etc.) by known methods, including, but not limited to, transfection (e.g., lipid-mediated, cationic polymer, calcium phosphate, dendrimers), electroporation or other methods of membrane disruption (e.g., nucleofection), viral delivery (e.g., lentivirus, retrovirus, adenovirus, adeno-associated virus (AAV)), microinjection, microprojectile bombardment ("gene gun"), fugene, direct sonic loading, cell squeezing, phototransfection, protoplast fusion, imparefection, magnetofection, exome-mediated transfer, lipid nanoparticle-mediated transfer, and any combination thereof.

[0361] In some embodiments, the method includes delivering one or more nucleic acids (e.g., nucleic acids encoding a Cas12i polypeptide, an RNA guide, donor DNA, etc.), one or more transcripts thereof, and / or a preformed RNA guide / Cas12i polypeptide complex to a cell, where a ternary complex is formed. In some embodiments, the RNA guide and the RNA encoding the Cas12i polypeptide are delivered together in a single composition. In some embodiments, the RNA guide and the RNA encoding the Cas12i polypeptide are delivered in separate compositions. In some embodiments, the RNA guide and the RNA encoding the Cas12i polypeptide delivered in separate compositions are delivered using the same delivery technology. In some embodiments, the RNA guide and the RNA encoding the Cas12i polypeptide delivered in separate compositions are delivered using different delivery technologies.

[0362] In some embodiments, the Cas12i component and the RNA guide component are delivered together. For example, the Cas12i component and the RNA guide component are packaged together in a single AAV particle. In another example, the Cas12i component and the RNA guide component are delivered together via lipid nanoparticles (LNPs). In some embodiments, the Cas12i component and the RNA guide component are delivered separately. For example, the Cas12i component and the RNA guide are packaged in separate AAV particles. In another example, the Cas12i component is delivered by a first delivery mechanism, and the RNA guide is delivered by a second delivery mechanism.

[0363] Exemplary intracellular delivery methods include, but are not limited to, viruses such as AAV, 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, plasmid or transposon delivery; particle-based methods such as the use of gene guns, magnetofection or magnetically assisted transfection, particle bombardment; and hybrid methods such as nucleofection. In some embodiments, the lipid nanoparticle comprises an mRNA encoding a Cas12i polypeptide, an RNA guide, or an mRNA and an RNA guide encoding a Cas12i polypeptide. In some embodiments, the mRNA encoding a Cas12i polypeptide is a transcript of the nucleotide sequence set forth in SEQ ID NO: 447 or SEQ ID NO: 481, or a variant thereof. In some embodiments, the application further provides cells produced by such methods, and organisms (e.g., animals, plants, or fungi) that contain or are produced from such cells.

[0364] D. Genetically Modified Cells Any of the gene editing systems disclosed herein can be delivered to a variety of cells. In some embodiments, the cell is an isolated cell. In some embodiments, the cell is in cell culture or a co-culture of two or more cell types. In some embodiments, the cell is ex vivo. In some embodiments, the cell is obtained from a living organism and maintained in cell culture. In some embodiments, the cell is a single-cell organism.

[0365] 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.

[0366] 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 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 rodent cell. In some embodiments, the cell is synthetically produced, often referred to as an artificial cell.

[0367] In some embodiments, the cells are 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 skilled in the art (see, for example, American Type Culture Collection (ATCC) (Manassas, Va.)). In some embodiments, the cells are immortal or immortalized cells.

[0368] In some embodiments, the cell is a primary 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 differentiated cell. For example, in some embodiments, the differentiated cell is a neural cell (e.g., a glial cell, such as an astrocyte, oligodendrocyte, microglia, or ependymal cell, or a neuron), 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, lymphocyte, neutrophil, eosinophil, basophil, macrophage, erythrocyte, or platelet), an epithelial cell, an immune cell (e.g., a lymphocyte, neutrophil, monocyte, or 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 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 cancer cell (e.g., a colorectal cancer cell, a renal cell carcinoma cell, a breast cancer cell, or a glioma cell). In some embodiments, the cell is a mammalian cell, e.g., a human 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.

[0369] Also within the scope of the present disclosure are any genetically modified cells produced using any of the gene editing systems disclosed herein. Such modified cells may contain a disrupted STMN2 gene.

[0370] Any of the gene editing systems, compositions, vectors, nucleic acids, RNA guides, and cells comprising the same disclosed herein can be used in therapy. The gene editing systems, compositions, vectors, nucleic acids, RNA guides, and cells disclosed herein can be used in methods of treating a disease or condition in a subject. Any suitable delivery or administration method known in the art can be used to deliver the compositions, vectors, nucleic acids, RNA guides, and cells disclosed herein. Such methods can include contacting a target sequence with the compositions, vectors, nucleic acids, or RNA guides disclosed herein. Such methods can include methods of editing STMN2 sequences disclosed herein. In some embodiments, cells engineered using the RNA guides disclosed herein are used for ex vivo gene therapy.

[0371] IV. Therapeutic uses Any of the gene editing systems disclosed herein or modified cells generated using such gene editing systems may be used to treat diseases associated with the STMN2 gene, such as neurodegenerative diseases (e.g., amyotrophic lateral sclerosis (ALS) or frontotemporal dementia (FTD)). Any suitable delivery or administration method known in the art may be used to deliver the compositions, vectors, nucleic acids, RNA guides, and cells disclosed herein. Such methods may include contacting a target sequence with a composition, vector, nucleic acid, or RNA guide disclosed herein. Such methods may include a method of editing a STMN2 sequence disclosed herein. In some embodiments, cells engineered using RNA guides disclosed herein are used for ex vivo gene therapy. In some embodiments, provided herein are methods for treating a target disease disclosed herein (e.g., a neurodegenerative 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 may be delivered to a specific tissue or specific cell type where gene editing is required. The gene editing system may 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 may be formulated to form a pharmaceutical composition, which may further include one or more pharma- ceutically acceptable carriers.

[0372] 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 the STMN2 gene. In some examples, the cells can be suspended in a suitable medium.

[0373] In some embodiments, compositions comprising gene editing systems or components thereof 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" refers to a discrete amount of a pharmaceutical composition (e.g., gene editing system or components thereof) administered to a subject, or a convenient fraction of such a dose, such as, for example, a half or a third of such a dose.

[0374] 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 components, including, but not limited to, suspending agents, stabilizing agents, or dispersing agents.

[0375] 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 sodium chloride water, 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 components of biodegradable polymer systems. Some compositions for sustained release or implantation may contain pharmaceutically acceptable polymers or hydrophobic materials, such as emulsions, ion exchange resins, poorly soluble polymers, or poorly soluble salts.

[0376] V. KITS AND THEIR USES The present disclosure also provides a kit that can be used to carry out the methods described herein, for example, for genetic modification of the STMN2 gene. In some embodiments, the kit includes an RNA guide and a Cas12i polypeptide. In some embodiments, the kit includes an RNA guide, a template DNA, and a Cas12i polypeptide. In some embodiments, the kit includes a polynucleotide encoding such a Cas12i polypeptide, optionally the polynucleotide is contained in a vector, for example, a vector described herein. In some embodiments, the kit includes a polynucleotide encoding an RNA guide disclosed herein. The Cas12i polypeptide (or a polynucleotide encoding a Cas12i polypeptide) (e.g., as a ribonucleoprotein) and the RNA guide can be packaged in the same or other containers in the kit, or in separate vials or other containers, and their contents can be mixed before use.

[0377] The Cas12i polypeptide, RNA guide, and template DNA may be packaged in the same or other containers within the kit, or may be packaged in separate vials or other containers, the contents of which may be mixed prior to use. In addition, the kit may optionally include buffers and / or instructions for use of the RNA guide, template DNA, and Cas12i polypeptide.

[0378] All references and publications cited herein are hereby incorporated by reference.

[0379] Additional Embodiments Additional embodiments are provided below and are also within the scope of the present disclosure.

[0380] Embodiment 1: A composition comprising an RNA guide, the RNA guide comprising (i) a spacer sequence that is substantially or fully complementary to a region on the non-PAM strand in the STMN2 gene (the complementary sequence of the target sequence), and (ii) a direct repeat sequence, wherein the target sequence is adjacent to a protospacer adjacent motif (PAM), and the PAM comprises the sequence 5'-NTTN-3'.

[0381] In embodiment 1, the target sequence can be within exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, or an intron of the STMN2 gene. In some examples, the STMN2 gene comprises the sequence of SEQ ID NO:454, the reverse complement of SEQ ID NO:454, a variant of SEQ ID NO:454, or the reverse complement of the variant of SEQ ID NO:454.

[0382] In embodiment 1, the spacer sequence is selected from the group consisting of: (a) nucleotides 1 to 16 of a sequence that is at least 90% identical to any one of SEQ ID NOs: 229 to 446 or 2497 to 4502; (b) nucleotides 1 to 17 of a sequence that is at least 90% identical to any one of SEQ ID NOs: 229 to 446 or 2497 to 4502; (c) nucleotides 1 to 18 of a sequence that is at least 90% identical to any one of SEQ ID NOs: 229 to 446 or 2497 to 4502; (d) nucleotides 1 to 19 of a sequence that is at least 90% identical to any one of SEQ ID NOs: 229 to 446 or 2497 to 4502;(f) nucleotides 1 to 21 of a sequence that is at least 90% identical to any one of SEQ ID NOs: 229 to 446 or 2497 to 4502; (g) nucleotides 1 to 22 of a sequence that is at least 90% identical to any one of SEQ ID NOs: 229 to 446 or 2497 to 4502; (h) nucleotides 1 to 23 of a sequence that is at least 90% identical to any one of SEQ ID NOs: 229 to 446 or 2497 to 4502; (i) nucleotides 1 to 24 of a sequence that is at least 90% identical to any one of SEQ ID NOs: 229 to 446 or 2497 to 4502; (j) nucleotides 1 to 25 of a sequence that is at least 90% identical to any one of SEQ ID NOs: 229 to 446 or 2497 to 4502 (k) nucleotides 1 to 26 of a sequence that is at least 90% identical to any one of SEQ ID NOs: 229 to 446 or 2497 to 4502; (l) nucleotides 1 to 27 of a sequence that is at least 90% identical to any one of SEQ ID NOs: 229 to 446 or 2497 to 4502; (m) nucleotides 1 to 28 of a sequence that is at least 90% identical to any one of SEQ ID NOs: 229 to 446 or 2497 to 4502; (n) nucleotides 1 to 29 of a sequence that is at least 90% identical to any one of SEQ ID NOs: 229 to 446 or 2497 to 4502; or (o) nucleotides 1 to 30 of a sequence that is at least 90% identical to any one of SEQ ID NOs: 229 to 446 or 2497 to 4502.

[0383] In any of the compositions of embodiment 1, the spacer sequence is selected from the group consisting of: (a) nucleotides 1 to 16 of any one of SEQ ID NOs: 229 to 446 or 2497 to 4502; (b) nucleotides 1 to 17 of any one of SEQ ID NOs: 229 to 446 or 2497 to 4502; (c) nucleotides 1 to 18 of any one of SEQ ID NOs: 229 to 446 or 2497 to 4502; (d) nucleotides 1 to 19 of any one of SEQ ID NOs: 229 to 446 or 2497 to 4502; (e) nucleotides 1 to 20 of any one of SEQ ID NOs: 229 to 446 or 2497 to 4502; (f) nucleotides 1 to 21 of any one of SEQ ID NOs: 229 to 446 or 2497 to 4502; (g) nucleotides 1 to 22 of any one of SEQ ID NOs: 229 to 446 or 2497 to 4502; (h) nucleotides 1 to 22 of any one of SEQ ID NOs: 229 to 446 or 2497 to 4502 or 2497 to 4502; (i) nucleotides 1 to 24 of any one of SEQ ID NOs: 229 to 446 or 2497 to 4502; (j) nucleotides 1 to 25 of any one of SEQ ID NOs: 229 to 446 or 2497 to 4502; (k) nucleotides 1 to 26 of any one of SEQ ID NOs: 229 to 446 or 2497 to 4502; (l) nucleotides 1 to 27 of any one of SEQ ID NOs: 229 to 446 or 2497 to 4502; (m) nucleotides 1 to 28 of any one of SEQ ID NOs: 229 to 446 or 2497 to 4502; (n) nucleotides 1 to 29 of any one of SEQ ID NOs: 229 to 446 or 2497 to 4502; or (o) nucleotides 1 to 30 of any one of SEQ ID NOs: 229 to 446 or 2497 to 4502.

[0384] In any of the compositions of embodiment 1, the direct repeat sequence may be any of the following: (a) nucleotides 1 to 36 of a sequence that is at least 90% identical to any one of SEQ ID NOs: 1 to 8; (b) nucleotides 2 to 36 of a sequence that is at least 90% identical to any one of SEQ ID NOs: 1 to 8; (c) nucleotides 3 to 36 of a sequence that is at least 90% identical to any one of SEQ ID NOs: 1 to 8; (d) nucleotides 4 to 36 of a sequence that is at least 90% identical to any one of SEQ ID NOs: 1 to 8; (e) nucleotides 5 to 36 of a sequence that is at least 90% identical to any one of SEQ ID NOs: 1 to 8; (f) nucleotides 6 to 36 of a sequence that is at least 90% identical to any one of SEQ ID NOs: 1 to 8; (g) nucleotides 7 to 36 of a sequence that is at least 90% identical to any one of SEQ ID NOs: 1 to 8; (h) nucleotides 8 to 36 of a sequence that is at least 90% identical to any one of SEQ ID NOs: 1 to 8. (i) nucleotides 9 to 36 of a sequence that is at least 90% identical to any one of SEQ ID NOs: 1 to 8; (j) nucleotides 10 to 36 of a sequence that is at least 90% identical to any one of SEQ ID NOs: 1 to 8; (k) nucleotides 11 to 36 of a sequence that is at least 90% identical to any one of SEQ ID NOs: 1 to 8; (l) nucleotides 12 to 36 of a sequence that is at least 90% identical to any one of SEQ ID NOs: 1 to 8; (m) SEQ ID NO: (n) nucleotides 14 to 36 of a sequence that is at least 90% identical to any one of the sequences of SEQ ID NOs: 1 to 8; (o) nucleotides 1 to 34 of a sequence that is at least 90% identical to the sequence of SEQ ID NO: 9; (p) nucleotides 2 to 34 of a sequence that is at least 90% identical to the sequence of SEQ ID NO: 9; (q) nucleotides 3 to 34 of a sequence that is at least 90% identical to the sequence of SEQ ID NO: 9;(r) nucleotides 4 to 34 of a sequence that is at least 90% identical to the sequence of SEQ ID NO:9, (s) nucleotides 5 to 34 of a sequence that is at least 90% identical to the sequence of SEQ ID NO:9, (t) nucleotides 6 to 34 of a sequence that is at least 90% identical to the sequence of SEQ ID NO:9, (u) nucleotides 7 to 34 of a sequence that is at least 90% identical to the sequence of SEQ ID NO:9, (v) nucleotides 8 to 34 of a sequence that is at least 90% identical to the sequence of SEQ ID NO:9, (w) nucleotides 9 to 34 of a sequence that is at least 90% identical to the sequence of SEQ ID NO:9, (x) nucleotides 10 to 34 of a sequence that is at least 90% identical to the sequence of SEQ ID NO:9, (y) nucleotides 11 to 34 of a sequence that is at least 90% identical to the sequence of SEQ ID NO:9, (z) nucleotides 12 to 34 of a sequence that is at least 90% identical to the sequence of SEQ ID NO:9, or (aa) a sequence that is at least 90% identical to the sequence of SEQ ID NO:10 or a portion thereof.

[0385] In some examples, the direct repeat sequence is selected from the group consisting of: (a) nucleotide 1 to nucleotide 36 of any one of SEQ ID NOs: 1 to 8; (b) nucleotide 2 to nucleotide 36 of any one of SEQ ID NOs: 1 to 8; (c) nucleotide 3 to nucleotide 36 of any one of SEQ ID NOs: 1 to 8; (d) nucleotide 4 to nucleotide 36 of any one of SEQ ID NOs: 1 to 8; (e) nucleotide 5 to nucleotide 36 of any one of SEQ ID NOs: 1 to 8; (f) nucleotide 6 to nucleotide 36 of any one of SEQ ID NOs: 1 to 8; (g) nucleotide 7 to nucleotide 36 of any one of SEQ ID NOs: 1 to 8; (h) nucleotide 8 to nucleotide 36 of any one of SEQ ID NOs: 1 to 8; (i) nucleotide 9 to nucleotide 36 of any one of SEQ ID NOs: 1 to 8; (j) nucleotide 10 to nucleotide 36 of any one of SEQ ID NOs: 1 to 8; (k) nucleotide 11 to nucleotide 36 of any one of SEQ ID NOs: 1 to 8; (m) nucleotides 13 to 36 of any one of SEQ ID NOs: 1 to 8, (n) nucleotides 14 to 36 of any one of SEQ ID NOs: 1 to 8, (o) nucleotides 1 to 34 of SEQ ID NO: 9, (p) nucleotides 2 to 34 of SEQ ID NO: 9, (q) nucleotides 3 to 34 of SEQ ID NO: 9, (r) nucleotides 4 to 34 of SEQ ID NO: 9, (s) nucleotides 5 to 34 of SEQ ID NO: 9, (t) nucleotides 6 to 34 of SEQ ID NO: 9, (u) nucleotides 7 to 34 of SEQ ID NO: 9, (v) nucleotides 8 to 34 of SEQ ID NO: 9, (w) nucleotides 9 to 34 of SEQ ID NO: 9, (x) nucleotides 10 to 34 of SEQ ID NO: 9, (y) nucleotides 11 to 34 of SEQ ID NO: 9, (z) nucleotides 12 to 34 of SEQ ID NO: 9, or (aa) SEQ ID NO: 10 or a portion thereof.

[0386] In some examples, the direct repeat sequence is selected from the group consisting of (a) nucleotides 1 to 36 of a sequence that is at least 90% identical to any one of SEQ ID NOs: 462 to 479, (b) nucleotides 2 to 36 of a sequence that is at least 90% identical to any one of SEQ ID NOs: 462 to 479, (c) nucleotides 3 to 36 of a sequence that is at least 90% identical to any one of SEQ ID NOs: 462 to 479, (d) nucleotides 4 to 36 of a sequence that is at least 90% identical to any one of SEQ ID NOs: 462 to 479, (e) nucleotides 5 to 36 of a sequence that is at least 90% identical to any one of SEQ ID NOs: 462 to 479, (f) nucleotides 6 to 36 of a sequence that is at least 90% identical to any one of SEQ ID NOs: 462 to 479, (g) nucleotides 7 to 36 of a sequence that is at least 90% identical to any one of SEQ ID NOs: 462 to 479, (h) nucleotides 8 to 8 of a sequence that is at least 90% identical to any one of SEQ ID NOs: 462 to 479, (i) nucleotides 9 to 9 of a sequence that is at least 90% identical to any one of SEQ ID NOs: 462 to 479, (ii) nucleotides 10 to 12 of a sequence that is at least 90% identical to any one of SEQ ID NOs: 462 to 479, (iii) nucleotides 13 to 14 of a sequence that is at least 90% identical to any one of SEQ ID NOs: 462 to 479, (iv) nucleotides 15 to 1 (i) nucleotides 8 to 36 of a sequence that is at least 90% identical to any one of SEQ ID NOs: 462 to 479; (j) nucleotides 10 to 36 of a sequence that is at least 90% identical to any one of SEQ ID NOs: 462 to 479; (k) nucleotides 11 to 36 of a sequence that is at least 90% identical to any one of SEQ ID NOs: 462 to 479; (l) nucleotides 12 to 36 of a sequence that is at least 90% identical to any one of SEQ ID NOs: 462 to 479; (m) nucleotides 13 to 36 of a sequence that is at least 90% identical to any one of SEQ ID NOs: 462 to 479; (n) nucleotides 14 to 36 of a sequence that is at least 90% identical to any one of SEQ ID NOs: 462 to 479; or (o) a sequence that is at least 90% identical to the sequence of SEQ ID NO: 480 or a portion thereof.

[0387] In some examples, the direct repeat sequence is: (a) nucleotide 1 to nucleotide 36 of any one of SEQ ID NOs: 462-479; (b) nucleotide 2 to nucleotide 36 of any one of SEQ ID NOs: 462-479; (c) nucleotide 3 to nucleotide 36 of any one of SEQ ID NOs: 462-479; (d) nucleotide 4 to nucleotide 36 of any one of SEQ ID NOs: 462-479; (e) nucleotide 5 to nucleotide 36 of any one of SEQ ID NOs: 462-479; (f) nucleotide 6 to nucleotide 36 of any one of SEQ ID NOs: 462-479; (g) nucleotide 7 to nucleotide 36 of any one of SEQ ID NOs: 462-479; (h) (i) nucleotides 8 to 36 of any one of SEQ ID NOs: 462 to 479; (j) nucleotides 10 to 36 of any one of SEQ ID NOs: 462 to 479; (k) nucleotides 11 to 36 of any one of SEQ ID NOs: 462 to 479; (l) nucleotides 12 to 36 of any one of SEQ ID NOs: 462 to 479; (m) nucleotides 13 to 36 of any one of SEQ ID NOs: 462 to 479; (n) nucleotides 14 to 36 of any one of SEQ ID NOs: 462 to 479; or (o) SEQ ID NO: 480 or a portion thereof.

[0388] In some examples, the direct repeat sequence is selected from the group consisting of (a) nucleotides 1 to 36 of a sequence that is at least 90% identical to SEQ ID NO: 485, (b) nucleotides 2 to 36 of a sequence that is at least 90% identical to SEQ ID NO: 485, (c) nucleotides 3 to 36 of a sequence that is at least 90% identical to SEQ ID NO: 485, (d) nucleotides 4 to 36 of a sequence that is at least 90% identical to SEQ ID NO: 485, (e) nucleotides 5 to 36 of a sequence that is at least 90% identical to SEQ ID NO: 485, (f) nucleotides 6 to 36 of a sequence that is at least 90% identical to SEQ ID NO: 485, (g) nucleotides 7 to 36 of a sequence that is at least 90% identical to SEQ ID NO: 485, (h) nucleotides 8 to 8 of a sequence that is at least 90% identical to SEQ ID NO: 485, (i) nucleotides 1 to 36 of a sequence that is at least 90% identical to SEQ ID NO: 485, (ii) nucleotides 1 to 36 of a sequence that is at least 90% identical to SEQ ID NO: 485, (iii) nucleotides 1 to 36 of a sequence that is at least 90% identical to SEQ ID NO: 485, (iv) nucleotides 1 to 36 of a sequence that is at least 90% identical to SEQ ID NO: 485, (v) nucleotides 2 to 36 of a sequence that is at least 90% identical to SEQ ID NO: 485, (vi) nucleotides 3 to 36 of a sequence that is at least 90% identical to SEQ ID NO: 485, (vi) nucleotides 485, (i) nucleotides 9 to 36 of a sequence that is at least 90% identical to SEQ ID NO: 485, (j) nucleotides 10 to 36 of a sequence that is at least 90% identical to SEQ ID NO: 485, (k) nucleotides 11 to 36 of a sequence that is at least 90% identical to SEQ ID NO: 485, (l) nucleotides 12 to 36 of a sequence that is at least 90% identical to SEQ ID NO: 485, (m) nucleotides 13 to 36 of a sequence that is at least 90% identical to SEQ ID NO: 485, (n) nucleotides 14 to 36 of a sequence that is at least 90% identical to SEQ ID NO: 485, or (o) a sequence that is at least 90% identical to SEQ ID NO: 486 or SEQ ID NO: 487, or a portion thereof.

[0389] In some examples, the direct repeat sequence comprises (a) nucleotides 1 to 36 of SEQ ID NO:485, (b) nucleotides 2 to 36 of SEQ ID NO:485, (c) nucleotides 3 to 36 of SEQ ID NO:485, (d) nucleotides 4 to 36 of SEQ ID NO:485, (e) nucleotides 5 to 36 of SEQ ID NO:485, (f) nucleotides 6 to 36 of SEQ ID NO:485, (g) nucleotides 7 to 36 of SEQ ID NO:485, (h) nucleotides 8 to 36 of SEQ ID NO:485, (i) nucleotides 9 to 36 of SEQ ID NO:485, (j) nucleotides 10 to 36 of SEQ ID NO:485, (k) nucleotides 11 to 36 of SEQ ID NO:485, (l) nucleotides 12 to 36 of SEQ ID NO:485, (m) nucleotides 13 to 36 of SEQ ID NO:485, (n) nucleotides 14 to 36 of SEQ ID NO:485, or (o) SEQ ID NO:486 or SEQ ID NO:487, or a portion thereof.

[0390] In some examples, the direct repeat sequence is selected from the group consisting of (a) nucleotides 1 to 36 of a sequence that is at least 90% identical to the sequence of SEQ ID NO:488 or SEQ ID NO:489, (b) nucleotides 2 to 36 of a sequence that is at least 90% identical to the sequence of SEQ ID NO:488 or SEQ ID NO:489, (c) nucleotides 3 to 36 of a sequence that is at least 90% identical to the sequence of SEQ ID NO:488 or SEQ ID NO:489, (d) nucleotides 4 to 36 of a sequence that is at least 90% identical to the sequence of SEQ ID NO:488 or SEQ ID NO:489, (e) nucleotides 5 to 36 of a sequence that is at least 90% identical to the sequence of SEQ ID NO:488 or SEQ ID NO:489, (f) nucleotides 6 to 36 of a sequence that is at least 90% identical to the sequence of SEQ ID NO:488 or SEQ ID NO:489, (g) nucleotides 7 to 36 of a sequence that is at least 90% identical to the sequence of SEQ ID NO:488 or SEQ ID NO:489, (h) nucleotides 8 to 36 of a sequence that is at least 90% identical to the sequence of SEQ ID NO:488 or SEQ ID NO:489. (i) nucleotides 9 to 36 of a sequence that is at least 90% identical to the sequence of SEQ ID NO: 488 or SEQ ID NO: 489; (j) nucleotides 10 to 36 of a sequence that is at least 90% identical to the sequence of SEQ ID NO: 488 or SEQ ID NO: 489; (k) nucleotides 11 to 36 of a sequence that is at least 90% identical to the sequence of SEQ ID NO: 488 or SEQ ID NO: 489; (l) nucleotides 12 to 36 of a sequence that is at least 90% identical to the sequence of SEQ ID NO: 488 or SEQ ID NO: 489. (m) nucleotides 13 to 36 of a sequence that is at least 90% identical to the sequence of SEQ ID NO:488 or SEQ ID NO:489; (n) nucleotides 14 to 36 of a sequence that is at least 90% identical to the sequence of SEQ ID NO:488 or SEQ ID NO:489; (o) nucleotides 15 to 36 of a sequence that is at least 90% identical to the sequence of SEQ ID NO:488 or SEQ ID NO:489; or (p) a sequence that is at least 90% identical to the sequence of SEQ ID NO:490 or a portion thereof.

[0391] In some examples, the direct repeat sequence is: (a) nucleotide 1 to nucleotide 36 of SEQ ID NO:488 or SEQ ID NO:489; (b) nucleotide 2 to nucleotide 36 of SEQ ID NO:488 or SEQ ID NO:489; (c) nucleotide 3 to nucleotide 36 of SEQ ID NO:488 or SEQ ID NO:489; (d) nucleotide 4 to nucleotide 36 of SEQ ID NO:488 or SEQ ID NO:489; (e) nucleotide 5 to nucleotide 36 of SEQ ID NO:488 or SEQ ID NO:489; (f) nucleotide 6 to nucleotide 36 of SEQ ID NO:488 or SEQ ID NO:489; (g) nucleotide 7 to nucleotide 36 of SEQ ID NO:488 or SEQ ID NO:489; (h) nucleotide 8 to nucleotide 36 of SEQ ID NO:488 or SEQ ID NO:489 (i) nucleotides 9 to 36 of SEQ ID NO:488 or SEQ ID NO:489, (j) nucleotides 10 to 36 of SEQ ID NO:488 or SEQ ID NO:489, (k) nucleotides 11 to 36 of SEQ ID NO:488 or SEQ ID NO:489, (l) nucleotides 12 to 36 of SEQ ID NO:488 or SEQ ID NO:489, (m) nucleotides 13 to 36 of SEQ ID NO:488 or SEQ ID NO:489, (n) nucleotides 14 to 36 of SEQ ID NO:488 or SEQ ID NO:489, (o) nucleotides 15 to 36 of SEQ ID NO:488 or SEQ ID NO:489, or (p) SEQ ID NO:490, or a portion thereof.

[0392] In some examples, the spacer sequence is substantially complementary to the complement of any one of SEQ ID NOs: 11-228 or 491-2496.

[0393] In any of the compositions of embodiment 1, the PAM may comprise the sequence 5'-ATTA-3', 5'-ATTT-3', 5'-ATTG-3', 5'-ATTC-3', 5'-TTTA-3', 5'-TTTT-3', 5'-TTTG-3', 5'-TTTC-3', 5'-GTTA-3', 5'-GTTT-3', 5'-GTTG-3', 5'-GTTC-3', 5'-CTTA-3', 5'-CTTT-3', 5'-CTTG-3', or 5'-CTTC-3'.

[0394] In some instances, the target sequence is immediately adjacent to the PAM sequence.

[0395] In some examples, the RNA guide has a sequence that is at least 90% identical to any one of SEQ ID NOs: 4505-4562.

[0396] In some examples, the RNA guide has the sequence of any one of SEQ ID NOs: 4505-4562.

[0397] Embodiment 2: The composition of embodiment 1 may further comprise a Cas12i polypeptide or a polyribonucleotide encoding a Cas12i polypeptide, which may be one of the following: (a) a Cas12i2 polypeptide comprising a sequence at least 90% identical to the sequence of SEQ ID NO:448, SEQ ID NO:449, SEQ ID NO:450, SEQ ID NO:451, SEQ ID NO:452, or SEQ ID NO:453; (b) a Cas12i4 polypeptide comprising a sequence at least 90% identical to the sequence of SEQ ID NO:482, SEQ ID NO:483, or SEQ ID NO:484; (c) a Cas12i1 polypeptide comprising a sequence at least 90% identical to the sequence of SEQ ID NO:4503; or (d) a Cas12i3 polypeptide comprising a sequence at least 90% identical to the sequence of SEQ ID NO:4504.

[0398] In specific examples, the Cas12i polypeptide is (a) a Cas12i2 polypeptide comprising the sequence of SEQ ID NO: 448, SEQ ID NO: 449, SEQ ID NO: 450, SEQ ID NO: 451, SEQ ID NO: 452, or SEQ ID NO: 453; (b) a Cas12i4 polypeptide comprising the sequence of SEQ ID NO: 482, SEQ ID NO: 483, or SEQ ID NO: 484; (c) a Cas12i1 polypeptide comprising the sequence of SEQ ID NO: 4503; or (d) a Cas12i3 polypeptide comprising the sequence of SEQ ID NO: 4504.

[0399] In any of the compositions of embodiment 2, the RNA guide and the Cas12i polypeptide may form a ribonucleoprotein complex. In some examples, the ribonucleoprotein complex binds to a target nucleic acid. In some examples, the composition is present in a cell.

[0400] In any of the compositions of embodiment 2, the RNA guide and the Cas12i polypeptide may be encoded in a vector, for example, an expression vector. In some examples, the RNA guide and the Cas12i polypeptide are encoded in a single vector. In other examples, the RNA guide is encoded in a first vector and the Cas12i polypeptide is encoded in a second vector.

[0401] Embodiment 3: A vector system comprising one or more vectors, wherein the one or more vectors encode an RNA guide and a Cas12i polypeptide as disclosed herein. In some examples, the vector system comprises a first vector encoding an RNA guide as disclosed herein and a second vector encoding a Cas12i polypeptide. The vectors may be expression vectors.

[0402] Embodiment 4: A composition comprising an RNA guide and a Cas12i polypeptide, wherein the RNA guide comprises (i) a spacer sequence that is substantially complementary or fully complementary to a region on the non-PAM strand in the STMN2 gene (the complementary sequence of the target sequence), and (ii) a direct repeat sequence.

[0403] In some examples, the target sequence is within exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, or an intron of the STMN2 gene, and the STMN2 gene can include the sequence of SEQ ID NO: 454, the reverse complement of SEQ ID NO: 454, a variant of the sequence of SEQ ID NO: 454, or the reverse complement of the variant of SEQ ID NO: 454.

[0404] In some examples, the spacer sequence is selected from the group consisting of: (a) nucleotides 1 to 16 of a sequence that is at least 90% identical to any one of SEQ ID NOs: 229 to 446 or 2497 to 4502; (b) nucleotides 1 to 17 of a sequence that is at least 90% identical to any one of SEQ ID NOs: 229 to 446 or 2497 to 4502; (c) nucleotides 1 to 18 of a sequence that is at least 90% identical to any one of SEQ ID NOs: 229 to 446 or 2497 to 4502; (d) (e) nucleotides 1 to 20 of a sequence that is at least 90% identical to any one of SEQ ID NOs: 229 to 446 or 2497 to 4502; (f) nucleotides 1 to 21 of a sequence that is at least 90% identical to any one of SEQ ID NOs: 229 to 446 or 2497 to 4502; (g) nucleotides 1 to 21 of a sequence that is at least 90% identical to any one of SEQ ID NOs: 229 to 446 or 2497 to 4502 (h) nucleotides 1 to 23 of a sequence that is at least 90% identical to any one of SEQ ID NOs: 229 to 446 or 2497 to 4502; (i) nucleotides 1 to 24 of a sequence that is at least 90% identical to any one of SEQ ID NOs: 229 to 446 or 2497 to 4502; (j) nucleotides 1 to 25 of a sequence that is at least 90% identical to any one of SEQ ID NOs: 229 to 446 or 2497 to 4502. (k) nucleotides 1 to 26 of a sequence that is at least 90% identical to any one of SEQ ID NOs: 229 to 446 or 2497 to 4502; (l) nucleotides 1 to 27 of a sequence that is at least 90% identical to any one of SEQ ID NOs: 229 to 446 or 2497 to 4502; (m) nucleotides 1 to 28 of a sequence that is at least 90% identical to any one of SEQ ID NOs: 229 to 446 or 2497 to 4502;(n) nucleotides 1 to 29 of a sequence that is at least 90% identical to any one of SEQ ID NOs: 229 to 446 or 2497 to 4502, or (o) nucleotides 1 to 30 of a sequence that is at least 90% identical to any one of SEQ ID NOs: 229 to 446 or 2497 to 4502.

[0405] In some examples, the spacer sequence is: (a) nucleotides 1 to 16 of any one of SEQ ID NOs: 229 to 446 or 2497 to 4502; (b) nucleotides 1 to 17 of any one of SEQ ID NOs: 229 to 446 or 2497 to 4502; (c) nucleotides 1 to 18 of any one of SEQ ID NOs: 229 to 446 or 2497 to 4502; or (d) nucleotides 1 to 18 of any one of SEQ ID NOs: 229 to 446 or 2497 to 4502. (e) nucleotides 1 to 20 of any one of SEQ ID NOs: 229 to 446 or 2497 to 4502; (f) nucleotides 1 to 21 of any one of SEQ ID NOs: 229 to 446 or 2497 to 4502; (g) nucleotides 1 to 22 of any one of SEQ ID NOs: 229 to 446 or 2497 to 4502; (h) nucleotides 1 to 23 of any one of SEQ ID NOs: 229 to 446 or 2497 to 4502 (i) nucleotides 1 to 24 of any one of SEQ ID NOs: 229 to 446 or 2497 to 4502; (j) nucleotides 1 to 25 of any one of SEQ ID NOs: 229 to 446 or 2497 to 4502; (k) nucleotides 1 to 26 of any one of SEQ ID NOs: 229 to 446 or 2497 to 4502; (l) nucleotides 1 to 27 of any one of SEQ ID NOs: 229 to 446 or 2497 to 4502; (m) nucleotides 1 to 28 of any one of SEQ ID NOs: 229 to 446 or 2497 to 4502; (n) nucleotides 1 to 29 of any one of SEQ ID NOs: 229 to 446 or 2497 to 4502; or (o) nucleotides 1 to 30 of any one of SEQ ID NOs: 229 to 446 or 2497 to 4502.

[0406] In some examples, the direct repeat sequence is: (a) nucleotides 1 to 36 of a sequence that is at least 90% identical to any one of SEQ ID NOs: 1 to 8; (b) nucleotides 2 to 36 of a sequence that is at least 90% identical to any one of SEQ ID NOs: 1 to 8; (c) nucleotides 3 to 36 of a sequence that is at least 90% identical to any one of SEQ ID NOs: 1 to 8; (d) nucleotides 4 to 36 of a sequence that is at least 90% identical to any one of SEQ ID NOs: 1 to 8; (e) nucleotides 5 to 36 of a sequence that is at least 90% identical to any one of SEQ ID NOs: 1 to 8; (f) SIQ (g) nucleotides 6 to 36 of a sequence that is at least 90% identical to any one of SEQ ID NOs: 1 to 8; (h) nucleotides 8 to 36 of a sequence that is at least 90% identical to any one of SEQ ID NOs: 1 to 8; (i) nucleotides 9 to 36 of a sequence that is at least 90% identical to any one of SEQ ID NOs: 1 to 8; (j) nucleotides 10 to 36 of a sequence that is at least 90% identical to any one of SEQ ID NOs: 1 to 8; (k) nucleotides 11 to 16 of a sequence that is at least 90% identical to any one of SEQ ID NOs: 1 to 8. (l) nucleotides 12 to 36 of a sequence that is at least 90% identical to any one of SEQ ID NOs: 1 to 8; (m) nucleotides 13 to 36 of a sequence that is at least 90% identical to any one of SEQ ID NOs: 1 to 8; (n) nucleotides 14 to 36 of a sequence that is at least 90% identical to any one of SEQ ID NOs: 1 to 8; (o) nucleotides 1 to 34 of a sequence that is at least 90% identical to SEQ ID NO: 9; (p) nucleotides 2 to 34 of a sequence that is at least 90% identical to SEQ ID NO: 9; (q) nucleotides 3 to 34 of a sequence that is at least 90% identical to SEQ ID NO: 9;(r) nucleotides 4 to 34 of a sequence that is at least 90% identical to the sequence of SEQ ID NO:9, (s) nucleotides 5 to 34 of a sequence that is at least 90% identical to the sequence of SEQ ID NO:9, (t) nucleotides 6 to 34 of a sequence that is at least 90% identical to the sequence of SEQ ID NO:9, (u) nucleotides 7 to 34 of a sequence that is at least 90% identical to the sequence of SEQ ID NO:9, (v) nucleotides 8 to 34 of a sequence that is at least 90% identical to the sequence of SEQ ID NO:9, (w) nucleotides 9 to 34 of a sequence that is at least 90% identical to the sequence of SEQ ID NO:9, (x) nucleotides 10 to 34 of a sequence that is at least 90% identical to the sequence of SEQ ID NO:9, (y) nucleotides 11 to 34 of a sequence that is at least 90% identical to the sequence of SEQ ID NO:9, (z) nucleotides 12 to 34 of a sequence that is at least 90% identical to the sequence of SEQ ID NO:9, or (aa) a sequence that is at least 90% identical to the sequence of SEQ ID NO:10 or a portion thereof.

[0407] In some examples, the direct repeat sequence is selected from the group consisting of: (a) nucleotide 1 to nucleotide 36 of any one of SEQ ID NOs: 1 to 8; (b) nucleotide 2 to nucleotide 36 of any one of SEQ ID NOs: 1 to 8; (c) nucleotide 3 to nucleotide 36 of any one of SEQ ID NOs: 1 to 8; (d) nucleotide 4 to nucleotide 36 of any one of SEQ ID NOs: 1 to 8; (e) nucleotide 5 to nucleotide 36 of any one of SEQ ID NOs: 1 to 8; (f) nucleotide 6 to nucleotide 36 of any one of SEQ ID NOs: 1 to 8; (g) nucleotide 7 to nucleotide 36 of any one of SEQ ID NOs: 1 to 8; (h) nucleotide 8 to nucleotide 36 of any one of SEQ ID NOs: 1 to 8; (i) nucleotide 9 to nucleotide 36 of any one of SEQ ID NOs: 1 to 8; (j) nucleotide 10 to nucleotide 36 of any one of SEQ ID NOs: 1 to 8; (k) nucleotide 11 to nucleotide 36 of any one of SEQ ID NOs: 1 to 8; (m) nucleotides 13 to 36 of any one of SEQ ID NOs: 1 to 8, (n) nucleotides 14 to 36 of any one of SEQ ID NOs: 1 to 8, (o) nucleotides 1 to 34 of SEQ ID NO: 9, (p) nucleotides 2 to 34 of SEQ ID NO: 9, (q) nucleotides 3 to 34 of SEQ ID NO: 9, (r) nucleotides 4 to 34 of SEQ ID NO: 9, (s) nucleotides 5 to 34 of SEQ ID NO: 9, (t) nucleotides 6 to 34 of SEQ ID NO: 9, (u) nucleotides 7 to 34 of SEQ ID NO: 9, (v) nucleotides 8 to 34 of SEQ ID NO: 9, (w) nucleotides 9 to 34 of SEQ ID NO: 9, (x) nucleotides 10 to 34 of SEQ ID NO: 9, (y) nucleotides 11 to 34 of SEQ ID NO: 9, (z) nucleotides 12 to 34 of SEQ ID NO: 9, or (aa) SEQ ID NO: 10 or a portion thereof.

[0408] In some examples, the direct repeat sequence is selected from the group consisting of (a) nucleotides 1 to 36 of a sequence that is at least 90% identical to any one of SEQ ID NOs: 462 to 479, (b) nucleotides 2 to 36 of a sequence that is at least 90% identical to any one of SEQ ID NOs: 462 to 479, (c) nucleotides 3 to 36 of a sequence that is at least 90% identical to any one of SEQ ID NOs: 462 to 479, (d) nucleotides 4 to 36 of a sequence that is at least 90% identical to any one of SEQ ID NOs: 462 to 479, (e) nucleotides 5 to 36 of a sequence that is at least 90% identical to any one of SEQ ID NOs: 462 to 479, (f) nucleotides 6 to 36 of a sequence that is at least 90% identical to any one of SEQ ID NOs: 462 to 479, (g) nucleotides 7 to 36 of a sequence that is at least 90% identical to any one of SEQ ID NOs: 462 to 479, (h) nucleotides 8 to 8 of a sequence that is at least 90% identical to any one of SEQ ID NOs: 462 to 479, (i) nucleotides 9 to 9 of a sequence that is at least 90% identical to any one of SEQ ID NOs: 462 to 479, (ii) nucleotides 10 to 12 of a sequence that is at least 90% identical to any one of SEQ ID NOs: 462 to 479, (iii) nucleotides 13 to 14 of a sequence that is at least 90% identical to any one of SEQ ID NOs: 462 to 479, (iv) nucleotides 15 to 1 (i) nucleotides 8 to 36 of a sequence that is at least 90% identical to any one of SEQ ID NOs: 462 to 479; (j) nucleotides 10 to 36 of a sequence that is at least 90% identical to any one of SEQ ID NOs: 462 to 479; (k) nucleotides 11 to 36 of a sequence that is at least 90% identical to any one of SEQ ID NOs: 462 to 479; (l) nucleotides 12 to 36 of a sequence that is at least 90% identical to any one of SEQ ID NOs: 462 to 479; (m) nucleotides 13 to 36 of a sequence that is at least 90% identical to any one of SEQ ID NOs: 462 to 479; (n) nucleotides 14 to 36 of a sequence that is at least 90% identical to any one of SEQ ID NOs: 462 to 479; or (o) a sequence that is at least 90% identical to the sequence of SEQ ID NO: 480 or a portion thereof.

[0409] In some examples, the direct repeat sequence is: (a) nucleotide 1 to nucleotide 36 of any one of SEQ ID NOs: 462-479; (b) nucleotide 2 to nucleotide 36 of any one of SEQ ID NOs: 462-479; (c) nucleotide 3 to nucleotide 36 of any one of SEQ ID NOs: 462-479; (d) nucleotide 4 to nucleotide 36 of any one of SEQ ID NOs: 462-479; (e) nucleotide 5 to nucleotide 36 of any one of SEQ ID NOs: 462-479; (f) nucleotide 6 to nucleotide 36 of any one of SEQ ID NOs: 462-479; (g) nucleotide 7 to nucleotide 36 of any one of SEQ ID NOs: 462-479; (h) (i) nucleotides 8 to 36 of any one of SEQ ID NOs: 462 to 479; (j) nucleotides 10 to 36 of any one of SEQ ID NOs: 462 to 479; (k) nucleotides 11 to 36 of any one of SEQ ID NOs: 462 to 479; (l) nucleotides 12 to 36 of any one of SEQ ID NOs: 462 to 479; (m) nucleotides 13 to 36 of any one of SEQ ID NOs: 462 to 479; (n) nucleotides 14 to 36 of any one of SEQ ID NOs: 462 to 479; or (o) SEQ ID NO: 480 or a portion thereof.

[0410] In some examples, the direct repeat sequence is selected from the group consisting of (a) nucleotides 1 to 36 of a sequence that is at least 90% identical to SEQ ID NO: 485, (b) nucleotides 2 to 36 of a sequence that is at least 90% identical to SEQ ID NO: 485, (c) nucleotides 3 to 36 of a sequence that is at least 90% identical to SEQ ID NO: 485, (d) nucleotides 4 to 36 of a sequence that is at least 90% identical to SEQ ID NO: 485, (e) nucleotides 5 to 36 of a sequence that is at least 90% identical to SEQ ID NO: 485, (f) nucleotides 6 to 36 of a sequence that is at least 90% identical to SEQ ID NO: 485, (g) nucleotides 7 to 36 of a sequence that is at least 90% identical to SEQ ID NO: 485, (h) nucleotides 8 to 8 of a sequence that is at least 90% identical to SEQ ID NO: 485, (i) nucleotides 1 to 36 of a sequence that is at least 90% identical to SEQ ID NO: 485, (ii) nucleotides 1 to 36 of a sequence that is at least 90% identical to SEQ ID NO: 485, (iii) nucleotides 1 to 36 of a sequence that is at least 90% identical to SEQ ID NO: 485, (iv) nucleotides 1 to 36 of a sequence that is at least 90% identical to SEQ ID NO: 485, (v) nucleotides 2 to 36 of a sequence that is at least 90% identical to SEQ ID NO: 485, (vi) nucleotides 3 to 36 of a sequence that is at least 90% identical to SEQ ID NO: 485, (vi) nucleotides 485, (i) nucleotides 9 to 36 of a sequence that is at least 90% identical to SEQ ID NO: 485, (j) nucleotides 10 to 36 of a sequence that is at least 90% identical to SEQ ID NO: 485, (k) nucleotides 11 to 36 of a sequence that is at least 90% identical to SEQ ID NO: 485, (l) nucleotides 12 to 36 of a sequence that is at least 90% identical to SEQ ID NO: 485, (m) nucleotides 13 to 36 of a sequence that is at least 90% identical to SEQ ID NO: 485, (n) nucleotides 14 to 36 of a sequence that is at least 90% identical to SEQ ID NO: 485, or (o) a sequence that is at least 90% identical to SEQ ID NO: 486 or SEQ ID NO: 487, or a portion thereof.

[0411] In some examples, the direct repeat sequence comprises (a) nucleotides 1 to 36 of SEQ ID NO:485, (b) nucleotides 2 to 36 of SEQ ID NO:485, (c) nucleotides 3 to 36 of SEQ ID NO:485, (d) nucleotides 4 to 36 of SEQ ID NO:485, (e) nucleotides 5 to 36 of SEQ ID NO:485, (f) nucleotides 6 to 36 of SEQ ID NO:485, (g) nucleotides 7 to 36 of SEQ ID NO:485, (h) nucleotides 8 to 36 of SEQ ID NO:485, (i) nucleotides 9 to 36 of SEQ ID NO:485, (j) nucleotides 10 to 36 of SEQ ID NO:485, (k) nucleotides 11 to 36 of SEQ ID NO:485, (l) nucleotides 12 to 36 of SEQ ID NO:485, (m) nucleotides 13 to 36 of SEQ ID NO:485, (n) nucleotides 14 to 36 of SEQ ID NO:485, or (o) SEQ ID NO:486 or SEQ ID NO:487, or a portion thereof.

[0412] In some examples, the direct repeat sequence is selected from the group consisting of (a) nucleotides 1 to 36 of a sequence that is at least 90% identical to the sequence of SEQ ID NO:488 or SEQ ID NO:489, (b) nucleotides 2 to 36 of a sequence that is at least 90% identical to the sequence of SEQ ID NO:488 or SEQ ID NO:489, (c) nucleotides 3 to 36 of a sequence that is at least 90% identical to the sequence of SEQ ID NO:488 or SEQ ID NO:489, (d) nucleotides 4 to 36 of a sequence that is at least 90% identical to the sequence of SEQ ID NO:488 or SEQ ID NO:489, (e) nucleotides 5 to 36 of a sequence that is at least 90% identical to the sequence of SEQ ID NO:488 or SEQ ID NO:489, (f) nucleotides 6 to 36 of a sequence that is at least 90% identical to the sequence of SEQ ID NO:488 or SEQ ID NO:489, (g) nucleotides 7 to 36 of a sequence that is at least 90% identical to the sequence of SEQ ID NO:488 or SEQ ID NO:489, (h) nucleotides 8 to 36 of a sequence that is at least 90% identical to the sequence of SEQ ID NO:488 or SEQ ID NO:489. (i) nucleotides 9 to 36 of a sequence that is at least 90% identical to the sequence of SEQ ID NO: 488 or SEQ ID NO: 489; (j) nucleotides 10 to 36 of a sequence that is at least 90% identical to the sequence of SEQ ID NO: 488 or SEQ ID NO: 489; (k) nucleotides 11 to 36 of a sequence that is at least 90% identical to the sequence of SEQ ID NO: 488 or SEQ ID NO: 489; (l) nucleotides 12 to 36 of a sequence that is at least 90% identical to the sequence of SEQ ID NO: 488 or SEQ ID NO: 489. (m) nucleotides 13 to 36 of a sequence that is at least 90% identical to the sequence of SEQ ID NO:488 or SEQ ID NO:489; (n) nucleotides 14 to 36 of a sequence that is at least 90% identical to the sequence of SEQ ID NO:488 or SEQ ID NO:489; (o) nucleotides 15 to 36 of a sequence that is at least 90% identical to the sequence of SEQ ID NO:488 or SEQ ID NO:489; or (p) a sequence that is at least 90% identical to the sequence of SEQ ID NO:490 or a portion thereof.

[0413] In some examples, the direct repeat sequence is: (a) nucleotide 1 to nucleotide 36 of SEQ ID NO:488 or SEQ ID NO:489; (b) nucleotide 2 to nucleotide 36 of SEQ ID NO:488 or SEQ ID NO:489; (c) nucleotide 3 to nucleotide 36 of SEQ ID NO:488 or SEQ ID NO:489; (d) nucleotide 4 to nucleotide 36 of SEQ ID NO:488 or SEQ ID NO:489; (e) nucleotide 5 to nucleotide 36 of SEQ ID NO:488 or SEQ ID NO:489; (f) nucleotide 6 to nucleotide 36 of SEQ ID NO:488 or SEQ ID NO:489; (g) nucleotide 7 to nucleotide 36 of SEQ ID NO:488 or SEQ ID NO:489; (h) nucleotide 8 to nucleotide 36 of SEQ ID NO:488 or SEQ ID NO:489 (i) nucleotides 9 to 36 of SEQ ID NO:488 or SEQ ID NO:489, (j) nucleotides 10 to 36 of SEQ ID NO:488 or SEQ ID NO:489, (k) nucleotides 11 to 36 of SEQ ID NO:488 or SEQ ID NO:489, (l) nucleotides 12 to 36 of SEQ ID NO:488 or SEQ ID NO:489, (m) nucleotides 13 to 36 of SEQ ID NO:488 or SEQ ID NO:489, (n) nucleotides 14 to 36 of SEQ ID NO:488 or SEQ ID NO:489, (o) nucleotides 15 to 36 of SEQ ID NO:488 or SEQ ID NO:489, or (p) SEQ ID NO:490, or a portion thereof.

[0414] In any of the compositions of embodiment 4, the spacer sequence may be substantially complementary to the complement of any one of SEQ ID NOs: 11-228 or 491-2496.

[0415] In some instances, the target sequence is adjacent to a protospacer adjacent motif (PAM), and the PAM comprises the sequence 5'-NTTN-3'. In some instances, the PAM comprises the sequence 5'-ATTA-3', 5'-ATTT-3', 5'-ATTG-3', 5'-ATTC-3', 5'-TTTA-3', 5'-TTTT-3', 5'-TTTG-3', 5'-TTTC-3', 5'-GTTA-3', 5'-GTTT-3', 5'-GTTG-3', 5'-GTTC-3', 5'-CTTA-3', 5'-CTTT-3', 5'-CTTG-3', or 5'-CTTC-3'.

[0416] In some cases, the target sequence is immediately adjacent to the PAM sequence. In some cases, the target sequence is within 1, 2, 3, 4, or 5 nucleotides of the PAM sequence.

[0417] In any of the compositions of embodiment 4, the Cas12i polypeptide is (a) a Cas12i2 polypeptide comprising a sequence at least 90% identical to the sequence of SEQ ID NO:448, SEQ ID NO:449, SEQ ID NO:450, SEQ ID NO:451, SEQ ID NO:452, or SEQ ID NO:453; (b) a Cas12i4 polypeptide comprising a sequence at least 90% identical to the sequence of SEQ ID NO:482, SEQ ID NO:483, or SEQ ID NO:484; (c) a Cas12i1 polypeptide comprising a sequence at least 90% identical to the sequence of SEQ ID NO:4503; or (d) a Cas12i3 polypeptide comprising a sequence at least 90% identical to the sequence of SEQ ID NO:4504.

[0418] In some examples, the Cas12i polypeptide is (a) a Cas12i2 polypeptide comprising the sequence of SEQ ID NO: 448, SEQ ID NO: 449, SEQ ID NO: 450, SEQ ID NO: 451, SEQ ID NO: 452, or SEQ ID NO: 453; (b) a Cas12i4 polypeptide comprising the sequence of SEQ ID NO: 482, SEQ ID NO: 483, or SEQ ID NO: 484; (c) a Cas12i1 polypeptide comprising the sequence of SEQ ID NO: 4503; or (d) a Cas12i3 polypeptide comprising the sequence of SEQ ID NO: 4504.

[0419] In any of the compositions of embodiment 4, the RNA guide and the Cas12i polypeptide may form a ribonucleoprotein complex. In some instances, the ribonucleoprotein complex binds to the target nucleic acid.

[0420] In any of the compositions of embodiment 4, the composition may be present intracellularly.

[0421] In any of the compositions of embodiment 4, the RNA guide and the Cas12i polypeptide may be encoded in a vector, for example, an expression vector. In some examples, the RNA guide and the Cas12i polypeptide are encoded in a single vector. In other examples, the RNA guide is encoded in a first vector and the Cas12i polypeptide is encoded in a second vector.

[0422] Embodiment 5: A vector system comprising one or more vectors, wherein the one or more vectors encode an RNA guide and a Cas12i polypeptide as disclosed herein. In some examples, the vector system comprises a first vector encoding an RNA guide as disclosed herein and a second vector encoding a Cas12i polypeptide. In some examples, the vector is an expression vector.

[0423] Embodiment 6: An RNA guide comprising: (i) a spacer sequence that is substantially complementary or completely complementary to a region on the non-PAM strand in the STMN2 gene (the complementary sequence of the target sequence); and (ii) a direct repeat sequence.

[0424] In some examples, the target sequence is within exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, or an intron of the STMN2 gene, and the STMN2 gene can include the sequence of SEQ ID NO: 454, the reverse complement of SEQ ID NO: 454, a variant of the sequence of SEQ ID NO: 454, or the reverse complement of the variant of SEQ ID NO: 454.

[0425] In some examples, the spacer sequence is selected from the group consisting of: (a) nucleotides 1 to 16 of a sequence that is at least 90% identical to any one of SEQ ID NOs: 229 to 446 or 2497 to 4502; (b) nucleotides 1 to 17 of a sequence that is at least 90% identical to any one of SEQ ID NOs: 229 to 446 or 2497 to 4502; (c) nucleotides 1 to 18 of a sequence that is at least 90% identical to any one of SEQ ID NOs: 229 to 446 or 2497 to 4502; (d) (e) nucleotides 1 to 20 of a sequence that is at least 90% identical to any one of SEQ ID NOs: 229 to 446 or 2497 to 4502; (f) nucleotides 1 to 21 of a sequence that is at least 90% identical to any one of SEQ ID NOs: 229 to 446 or 2497 to 4502; (g) nucleotides 1 to 21 of a sequence that is at least 90% identical to any one of SEQ ID NOs: 229 to 446 or 2497 to 4502 (h) nucleotides 1 to 23 of a sequence that is at least 90% identical to any one of SEQ ID NOs: 229 to 446 or 2497 to 4502; (i) nucleotides 1 to 24 of a sequence that is at least 90% identical to any one of SEQ ID NOs: 229 to 446 or 2497 to 4502; (j) nucleotides 1 to 25 of a sequence that is at least 90% identical to any one of SEQ ID NOs: 229 to 446 or 2497 to 4502. (k) nucleotides 1 to 26 of a sequence that is at least 90% identical to any one of SEQ ID NOs: 229 to 446 or 2497 to 4502; (l) nucleotides 1 to 27 of a sequence that is at least 90% identical to any one of SEQ ID NOs: 229 to 446 or 2497 to 4502; (m) nucleotides 1 to 28 of a sequence that is at least 90% identical to any one of SEQ ID NOs: 229 to 446 or 2497 to 4502;(n) nucleotides 1 to 29 of a sequence that is at least 90% identical to any one of SEQ ID NOs: 229 to 446 or 2497 to 4502, or (o) nucleotides 1 to 30 of a sequence that is at least 90% identical to any one of SEQ ID NOs: 229 to 446 or 2497 to 4502.

[0426] In some examples, the spacer sequence is: (a) nucleotides 1 to 16 of any one of SEQ ID NOs: 229 to 446 or 2497 to 4502; (b) nucleotides 1 to 17 of any one of SEQ ID NOs: 229 to 446 or 2497 to 4502; (c) nucleotides 1 to 18 of any one of SEQ ID NOs: 229 to 446 or 2497 to 4502; or (d) nucleotides 1 to 18 of any one of SEQ ID NOs: 229 to 446 or 2497 to 4502. (e) nucleotides 1 to 20 of any one of SEQ ID NOs: 229 to 446 or 2497 to 4502; (f) nucleotides 1 to 21 of any one of SEQ ID NOs: 229 to 446 or 2497 to 4502; (g) nucleotides 1 to 22 of any one of SEQ ID NOs: 229 to 446 or 2497 to 4502; (h) nucleotides 1 to 23 of any one of SEQ ID NOs: 229 to 446 or 2497 to 4502 (i) nucleotides 1 to 24 of any one of SEQ ID NOs: 229 to 446 or 2497 to 4502; (j) nucleotides 1 to 25 of any one of SEQ ID NOs: 229 to 446 or 2497 to 4502; (k) nucleotides 1 to 26 of any one of SEQ ID NOs: 229 to 446 or 2497 to 4502; (l) nucleotides 1 to 27 of any one of SEQ ID NOs: 229 to 446 or 2497 to 4502; (m) nucleotides 1 to 28 of any one of SEQ ID NOs: 229 to 446 or 2497 to 4502; (n) nucleotides 1 to 29 of any one of SEQ ID NOs: 229 to 446 or 2497 to 4502; or (o) nucleotides 1 to 30 of any one of SEQ ID NOs: 229 to 446 or 2497 to 4502.

[0427] In some examples, the direct repeat sequence is selected from the group consisting of: (a) nucleotides 1 to 36 of a sequence that is at least 90% identical to any one of SEQ ID NOs: 1 to 8; (b) nucleotides 2 to 36 of a sequence that is at least 90% identical to any one of SEQ ID NOs: 1 to 8; (c) nucleotides 3 to 36 of a sequence that is at least 90% identical to any one of SEQ ID NOs: 1 to 8; (d) nucleotides 4 to 36 of a sequence that is at least 90% identical to any one of SEQ ID NOs: 1 to 8; (e) nucleotides 5 to 36 of a sequence that is at least 90% identical to any one of SEQ ID NOs: 1 to 8; (f) an IEQ (g) nucleotides 6 to 36 of a sequence that is at least 90% identical to any one of SEQ ID NOs: 1 to 8; (h) nucleotides 8 to 36 of a sequence that is at least 90% identical to any one of SEQ ID NOs: 1 to 8; (i) nucleotides 9 to 36 of a sequence that is at least 90% identical to any one of SEQ ID NOs: 1 to 8; (j) nucleotides 10 to 36 of a sequence that is at least 90% identical to any one of SEQ ID NOs: 1 to 8; (k) nucleotides 11 to 16 of a sequence that is at least 90% identical to any one of SEQ ID NOs: 1 to 8. (l) nucleotides 12 to 36 of a sequence that is at least 90% identical to any one of SEQ ID NOs: 1 to 8; (m) nucleotides 13 to 36 of a sequence that is at least 90% identical to any one of SEQ ID NOs: 1 to 8; (n) nucleotides 14 to 36 of a sequence that is at least 90% identical to any one of SEQ ID NOs: 1 to 8; (o) nucleotides 1 to 34 of a sequence that is at least 90% identical to SEQ ID NO: 9; (p) nucleotides 2 to 34 of a sequence that is at least 90% identical to SEQ ID NO: 9; (q) nucleotides 3 to 34 of a sequence that is at least 90% identical to SEQ ID NO: 9;(r) nucleotides 4 to 34 of a sequence that is at least 90% identical to the sequence of SEQ ID NO:9, (s) nucleotides 5 to 34 of a sequence that is at least 90% identical to the sequence of SEQ ID NO:9, (t) nucleotides 6 to 34 of a sequence that is at least 90% identical to the sequence of SEQ ID NO:9, (u) nucleotides 7 to 34 of a sequence that is at least 90% identical to the sequence of SEQ ID NO:9, (v) nucleotides 8 to 34 of a sequence that is at least 90% identical to the sequence of SEQ ID NO:9, (w) nucleotides 9 to 34 of a sequence that is at least 90% identical to the sequence of SEQ ID NO:9, (x) nucleotides 10 to 34 of a sequence that is at least 90% identical to the sequence of SEQ ID NO:9, (y) nucleotides 11 to 34 of a sequence that is at least 90% identical to the sequence of SEQ ID NO:9, (z) nucleotides 12 to 34 of a sequence that is at least 90% identical to the sequence of SEQ ID NO:9, or (aa) a sequence that is at least 90% identical to the sequence of SEQ ID NO:10 or a portion thereof.

[0428] In some examples, the direct repeat sequence is selected from the group consisting of: (a) nucleotide 1 to nucleotide 36 of any one of SEQ ID NOs: 1 to 8; (b) nucleotide 2 to nucleotide 36 of any one of SEQ ID NOs: 1 to 8; (c) nucleotide 3 to nucleotide 36 of any one of SEQ ID NOs: 1 to 8; (d) nucleotide 4 to nucleotide 36 of any one of SEQ ID NOs: 1 to 8; (e) nucleotide 5 to nucleotide 36 of any one of SEQ ID NOs: 1 to 8; (f) nucleotide 6 to nucleotide 36 of any one of SEQ ID NOs: 1 to 8; (g) nucleotide 7 to nucleotide 36 of any one of SEQ ID NOs: 1 to 8; (h) nucleotide 8 to nucleotide 36 of any one of SEQ ID NOs: 1 to 8; (i) nucleotide 9 to nucleotide 36 of any one of SEQ ID NOs: 1 to 8; (j) nucleotide 10 to nucleotide 36 of any one of SEQ ID NOs: 1 to 8; (k) nucleotide 11 to nucleotide 36 of any one of SEQ ID NOs: 1 to 8; (m) nucleotides 13 to 36 of any one of SEQ ID NOs: 1 to 8, (n) nucleotides 14 to 36 of any one of SEQ ID NOs: 1 to 8, (o) nucleotides 1 to 34 of SEQ ID NO: 9, (p) nucleotides 2 to 34 of SEQ ID NO: 9, (q) nucleotides 3 to 34 of SEQ ID NO: 9, (r) nucleotides 4 to 34 of SEQ ID NO: 9, (s) nucleotides 5 to 34 of SEQ ID NO: 9, (t) nucleotides 6 to 34 of SEQ ID NO: 9, (u) nucleotides 7 to 34 of SEQ ID NO: 9, (v) nucleotides 8 to 34 of SEQ ID NO: 9, (w) nucleotides 9 to 34 of SEQ ID NO: 9, (x) nucleotides 10 to 34 of SEQ ID NO: 9, (y) nucleotides 11 to 34 of SEQ ID NO: 9, (z) nucleotides 12 to 34 of SEQ ID NO: 9, or (aa) SEQ ID NO: 10 or a portion thereof.

[0429] In some examples, the direct repeat sequence is selected from the group consisting of (a) nucleotides 1 to 36 of a sequence that is at least 90% identical to any one of SEQ ID NOs: 462 to 479, (b) nucleotides 2 to 36 of a sequence that is at least 90% identical to any one of SEQ ID NOs: 462 to 479, (c) nucleotides 3 to 36 of a sequence that is at least 90% identical to any one of SEQ ID NOs: 462 to 479, (d) nucleotides 4 to 36 of a sequence that is at least 90% identical to any one of SEQ ID NOs: 462 to 479, (e) nucleotides 5 to 36 of a sequence that is at least 90% identical to any one of SEQ ID NOs: 462 to 479, (f) nucleotides 6 to 36 of a sequence that is at least 90% identical to any one of SEQ ID NOs: 462 to 479, (g) nucleotides 7 to 36 of a sequence that is at least 90% identical to any one of SEQ ID NOs: 462 to 479, (h) nucleotides 8 to 8 of a sequence that is at least 90% identical to any one of SEQ ID NOs: 462 to 479, (i) nucleotides 9 to 9 of a sequence that is at least 90% identical to any one of SEQ ID NOs: 462 to 479, (ii) nucleotides 10 to 12 of a sequence that is at least 90% identical to any one of SEQ ID NOs: 462 to 479, (iii) nucleotides 13 to 14 of a sequence that is at least 90% identical to any one of SEQ ID NOs: 462 to 479, (iv) nucleotides 15 to 1 (i) nucleotides 8 to 36 of a sequence that is at least 90% identical to any one of SEQ ID NOs: 462 to 479; (j) nucleotides 10 to 36 of a sequence that is at least 90% identical to any one of SEQ ID NOs: 462 to 479; (k) nucleotides 11 to 36 of a sequence that is at least 90% identical to any one of SEQ ID NOs: 462 to 479; (l) nucleotides 12 to 36 of a sequence that is at least 90% identical to any one of SEQ ID NOs: 462 to 479; (m) nucleotides 13 to 36 of a sequence that is at least 90% identical to any one of SEQ ID NOs: 462 to 479; (n) nucleotides 14 to 36 of a sequence that is at least 90% identical to any one of SEQ ID NOs: 462 to 479; or (o) a sequence that is at least 90% identical to the sequence of SEQ ID NO: 480 or a portion thereof.

[0430] In some examples, the direct repeat sequence is: (a) nucleotide 1 to nucleotide 36 of any one of SEQ ID NOs: 462-479; (b) nucleotide 2 to nucleotide 36 of any one of SEQ ID NOs: 462-479; (c) nucleotide 3 to nucleotide 36 of any one of SEQ ID NOs: 462-479; (d) nucleotide 4 to nucleotide 36 of any one of SEQ ID NOs: 462-479; (e) nucleotide 5 to nucleotide 36 of any one of SEQ ID NOs: 462-479; (f) nucleotide 6 to nucleotide 36 of any one of SEQ ID NOs: 462-479; (g) nucleotide 7 to nucleotide 36 of any one of SEQ ID NOs: 462-479; (h) (i) nucleotides 8 to 36 of any one of SEQ ID NOs: 462 to 479; (j) nucleotides 10 to 36 of any one of SEQ ID NOs: 462 to 479; (k) nucleotides 11 to 36 of any one of SEQ ID NOs: 462 to 479; (l) nucleotides 12 to 36 of any one of SEQ ID NOs: 462 to 479; (m) nucleotides 13 to 36 of any one of SEQ ID NOs: 462 to 479; (n) nucleotides 14 to 36 of any one of SEQ ID NOs: 462 to 479; or (o) SEQ ID NO: 480 or a portion thereof.

[0431] In some examples, the direct repeat sequence is selected from the group consisting of (a) nucleotides 1 to 36 of a sequence that is at least 90% identical to SEQ ID NO: 485, (b) nucleotides 2 to 36 of a sequence that is at least 90% identical to SEQ ID NO: 485, (c) nucleotides 3 to 36 of a sequence that is at least 90% identical to SEQ ID NO: 485, (d) nucleotides 4 to 36 of a sequence that is at least 90% identical to SEQ ID NO: 485, (e) nucleotides 5 to 36 of a sequence that is at least 90% identical to SEQ ID NO: 485, (f) nucleotides 6 to 36 of a sequence that is at least 90% identical to SEQ ID NO: 485, (g) nucleotides 7 to 36 of a sequence that is at least 90% identical to SEQ ID NO: 485, (h) nucleotides 8 to 8 of a sequence that is at least 90% identical to SEQ ID NO: 485, (i) nucleotides 1 to 36 of a sequence that is at least 90% identical to SEQ ID NO: 485, (ii) nucleotides 1 to 36 of a sequence that is at least 90% identical to SEQ ID NO: 485, (iii) nucleotides 1 to 36 of a sequence that is at least 90% identical to SEQ ID NO: 485, (iv) nucleotides 1 to 36 of a sequence that is at least 90% identical to SEQ ID NO: 485, (v) nucleotides 2 to 36 of a sequence that is at least 90% identical to SEQ ID NO: 485, (vi) nucleotides 3 to 36 of a sequence that is at least 90% identical to SEQ ID NO: 485, (vi) nucleotides 485, (i) nucleotides 9 to 36 of a sequence that is at least 90% identical to SEQ ID NO: 485, (j) nucleotides 10 to 36 of a sequence that is at least 90% identical to SEQ ID NO: 485, (k) nucleotides 11 to 36 of a sequence that is at least 90% identical to SEQ ID NO: 485, (l) nucleotides 12 to 36 of a sequence that is at least 90% identical to SEQ ID NO: 485, (m) nucleotides 13 to 36 of a sequence that is at least 90% identical to SEQ ID NO: 485, (n) nucleotides 14 to 36 of a sequence that is at least 90% identical to SEQ ID NO: 485, or (o) a sequence that is at least 90% identical to SEQ ID NO: 486 or SEQ ID NO: 487, or a portion thereof.

[0432] In some examples, the direct repeat sequence comprises (a) nucleotides 1 to 36 of SEQ ID NO:485, (b) nucleotides 2 to 36 of SEQ ID NO:485, (c) nucleotides 3 to 36 of SEQ ID NO:485, (d) nucleotides 4 to 36 of SEQ ID NO:485, (e) nucleotides 5 to 36 of SEQ ID NO:485, (f) nucleotides 6 to 36 of SEQ ID NO:485, (g) nucleotides 7 to 36 of SEQ ID NO:485, (h) nucleotides 8 to 36 of SEQ ID NO:485, (i) nucleotides 9 to 36 of SEQ ID NO:485, (j) nucleotides 10 to 36 of SEQ ID NO:485, (k) nucleotides 11 to 36 of SEQ ID NO:485, (l) nucleotides 12 to 36 of SEQ ID NO:485, (m) nucleotides 13 to 36 of SEQ ID NO:485, (n) nucleotides 14 to 36 of SEQ ID NO:485, or (o) SEQ ID NO:486 or SEQ ID NO:487, or a portion thereof.

[0433] In some examples, the direct repeat sequence is selected from the group consisting of (a) nucleotides 1 to 36 of a sequence that is at least 90% identical to the sequence of SEQ ID NO:488 or SEQ ID NO:489, (b) nucleotides 2 to 36 of a sequence that is at least 90% identical to the sequence of SEQ ID NO:488 or SEQ ID NO:489, (c) nucleotides 3 to 36 of a sequence that is at least 90% identical to the sequence of SEQ ID NO:488 or SEQ ID NO:489, (d) nucleotides 4 to 36 of a sequence that is at least 90% identical to the sequence of SEQ ID NO:488 or SEQ ID NO:489, (e) nucleotides 5 to 36 of a sequence that is at least 90% identical to the sequence of SEQ ID NO:488 or SEQ ID NO:489, (f) nucleotides 6 to 36 of a sequence that is at least 90% identical to the sequence of SEQ ID NO:488 or SEQ ID NO:489, (g) nucleotides 7 to 36 of a sequence that is at least 90% identical to the sequence of SEQ ID NO:488 or SEQ ID NO:489, (h) nucleotides 8 to 36 of a sequence that is at least 90% identical to the sequence of SEQ ID NO:488 or SEQ ID NO:489. (i) nucleotides 9 to 36 of a sequence that is at least 90% identical to the sequence of SEQ ID NO: 488 or SEQ ID NO: 489; (j) nucleotides 10 to 36 of a sequence that is at least 90% identical to the sequence of SEQ ID NO: 488 or SEQ ID NO: 489; (k) nucleotides 11 to 36 of a sequence that is at least 90% identical to the sequence of SEQ ID NO: 488 or SEQ ID NO: 489; (l) nucleotides 12 to 36 of a sequence that is at least 90% identical to the sequence of SEQ ID NO: 488 or SEQ ID NO: 489. (m) nucleotides 13 to 36 of a sequence that is at least 90% identical to the sequence of SEQ ID NO:488 or SEQ ID NO:489; (n) nucleotides 14 to 36 of a sequence that is at least 90% identical to the sequence of SEQ ID NO:488 or SEQ ID NO:489; (o) nucleotides 15 to 36 of a sequence that is at least 90% identical to the sequence of SEQ ID NO:488 or SEQ ID NO:489; or (p) a sequence that is at least 90% identical to the sequence of SEQ ID NO:490 or a portion thereof.

[0434] In some examples, the direct repeat sequence is: (a) nucleotide 1 to nucleotide 36 of SEQ ID NO:488 or SEQ ID NO:489; (b) nucleotide 2 to nucleotide 36 of SEQ ID NO:488 or SEQ ID NO:489; (c) nucleotide 3 to nucleotide 36 of SEQ ID NO:488 or SEQ ID NO:489; (d) nucleotide 4 to nucleotide 36 of SEQ ID NO:488 or SEQ ID NO:489; (e) nucleotide 5 to nucleotide 36 of SEQ ID NO:488 or SEQ ID NO:489; (f) nucleotide 6 to nucleotide 36 of SEQ ID NO:488 or SEQ ID NO:489; (g) nucleotide 7 to nucleotide 36 of SEQ ID NO:488 or SEQ ID NO:489; (h) nucleotide 8 to nucleotide 36 of SEQ ID NO:488 or SEQ ID NO:489 (i) nucleotides 9 to 36 of SEQ ID NO:488 or SEQ ID NO:489, (j) nucleotides 10 to 36 of SEQ ID NO:488 or SEQ ID NO:489, (k) nucleotides 11 to 36 of SEQ ID NO:488 or SEQ ID NO:489, (l) nucleotides 12 to 36 of SEQ ID NO:488 or SEQ ID NO:489, (m) nucleotides 13 to 36 of SEQ ID NO:488 or SEQ ID NO:489, (n) nucleotides 14 to 36 of SEQ ID NO:488 or SEQ ID NO:489, (o) nucleotides 15 to 36 of SEQ ID NO:488 or SEQ ID NO:489, or (p) SEQ ID NO:490, or a portion thereof.

[0435] In any of the RNA guides of embodiment 6, the spacer sequence may be substantially complementary to the complement of any one of SEQ ID NOs: 11-228 or 491-2496.

[0436] In any of the RNA guides of embodiment 6, the target sequence may be adjacent to a protospacer adjacent motif (PAM), the PAM comprising the sequence 5'-NTTN-3', where N is any nucleotide. In some examples, the PAM comprises the sequence 5'-ATTA-3', 5'-ATTT-3', 5'-ATTG-3', 5'-ATTC-3', 5'-TTTA-3', 5'-TTTT-3', 5'-TTTG-3', 5'-TTTC-3', 5'-GTTA-3', 5'-GTTT-3', 5'-GTTG-3', 5'-GTTC-3', 5'-CTTA-3', 5'-CTTT-3', 5'-CTTG-3', or 5'-CTTC-3'.

[0437] In some instances, the target sequence is immediately adjacent to the PAM sequence, while in other instances, the target sequence is within 1, 2, 3, 4, or 5 nucleotides of the PAM sequence.

[0438] In some examples, the RNA guide has a sequence that is at least 90% identical to any one of SEQ ID NOs: 4505 to 4562. In specific examples, the RNA guide has a sequence that is at least 90% identical to any one of SEQ ID NOs: 4505 to 4562.

[0439] Embodiment 7: A nucleic acid encoding an RNA guide as described herein.

[0440] Embodiment 8: A vector comprising such an RNA guide as described herein.

[0441] Embodiment 9: A cell comprising a composition, RNA guide, nucleic acid, or vector described herein. In some examples, the cell is a eukaryotic cell, an animal cell, a mammalian cell, a human cell, a primary cell, a cell line, a stem cell, a neuron, or a T cell.

[0442] Embodiment 10: A kit comprising a composition, RNA guide, nucleic acid, or vector described herein.

[0443] Embodiment 11: A method of editing an STMN2 sequence, comprising contacting the STMN2 sequence with a composition or an RNA guide described herein. In some examples, the method is performed in vitro. In other examples, the method is performed ex vivo.

[0444] In some instances, the STMN2 sequence is intracellular.

[0445] In some instances, the composition or RNA guide induces a deletion in the STMN2 sequence. In some instances, the deletion is adjacent to a 5'-NTTN-3' sequence, where N is any nucleotide. In some specific instances, the deletion is downstream of the 5'-NTTN-3' sequence. In some specific instances, the deletion is up to about 40 nucleotides in length. In some instances, the deletion is about 4 nucleotides to 40 nucleotides, about 4 nucleotides to 25 nucleotides, about 10 nucleotides to 25 nucleotides, or about 10 nucleotides to 15 nucleotides in length.

[0446] In some instances, the deletion begins within about 5 nucleotides to about 15 nucleotides, about 5 nucleotides to about 10 nucleotides, or about 10 nucleotides to about 15 nucleotides of the 5'-NTTN-3' sequence.

[0447] In some examples, the deletion begins within about 5 nucleotides to about 15 nucleotides, about 5 nucleotides to about 10 nucleotides, or about 10 nucleotides to about 15 nucleotides downstream of the 5'-NTTN-3' sequence.

[0448] In some instances, the deletion ends within about 20 nucleotides to about 30 nucleotides, about 20 nucleotides to about 25 nucleotides, or about 25 nucleotides to about 30 nucleotides of the 5'-NTTN-3' sequence.

[0449] In some instances, the deletion ends within about 20 nucleotides to about 30 nucleotides, about 20 nucleotides to about 25 nucleotides, about 25 nucleotides to about 30 nucleotides downstream of the 5'-NTTN-3' sequence.

[0450] In some examples, the deletion begins within about 5 to about 15 nucleotides downstream of the 5'-NTTN-3' sequence and ends within about 20 to about 30 nucleotides downstream of the 5'-NTTN-3' sequence.

[0451] In some examples, the deletion begins within about 5 to about 15 nucleotides downstream of the 5'-NTTN-3' sequence and ends within about 20 to about 25 nucleotides downstream of the 5'-NTTN-3' sequence.

[0452] In some examples, the deletion begins within about 5 to about 15 nucleotides downstream of the 5'-NTTN-3' sequence and ends within about 25 to about 30 nucleotides downstream of the 5'-NTTN-3' sequence.

[0453] In some examples, the deletion begins within about 5 to about 10 nucleotides downstream of the 5'-NTTN-3' sequence and ends within about 20 to about 30 nucleotides downstream of the 5'-NTTN-3' sequence.

[0454] In some examples, the deletion begins within about 5 to about 10 nucleotides downstream of the 5'-NTTN-3' sequence and ends within about 20 to about 25 nucleotides downstream of the 5'-NTTN-3' sequence.

[0455] In some examples, the deletion begins within about 5 to about 10 nucleotides downstream of the 5'-NTTN-3' sequence and ends within about 25 to about 30 nucleotides downstream of the 5'-NTTN-3' sequence.

[0456] In some examples, the deletion begins within about 10 to about 15 nucleotides downstream of the 5'-NTTN-3' sequence and ends within about 20 to about 30 nucleotides downstream of the 5'-NTTN-3' sequence.

[0457] In some examples, the deletion begins within about 10 to about 15 nucleotides downstream of the 5'-NTTN-3' sequence and ends within about 20 to about 25 nucleotides downstream of the 5'-NTTN-3' sequence.

[0458] In some examples, the deletion begins within about 10 to about 15 nucleotides downstream of the 5'-NTTN-3' sequence and ends within about 25 to about 30 nucleotides downstream of the 5'-NTTN-3' sequence.

[0459] In some examples, the 5'-NTTN-3' sequence is 5'-CTTT-3', 5'-CTTC-3', 5'-GTTT-3', 5'-GTTC-3', 5'-TTTC-3', 5'-GTTA-3', or 5'-GTTG-3'.

[0460] In some cases, the deletion overlaps with a mutation in the STMN2 sequence. In some cases, the deletion overlaps with an insertion in the STMN2 sequence. In some cases, the deletion removes a repeat expansion of the STMN2 sequence or a portion thereof. In some cases, the deletion disrupts one or both alleles of the STMN2 sequence.

[0461] In any of the compositions, RNA guides, nucleic acids, vectors, cells, kits, or methods of embodiments 1-11 described herein, the RNA guide may comprise the sequence of any one of SEQ ID NOs: 4505-4562.

[0462] Embodiment 12: A method of treating a neurodegenerative disease in a subject (e.g., amyotrophic lateral sclerosis (ALS) or frontotemporal dementia (FTD)), the method comprising administering to the subject a composition, RNA guide, or cell described herein.

[0463] In any of the compositions, RNA guides, cells, kits, or methods described herein, the RNA guides and / or polyribonucleotides encoding Cas12i polypeptides are contained within lipid nanoparticles. In some examples, the RNA guides and polyribonucleotides encoding Cas12i polypeptides are contained within the same lipid nanoparticle. In other examples, the RNA guides and polyribonucleotides encoding Cas12i polypeptides are contained within separate lipid nanoparticles.

[0464] Embodiment 13: An RNA guide comprising: (i) a spacer sequence that is complementary to a target site in the STMN2 gene, wherein the target site is on the non-PAM strand and is complementary to the target sequence; and (ii) a direct repeat sequence.

[0465] In some examples, the direct repeat sequence is selected from the group consisting of (a) nucleotides 1 to 36 of a sequence that is at least 90% identical to any one of SEQ ID NOs: 1 to 8, (b) nucleotides 2 to 36 of a sequence that is at least 90% identical to any one of SEQ ID NOs: 1 to 8, (c) nucleotides 3 to 36 of a sequence that is at least 90% identical to any one of SEQ ID NOs: 1 to 8, (d) nucleotides 4 to 36 of a sequence that is at least 90% identical to any one of SEQ ID NOs: 1 to 8, (e) nucleotides 5 to 36 of a sequence that is at least 90% identical to any one of SEQ ID NOs: 1 to 8, (f) nucleotides 6 to 36 of a sequence that is at least 90% identical to any one of SEQ ID NOs: 1 to 8, (g) nucleotides 7 to 36 of a sequence that is at least 90% identical to any one of SEQ ID NOs: 1 to 8, (h) nucleotides 8 to 36 of a sequence that is at least 90% identical to any one of SEQ ID NOs: 1 to 8, (i) a sequence that is at least 90% identical to any one of SEQ ID NOs: 1 to 8, (ii) a sequence that is at least 90% identical to any one of SEQ ID NOs: 1 to 8, (iii) a sequence that is at least 90% identical to any one of SEQ ID NOs: 1 to 8, (iv) a sequence that is at least 90% identical to any one of SEQ ID NOs: 1 to 8, (v) a sequence that is at least 90% identical to any one of SEQ ID NOs: 1 to 8, (vi) a sequence that is at least 90% identical to any one of SEQ ID NOs: 1 i) nucleotides 9 to 36 of a sequence that is at least 90% identical to any one of SEQ ID NOs: 1 to 8; (j) nucleotides 10 to 36 of a sequence that is at least 90% identical to any one of SEQ ID NOs: 1 to 8; (k) nucleotides 11 to 36 of a sequence that is at least 90% identical to any one of SEQ ID NOs: 1 to 8; (l) nucleotides 12 to 36 of a sequence that is at least 90% identical to any one of SEQ ID NOs: 1 to 8; (m) nucleotides 13 to 36 of a sequence that is at least 90% identical to any one of SEQ ID NOs: 1 to 8; (n) nucleotides 14 to 36 of a sequence that is at least 90% identical to any one of SEQ ID NOs: 1 to 8; (o) nucleotides 1 to 34 of a sequence that is at least 90% identical to SEQ ID NO: 9; (p) nucleotides 2 to 34 of a sequence that is at least 90% identical to SEQ ID NO: 9; (q) nucleotides 3 to 34 of a sequence that is at least 90% identical to SEQ ID NO: 9;(r) nucleotides 4 to 34 of a sequence that is at least 90% identical to the sequence of SEQ ID NO:9, (s) nucleotides 5 to 34 of a sequence that is at least 90% identical to the sequence of SEQ ID NO:9, (t) nucleotides 6 to 34 of a sequence that is at least 90% identical to the sequence of SEQ ID NO:9, (u) nucleotides 7 to 34 of a sequence that is at least 90% identical to the sequence of SEQ ID NO:9, (v) nucleotides 8 to 34 of a sequence that is at least 90% identical to the sequence of SEQ ID NO:9, (w) nucleotides 9 to 34 of a sequence that is at least 90% identical to the sequence of SEQ ID NO:9, (x) nucleotides 10 to 34 of a sequence that is at least 90% identical to the sequence of SEQ ID NO:9, (y) nucleotides 11 to 34 of a sequence that is at least 90% identical to the sequence of SEQ ID NO:9, (z) nucleotides 12 to 34 of a sequence that is at least 90% identical to the sequence of SEQ ID NO:9, or (aa) a sequence that is at least 90% identical to the sequence of SEQ ID NO:10 or a portion thereof.

[0466] In some examples, the direct repeat sequence is selected from the group consisting of: (a) nucleotide 1 to nucleotide 36 of any one of SEQ ID NOs: 1 to 8; (b) nucleotide 2 to nucleotide 36 of any one of SEQ ID NOs: 1 to 8; (c) nucleotide 3 to nucleotide 36 of any one of SEQ ID NOs: 1 to 8; (d) nucleotide 4 to nucleotide 36 of any one of SEQ ID NOs: 1 to 8; (e) nucleotide 5 to nucleotide 36 of any one of SEQ ID NOs: 1 to 8; (f) nucleotide 6 to nucleotide 36 of any one of SEQ ID NOs: 1 to 8; (g) nucleotide 7 to nucleotide 36 of any one of SEQ ID NOs: 1 to 8; (h) nucleotide 8 to nucleotide 36 of any one of SEQ ID NOs: 1 to 8; (i) nucleotide 9 to nucleotide 36 of any one of SEQ ID NOs: 1 to 8; (j) nucleotide 10 to nucleotide 36 of any one of SEQ ID NOs: 1 to 8; (k) nucleotide 11 to nucleotide 36 of any one of SEQ ID NOs: 1 to 8; (m) nucleotides 13 to 36 of any one of SEQ ID NOs: 1 to 8, (n) nucleotides 14 to 36 of any one of SEQ ID NOs: 1 to 8, (o) nucleotides 1 to 34 of SEQ ID NO: 9, (p) nucleotides 2 to 34 of SEQ ID NO: 9, (q) nucleotides 3 to 34 of SEQ ID NO: 9, (r) nucleotides 4 to 34 of SEQ ID NO: 9, (s) nucleotides 5 to 34 of SEQ ID NO: 9, (t) nucleotides 6 to 34 of SEQ ID NO: 9, (u) nucleotides 7 to 34 of SEQ ID NO: 9, (v) nucleotides 8 to 34 of SEQ ID NO: 9, (w) nucleotides 9 to 34 of SEQ ID NO: 9, (x) nucleotides 10 to 34 of SEQ ID NO: 9, (y) nucleotides 11 to 34 of SEQ ID NO: 9, (z) nucleotides 12 to 34 of SEQ ID NO: 9, or (aa) SEQ ID NO: 10 or a portion thereof.

[0467] In some examples, the direct repeat sequence is selected from the group consisting of (a) nucleotides 1 to 36 of a sequence that is at least 90% identical to any one of SEQ ID NOs: 462 to 479, (b) nucleotides 2 to 36 of a sequence that is at least 90% identical to any one of SEQ ID NOs: 462 to 479, (c) nucleotides 3 to 36 of a sequence that is at least 90% identical to any one of SEQ ID NOs: 462 to 479, (d) nucleotides 4 to 36 of a sequence that is at least 90% identical to any one of SEQ ID NOs: 462 to 479, (e) nucleotides 5 to 36 of a sequence that is at least 90% identical to any one of SEQ ID NOs: 462 to 479, (f) nucleotides 6 to 36 of a sequence that is at least 90% identical to any one of SEQ ID NOs: 462 to 479, (g) nucleotides 7 to 36 of a sequence that is at least 90% identical to any one of SEQ ID NOs: 462 to 479, (h) nucleotides 8 to 8 of a sequence that is at least 90% identical to any one of SEQ ID NOs: 462 to 479, (i) nucleotides 9 to 9 of a sequence that is at least 90% identical to any one of SEQ ID NOs: 462 to 479, (ii) nucleotides 10 to 12 of a sequence that is at least 90% identical to any one of SEQ ID NOs: 462 to 479, (iii) nucleotides 13 to 14 of a sequence that is at least 90% identical to any one of SEQ ID NOs: 462 to 479, (iv) nucleotides 15 to 1 (i) nucleotides 8 to 36 of a sequence that is at least 90% identical to any one of SEQ ID NOs: 462 to 479; (j) nucleotides 10 to 36 of a sequence that is at least 90% identical to any one of SEQ ID NOs: 462 to 479; (k) nucleotides 11 to 36 of a sequence that is at least 90% identical to any one of SEQ ID NOs: 462 to 479; (l) nucleotides 12 to 36 of a sequence that is at least 90% identical to any one of SEQ ID NOs: 462 to 479; (m) nucleotides 13 to 36 of a sequence that is at least 90% identical to any one of SEQ ID NOs: 462 to 479; (n) nucleotides 14 to 36 of a sequence that is at least 90% identical to any one of SEQ ID NOs: 462 to 479; or (o) a sequence that is at least 90% identical to the sequence of SEQ ID NO: 480 or a portion thereof.

[0468] In some examples, the direct repeat sequence is: (a) nucleotide 1 to nucleotide 36 of any one of SEQ ID NOs: 462-479; (b) nucleotide 2 to nucleotide 36 of any one of SEQ ID NOs: 462-479; (c) nucleotide 3 to nucleotide 36 of any one of SEQ ID NOs: 462-479; (d) nucleotide 4 to nucleotide 36 of any one of SEQ ID NOs: 462-479; (e) nucleotide 5 to nucleotide 36 of any one of SEQ ID NOs: 462-479; (f) nucleotide 6 to nucleotide 36 of any one of SEQ ID NOs: 462-479; (g) nucleotide 7 to nucleotide 36 of any one of SEQ ID NOs: 462-479; (h) (i) nucleotides 8 to 36 of any one of SEQ ID NOs: 462 to 479; (j) nucleotides 10 to 36 of any one of SEQ ID NOs: 462 to 479; (k) nucleotides 11 to 36 of any one of SEQ ID NOs: 462 to 479; (l) nucleotides 12 to 36 of any one of SEQ ID NOs: 462 to 479; (m) nucleotides 13 to 36 of any one of SEQ ID NOs: 462 to 479; (n) nucleotides 14 to 36 of any one of SEQ ID NOs: 462 to 479; or (o) SEQ ID NO: 480 or a portion thereof.

[0469] In some examples, the direct repeat sequence is selected from the group consisting of (a) nucleotides 1 to 36 of a sequence that is at least 90% identical to SEQ ID NO: 485, (b) nucleotides 2 to 36 of a sequence that is at least 90% identical to SEQ ID NO: 485, (c) nucleotides 3 to 36 of a sequence that is at least 90% identical to SEQ ID NO: 485, (d) nucleotides 4 to 36 of a sequence that is at least 90% identical to SEQ ID NO: 485, (e) nucleotides 5 to 36 of a sequence that is at least 90% identical to SEQ ID NO: 485, (f) nucleotides 6 to 36 of a sequence that is at least 90% identical to SEQ ID NO: 485, (g) nucleotides 7 to 36 of a sequence that is at least 90% identical to SEQ ID NO: 485, (h) nucleotides 8 to 8 of a sequence that is at least 90% identical to SEQ ID NO: 485, (i) nucleotides 1 to 36 of a sequence that is at least 90% identical to SEQ ID NO: 485, (ii) nucleotides 1 to 36 of a sequence that is at least 90% identical to SEQ ID NO: 485, (iii) nucleotides 1 to 36 of a sequence that is at least 90% identical to SEQ ID NO: 485, (iv) nucleotides 1 to 36 of a sequence that is at least 90% identical to SEQ ID NO: 485, (v) nucleotides 2 to 36 of a sequence that is at least 90% identical to SEQ ID NO: 485, (vi) nucleotides 3 to 36 of a sequence that is at least 90% identical to SEQ ID NO: 485, (vi) nucleotides 485, (i) nucleotides 9 to 36 of a sequence that is at least 90% identical to SEQ ID NO: 485, (j) nucleotides 10 to 36 of a sequence that is at least 90% identical to SEQ ID NO: 485, (k) nucleotides 11 to 36 of a sequence that is at least 90% identical to SEQ ID NO: 485, (l) nucleotides 12 to 36 of a sequence that is at least 90% identical to SEQ ID NO: 485, (m) nucleotides 13 to 36 of a sequence that is at least 90% identical to SEQ ID NO: 485, (n) nucleotides 14 to 36 of a sequence that is at least 90% identical to SEQ ID NO: 485, or (o) a sequence that is at least 90% identical to SEQ ID NO: 486 or SEQ ID NO: 487, or a portion thereof.

[0470] In some examples, the direct repeat sequence comprises (a) nucleotides 1 to 36 of SEQ ID NO:485, (b) nucleotides 2 to 36 of SEQ ID NO:485, (c) nucleotides 3 to 36 of SEQ ID NO:485, (d) nucleotides 4 to 36 of SEQ ID NO:485, (e) nucleotides 5 to 36 of SEQ ID NO:485, (f) nucleotides 6 to 36 of SEQ ID NO:485, (g) nucleotides 7 to 36 of SEQ ID NO:485, (h) nucleotides 8 to 36 of SEQ ID NO:485, (i) nucleotides 9 to 36 of SEQ ID NO:485, (j) nucleotides 10 to 36 of SEQ ID NO:485, (k) nucleotides 11 to 36 of SEQ ID NO:485, (l) nucleotides 12 to 36 of SEQ ID NO:485, (m) nucleotides 13 to 36 of SEQ ID NO:485, (n) nucleotides 14 to 36 of SEQ ID NO:485, or (o) SEQ ID NO:486 or SEQ ID NO:487, or a portion thereof.

[0471] In some examples, the direct repeat sequence is selected from the group consisting of (a) nucleotides 1 to 36 of a sequence that is at least 90% identical to the sequence of SEQ ID NO:488 or SEQ ID NO:489, (b) nucleotides 2 to 36 of a sequence that is at least 90% identical to the sequence of SEQ ID NO:488 or SEQ ID NO:489, (c) nucleotides 3 to 36 of a sequence that is at least 90% identical to the sequence of SEQ ID NO:488 or SEQ ID NO:489, (d) nucleotides 4 to 36 of a sequence that is at least 90% identical to the sequence of SEQ ID NO:488 or SEQ ID NO:489, (e) nucleotides 5 to 36 of a sequence that is at least 90% identical to the sequence of SEQ ID NO:488 or SEQ ID NO:489, (f) nucleotides 6 to 36 of a sequence that is at least 90% identical to the sequence of SEQ ID NO:488 or SEQ ID NO:489, (g) nucleotides 7 to 36 of a sequence that is at least 90% identical to the sequence of SEQ ID NO:488 or SEQ ID NO:489, (h) nucleotides 8 to 36 of a sequence that is at least 90% identical to the sequence of SEQ ID NO:488 or SEQ ID NO:489. (i) nucleotides 9 to 36 of a sequence that is at least 90% identical to the sequence of SEQ ID NO: 488 or SEQ ID NO: 489; (j) nucleotides 10 to 36 of a sequence that is at least 90% identical to the sequence of SEQ ID NO: 488 or SEQ ID NO: 489; (k) nucleotides 11 to 36 of a sequence that is at least 90% identical to the sequence of SEQ ID NO: 488 or SEQ ID NO: 489; (l) nucleotides 12 to 36 of a sequence that is at least 90% identical to the sequence of SEQ ID NO: 488 or SEQ ID NO: 489. (m) nucleotides 13 to 36 of a sequence that is at least 90% identical to the sequence of SEQ ID NO:488 or SEQ ID NO:489; (n) nucleotides 14 to 36 of a sequence that is at least 90% identical to the sequence of SEQ ID NO:488 or SEQ ID NO:489; (o) nucleotides 15 to 36 of a sequence that is at least 90% identical to the sequence of SEQ ID NO:488 or SEQ ID NO:489; or (p) a sequence that is at least 90% identical to the sequence of SEQ ID NO:490 or a portion thereof.

[0472] In some examples, the direct repeat sequence is: (a) nucleotide 1 to nucleotide 36 of SEQ ID NO:488 or SEQ ID NO:489; (b) nucleotide 2 to nucleotide 36 of SEQ ID NO:488 or SEQ ID NO:489; (c) nucleotide 3 to nucleotide 36 of SEQ ID NO:488 or SEQ ID NO:489; (d) nucleotide 4 to nucleotide 36 of SEQ ID NO:488 or SEQ ID NO:489; (e) nucleotide 5 to nucleotide 36 of SEQ ID NO:488 or SEQ ID NO:489; (f) nucleotide 6 to nucleotide 36 of SEQ ID NO:488 or SEQ ID NO:489; (g) nucleotide 7 to nucleotide 36 of SEQ ID NO:488 or SEQ ID NO:489; (h) nucleotide 8 to nucleotide 36 of SEQ ID NO:488 or SEQ ID NO:489 (i) nucleotides 9 to 36 of SEQ ID NO:488 or SEQ ID NO:489, (j) nucleotides 10 to 36 of SEQ ID NO:488 or SEQ ID NO:489, (k) nucleotides 11 to 36 of SEQ ID NO:488 or SEQ ID NO:489, (l) nucleotides 12 to 36 of SEQ ID NO:488 or SEQ ID NO:489, (m) nucleotides 13 to 36 of SEQ ID NO:488 or SEQ ID NO:489, (n) nucleotides 14 to 36 of SEQ ID NO:488 or SEQ ID NO:489, (o) nucleotides 15 to 36 of SEQ ID NO:488 or SEQ ID NO:489, or (p) SEQ ID NO:490, or a portion thereof.

[0473] In some instances, each of the first three nucleotides of the RNA guide comprises a 2'-O-methyl phosphorothioate modification.

[0474] In some instances, each of the last four nucleotides of the RNA guide comprises a 2'-O-methyl phosphorothioate modification.

[0475] In some instances, the first to last, second to last, and third to last nucleotide of the RNA guide each comprise a 2'-O-methyl phosphorothioate modification, and the last nucleotide of the RNA guide is unmodified.

[0476] Embodiment 14: A nucleic acid encoding an RNA guide as described herein.

[0477] Embodiment 15: A vector comprising a nucleic acid as described herein.

[0478] Embodiment 16: A vector system comprising (i) an RNA guide of embodiment 13 described herein, and (ii) one or more vectors encoding a Cas12i polypeptide. In some examples, the vector system comprises a first vector encoding an RNA guide and a second vector encoding a Cas12i polypeptide.

[0479] Embodiment 17: A cell comprising an RNA guide, nucleic acid, vector, or vector system of any of embodiments 13 to 16 described herein. In some examples, the cell is a eukaryotic cell, an animal cell, a mammalian cell, a human cell, a primary cell, a cell line, a stem cell, a neuron, or a T cell.

[0480] Embodiment 18: A kit comprising an RNA guide, a nucleic acid, a vector, or a vector system according to embodiments 13 to 16 described herein.

[0481] Embodiment 19: A method of editing an STMN2 sequence, comprising contacting the STMN2 sequence with an RNA guide of embodiment 13 described herein. In some examples, the STMN2 sequence is within a cell.

[0482] In some instances, the RNA guide induces an indel (e.g., an insertion or deletion) in the STMN2 sequence.

[0483] Embodiment 20: A method of treating a neurodegenerative disease in a subject (e.g., amyotrophic lateral sclerosis (ALS) or frontotemporal dementia (FTD)), the method comprising administering to the subject an RNA guide of embodiment 13 described herein.

[0484] General TechnologyThe practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology, which are within the skill of the art. Such techniques are described in Molecular Cloning: A Laboratory Manual, second edition (Sambrook, et al., 1989) Cold Spring Harbor Press, Oligonucleotide Synthesis (MJ Gait, ed. 1984), Methods in Molecular Biology, Humana Press, Cell Biology: A Laboratory Notebook (JECellis, ed., 1989) Academic Press, Animal Cell Culture (RIFreshney, ed. 1987), Introduction to Cell and Tissue Culture (JP Mather and PE Roberts, 1998) Plenum Press, Cell and Tissue Culture: Laboratory Procedures (A. Doyle, JBGriffiths, and DG Newell, eds. 1993-8) J. Wiley and Sons, Methods in Enzymology (Academic Press, Inc.), Handbook of Experimental Immunology (DM Weir and CC Blackwell, eds.): Gene Transfer Vectors for Mammalian Cells (JMMiller and MP Calos, eds., 1987), Current Protocols in Molecular Biology (FMAusubel, et al. eds. 1987), PCR: The Polymerase Chain Reaction, (Mullis, et al., eds. 1994), Current Protocols in Immunology (JEColigan et al., eds., (1991), Short Protocols in Molecular Biology (Wiley and Sons, 1999), Immunobiology (C.A. Janeway and P. Travers, 1997), Antibodies (P. Finch, 1997), Antibodies: a practice approach (D. Catty., ed., IRL Press, 1988 - 1989), Monoclonal antibodies: a practical approach (P. Shepherd and C. Dean, eds., Oxford University Press, 2000), Using antibodies: a laboratory manual (E. Harlow and D. Lane (Cold Spring Harbor Laboratory Press, 1999), The Antibodies (M. Zanetti and J.D. Capra, eds. Harwood Academic Publishers, 1995), DNA Cloning: A practical Approach, Volumes I and II (D.N. Glover ed. 1985), Nucleic Acid Hybridization (B.D. Hames & S.J. Higgins eds. (1985>>, Transcription and Translation (B.D. Hames & S.J. Higgins, eds. (1984>>, Animal Cell Culture (R.I. Freshney, ed. (1986>>, Immobilized Cells and Enzymes (lRL Press, (1986>>, and are fully described in the literature such as B. Perbal, A practical Guide To Molecular Cloning (1984), F.M. Ausubel et al. (eds.).

[0485] Without further elaboration, it is believed that one skilled in the art can utilize the present disclosure to its fullest extent based on the above description. Accordingly, the following specific embodiments are to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way. All publications cited herein are incorporated by reference for the purpose or subject matter referred to herein. EXAMPLES

[0486] The following examples are provided to further illustrate some embodiments of the present disclosure, but are not intended to limit the scope of the disclosure, and it will be understood that by their illustrative nature, other procedures, methodologies, or techniques known to those skilled in the art may be used instead.

[0487] Example 1 - Targeting STMN2 intron 1 with variant Cas12i2 This example describes indel assessment for multiple targets in the STMN2 gene in cells following transfection with a plasmid encoding a variant Cas12i2 (SEQ ID NO: 450) and an RNA guide.

[0488] The variant Cas12i2 polypeptide was cloned into a plasmid containing a CMV promoter. A fragment encoding an RNA guide targeting the STMN2 intron 1 gene was cloned into a pUC19 backbone (New England Biolabs). The plasmid was then maxiprepped and diluted. The crRNA, target, and PAM sequences are listed in Table 6.

[0489] [Table 8-1]

[0490] [Table 8-2]

[0491] [Table 8-3]

[0492]

Table 8-4

[0493] Approximately 16 hours prior to transfection, 25,000 HEK293T cells in DMEM / 10% FBS+Pen / Strep (D10 media) were seeded into each well of a 96-well plate. On the day of transfection, cells were 70-90% confluent. For each well to be transfected, a mixture of LIPOFECTAMINE® 2000 transfection reagent (ThermoFisher) and Opti-MEM® reduced serum medium (ThermoFisher) was prepared and incubated at room temperature for 5 minutes (Solution 1). After incubation, the LIPOFECTAMINE® 2000:Opti-MEM® (transfection reagent (ThermoFisher):reduced serum medium (ThermoFisher)) mixture was added to a separate mixture containing the nuclease plasmid, RNA guide plasmid, and Opti-MEM® reduced serum medium (ThermoFisher) (Solution 2). For the negative control, no RNA guide plasmid was included in solution 2. Solutions 1 and 2 were mixed by pipetting 8 times and then incubated at room temperature for 25 minutes. After incubation, solution 1 and 2 mixture was added dropwise to each well of the 96-well plate containing cells. 72 hours after transfection, cells were trypsinized by adding TRYPLE™ (recombinant cell dissociation enzyme, ThermoFisher) to the center of each well and incubating at 37°C for about 5 minutes. D10 medium was then added to each well and mixed to resuspend the cells. The resuspended cells were centrifuged at 500xg for 10 minutes to obtain a pellet, and the supernatant was discarded. QUICKEXTRACT™ (DNA extraction solution, Lucigen) extraction reagent was added thoroughly to each well to lyse the pelleted cells. The cells were incubated at 65°C for 15 minutes, 68°C for 15 minutes, and 98°C for 10 minutes.

[0494] Samples for NGS were prepared by two rounds of PCR. The first round (PCR1) was used to amplify specific genomic regions depending on the target. Round 2 PCR (PCR2) was performed to add Illumina adapters and indexes. Reactions were then pooled and purified by column purification. Sequencing runs were performed with the NEXTSEQ™ (Illumina) 500 / 550 High Throughput v2.5 kit for 300 cycles.

[0495] As shown in FIG. 1, RNA guides 1, 3, 4, 5, 6, 8, 9, 10, 11, 12, 13, 14, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 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, and 58 all yielded measurable indel activity, defined as >1% and >0.2% above the background rate of non-RNA guide controls.

[0496] RNA guides 4, 8, 55, and 57 resulted in the disruption of more than 15% of cryptic splice sites in intron 1 (Figure 2A), where a disruption is defined as an insertion or deletion at one or more bases of the cryptic splice site. 97% of indels generated by RNA guide 4 resulted in the disruption of a cryptic splice site in intron 1, where a disruption is defined as an insertion or deletion at one or more bases of the cryptic splice site.

[0497] RNA guides 12, 46, 47, 48, and 49 resulted in more than 15% disruption of at least one of the three TDP-43 binding motifs in intron 1 (Figure 2B), where a disruption is defined as an insertion or deletion of one or more bases in the TDP-43 binding motif. 97% of the indels generated by RNA guide 12 resulted in disruption of at least one of the three TDP-43 binding motifs in intron 1, where a disruption is defined as an insertion or deletion of one or more bases in the TDP-43 binding motif.

[0498] RNA guides 17 and 18 resulted in more than 15% disruption of the premature polyadenylation signal in intron 1 (Figure 2C), where a disruption is defined as an insertion or deletion of one or more bases of the polyadenylation signal. 88% of the indels generated by RNA guide 17 resulted in disruption of the premature polyadenylation signal in intron 1, where a disruption is defined as an insertion or deletion of one or more bases of the premature polyadenylation site. 93% of the indels generated by RNA guide 18 resulted in disruption of the premature polyadenylation signal in intron 1, where a disruption is defined as an insertion or deletion of one or more bases of the premature polyadenylation site.

[0499] Figure 3 shows the position where each of the RNA guides binds to intron 1 of STMN2 relative to the location of the cryptic splice site, the TDP-43 binding motif, and the early polyadenylation signal. Darker grey reflects RNA guides that show indels in more than 30% of the NGS reads, and lighter grey reflects RNA guides that show indels in less than 30% of the NGS reads. Thus, this example shows that the Cas12i2 guide was able to edit intron 1 of STMN2 and disrupt the cryptic splice site, the TDP-43 binding motif, and the early polyadenylation signal.

[0500] Example 2 - Targeting STMN2 intron 1 with variant Cas12i2 in SH-SY%Y cells This example describes indel assessment for multiple targets in the STMN2 gene in neuroblastoma cell lines following transfection with a plasmid encoding a variant Cas12i2 (SEQ ID NO: 450) and an RNA guide targeting a cryptic splice site in intron 1.

[0501] Variant Cas12i2 polypeptides and RNA guides 4, 5, 8, 9, 55, 56, 57, and 58 in Table 6 were cloned, purified, and diluted as described in Example 1. Approximately 16 hours prior to transfection, 25,000 SH-SY5Y cells in EMEM:F12 / 10% FBS+Pen / Strep (EF12-10 medium) were seeded into each well of a 96-well plate. On the day of transfection, cells were 70-90% confluent. For each well to be transfected, a mixture of LIPOFECTAMINE® 2000 Transfection Reagent (ThermoFisher) and Opti-MEM® Reduced Serum Medium (ThermoFisher) was prepared and incubated at room temperature for 5 minutes (solution 1). After incubation, the LIPOFECTAMINE® 2000:Opti-MEM® (transfection reagent (ThermoFisher):reduced serum medium (ThermoFisher)) mixture was added to a separate mixture containing the nuclease plasmid, the RNA guide plasmid, and Opti-MEM® reduced serum medium (ThermoFisher) (solution 2). For the negative control, the RNA guide plasmid was not included in solution 2. Solutions 1 and 2 were mixed with a pipette 8 times and then incubated at room temperature for 25 minutes. After incubation, the solution 1 and 2 mixture was added dropwise to each well of the 96-well plate containing the cells. 72 hours after transfection, the cells were trypsinized by adding TRYPLE™ (recombinant cell dissociation enzyme, ThermoFisher) to the center of each well and incubating at 37° C. for approximately 5 minutes. EF12-10 medium was then added to each well and mixed to resuspend the cells. The resuspended cells were centrifuged at 500xg for 10 minutes to obtain a pellet, and the supernatant was discarded. QUICKEXTRACT™ (DNA Extraction Solution, Lucigen) Extraction Reagent was added thoroughly to each well to lyse the pelleted cells. The cells were incubated at 65°C for 15 minutes, 68°C for 15 minutes, and 98°C for 10 minutes.

[0502] Samples for NGS were prepared by two rounds of PCR. The first round (PCR1) was used to amplify specific genomic regions depending on the target. Round 2 PCR (PCR2) was performed to add Illumina adapters and indexes. Reactions were then pooled and purified by column purification. Sequencing runs were performed with the NEXTSEQ™ (Illumina) 500 / 550 High Throughput v2.5 kit for 300 cycles.

[0503] Figure 4 shows the indel activity of the tested RNA guides in SH-SY5Y cells. Guide 4 showed 0.56% disruption of splice site motifs and 2.0% overall edits, with more than 25% of the total edits disrupting splice sites. Guide 5 showed 0.12% disruption of splice site motifs and 1.5% overall edits, with less than 10% of the total edits disrupting splice sites. Guide 8 showed 0.62% disruption of splice site motifs and 2.4% overall edits, with more than 25% of the total edits disrupting splice sites. Guide 9 showed 0.34% disruption of splice site motifs and 3.8% overall edits, with less than 10% of the total edits disrupting splice sites. Guide 55 showed 2.2% disruption of splice site motifs and 4.9% overall edits, with more than 40% of the total edits disrupting splice sites. Guide 56 showed 2.3% disruption of splice site motifs and 4.9% overall edits, with over 45% of all edits disrupting splice sites. Guide 57 showed 0% disruption of splice site motifs and 1.6% overall edits. Guide 58 showed 0.49% disruption of splice site motifs and 3.3% overall edits, with over 10% of all edits disrupting splice sites.

[0504] FIG. 5A is a plot comparing the indel activity (indel %) demonstrated in HEK293T cells and SH-SY5Y cells from Examples 1 and 2, respectively. FIG. 5B is a plot comparing the splice site motif disruption demonstrated in HEK293T cells and SH-SY5Y cells from Examples 1 and 2, respectively. As shown in FIG. 5A, guide 55 and guide 9 demonstrated the highest % indels across the two cell types. Guide 56 demonstrated the highest % indels in SH-SY5Y cells, but low % indels in HEK293T cells. Guide 55 similarly resulted in the highest splice site motif disruption in the two cell types (FIG. 5B).

[0505] Thus, this example shows that cryptic splice sites in intron 1 of STMN2 can be targeted by Cas12i2 and multiple RNA guides in multiple cell types.

[0506] Other embodiments All of the features disclosed herein may be combined in any combination. Each feature disclosed herein may be replaced by an alternative feature serving the same, equivalent, or similar purpose. Thus, unless expressly stated otherwise, each feature disclosed is only one example of a generic series of equivalent or similar features.

[0507] From the above description, those skilled in the art can easily ascertain the essential features of the present disclosure, and can make various changes and modifications to the present disclosure to adapt the present invention to various uses and conditions without departing from the spirit and scope thereof. Accordingly, other embodiments are also within the scope of the claims.

[0508] Equivalent While several embodiments of the invention have been described and illustrated herein, those skilled in the art will readily envision various other means and / or structures for performing the functions described herein and / or obtaining one or more of the results and / or advantages described herein, and each of such variations and / or modifications are deemed to be within the scope of the embodiments of the invention described herein. More generally, all parameters, dimensions, materials, and configurations described herein are intended to be exemplary, and those skilled in the art will readily appreciate that the actual parameters, dimensions, materials, and / or configurations will depend on the particular application or applications for which the teachings of the invention are used. Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation many equivalents to the specific embodiments of the invention described herein. Thus, the above-described embodiments are presented by way of example only, and it should be understood that within the scope of the appended claims and their equivalents, the embodiments of the invention may be practiced otherwise than as specifically described and claimed. The inventive embodiments of the present disclosure are directed to each individual feature, system, article, material, kit, and / or method described herein. In addition, any combination of two or more such features, systems, articles, materials, kits, and / or methods is included within the inventive scope of the present disclosure, if such features, systems, articles, materials, kits, and / or methods are not mutually inconsistent.

[0509] All definitions and uses herein should be understood to take precedence over dictionary definitions, definitions in documents incorporated by reference, and / or ordinary meanings of the defined terms.

[0510] All references, patents, and patent applications disclosed herein are incorporated by reference with respect to the subject matter for which each is cited, which in some cases may include the entire document.

[0511] As used herein in the specification and claims, unless clearly indicated to the contrary, the indefinite articles "a" and "an" should be understood to mean "at least one."

[0512] As used herein in the specification and claims, the term "and / or" should be understood to mean "either or both" of the elements so conjoined, e.g., elements that are present conjunctively in some cases and disjunctively in other cases. Multiple elements listed with "and / or" should be interpreted in the same manner, i.e., "one or more" of the elements so conjoined. Other elements other than the elements specifically identified by the "and / or" clause may optionally be present, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, a reference to "A and / or B", when used with an open-ended term such as "comprising", may refer in one embodiment to only A (optionally including elements other than B), in another embodiment to only B (optionally including elements other than A), in yet another embodiment to both A and B (optionally including other elements), etc.

[0513] As used herein in the specification and claims, "or" should be understood to have the same meaning as "and / or" as defined above. For example, when separating items in a list, "or" or "and / or" shall be interpreted as inclusive, i.e., including at least one of, but also including more than one, and optionally including additional unlisted items, of some elements or a list of elements. Only where terms clearly indicate to the contrary, e.g., "only one of" or "exactly one of," or when used in the claims, "consisting of" refers to the inclusion of exactly one element of some elements or a list of elements. In general, as used herein, the term "or" shall only be interpreted as indicating exclusive alternatives (i.e., "one or the other, but not both") when preceded by terms of exclusivity, e.g., "either," "one of," "only one of," or "exactly one of." When used in the claims, "consisting essentially of" shall have its ordinary meaning as used in the field of patent law.

[0514] As used herein in the specification and claims, the phrase "at least one" in reference to a list of one or more elements should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed in the list of elements, and not excluding any combination of elements in the list of elements. This definition also allows for the optional presence of elements other than those specifically identified in the list of elements to which the phrase "at least one" refers, whether or not related to those specifically identified elements. Thus, as a non-limiting example, "at least one of A and B" (or, equivalently, "at least one of A or B" or, equivalently, "at least one of A and / or B") can refer in one embodiment to at least one A, optionally including two or more As, and no B (optionally including elements other than B); in another embodiment to at least one B, optionally including two or more Bs, and no A (optionally including elements other than A); in yet another embodiment to at least one A, optionally including two or more As, and at least one B, optionally including two or more Bs (optionally including other elements); and so forth.

[0515] Also, unless expressly indicated to the contrary, in any method claimed herein that includes two or more steps or actions, the order of the method steps or actions should be understood as not necessarily limited to the order in which the method steps or actions are recited.

Claims

1. A gene editing system for gene editing of the stathmin 2 (STMN2) gene, comprising: (i) a Cas12i2 polypeptide or a first nucleic acid encoding the Cas12i2 polypeptide, wherein the Cas12i2 polypeptide comprises an amino acid sequence at least 95% identical to SEQ ID NO: 448 and comprises one or more mutations relative to SEQ ID NO: 448; and (ii) a gene editing system comprising an RNA guide or a second nucleic acid encoding the RNA guide, wherein the RNA guide comprises a spacer sequence specific for a target sequence within a STMN2 gene, the target sequence being adjacent to a protospacer adjacent motif (PAM), the PAM comprising a 5'-TTN-3' motif, and the 5'-TTN-3' motif is located 5' to the target sequence.

2. 2. The gene editing system of claim 1, wherein the one or more mutations in the Cas12i2 polypeptide are at positions D581, G624, F626, P868, I926, V1030, E1035, and / or S1046 of SEQ ID NO:

448.

3. 3. The gene editing system of Claim 2, wherein the one or more mutations are amino acid substitutions that are D581R, G624R, F626R, P868T, I926R, V1030G, E1035R, S1046G, or a combination thereof.

4. The Cas12i2 polypeptide is (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; The gene editing system of claim 3, comprising:

5. The Cas12i2 polypeptide, (i) D581R, D911R, I926R, and V1030G amino acid substitutions; (ii) D581R, I926R, and V1030G amino acid substitutions; (iii) D581R, I926R, V1030G, and S1046G amino acid substitutions; (iv) the amino acid substitutions D581R, G624R, F626R, I926R, V1030G, E1035R, and S1046G, or (v) D581R, G624R, F626R, P868T, I926R, V1030G, E1035R, and S1046G amino acid substitutions; The gene editing system of claim 4, comprising:

6. 2. The gene editing system of claim 1, wherein the Cas12i2 polypeptide comprises the amino acid sequence of SEQ ID NO: 450, 453, 449, 451, or 452.

7. 2. The gene editing system of claim 1, comprising the first nucleic acid encoding the Cas12i2 polypeptide.

8. 8. The gene editing system of claim 7, wherein the first nucleic acid is messenger RNA (mRNA) or is contained within a viral vector.

9. 2. The gene editing system of Claim 1, wherein the RNA guide comprises the sequence of any one of SEQ ID NOs: 4505, 4506, 4507, 4508, 4509, 4510, 4511, 4512, 4513, 4514, 4515, 4554, 4555, 4556, 4557, 4558, 4559, 4560, 4561, and 4562.

10. The gene editing system of claim 1, wherein the spacer sequence is 20 to 30 nucleotides in length.

11. 2. The gene editing system of claim 1, wherein the RNA guide comprises the spacer sequence and a direct repeat sequence, and the direct repeat sequence is 23 to 36 nucleotides in length.

12. The gene editing system of claim 11, wherein the direct repeat sequence is at least 90% identical to any one of SEQ ID NOs: 1 to 10, or a fragment thereof that is at least 23 nucleotides in length.

13. The gene editing system of claim 12, wherein the direct repeat sequence is 5'-AGAAAUCCGUCUUUCAUUGACGG-3' (sequence number 10).

14. 2. The gene editing system of Claim 1, wherein the system comprises the second nucleic acid encoding the RNA guide, and wherein the second nucleic acid encoding the RNA guide is located in a viral vector.

15. The gene editing system according to claim 1, wherein the gene editing system comprises a viral vector comprising the first nucleic acid encoding the Cas12i2 polypeptide and the second nucleic acid encoding the RNA guide; the gene editing system comprises the first nucleic acid encoding the Cas12i2 polypeptide located on a first vector and the second nucleic acid encoding the RNA guide located on a second vector; or The gene editing system comprises an mRNA encoding the Cas12i2 polypeptide and the RNA guide. The gene editing system of claim 1.

16. A gene editing system for gene editing of the stathmin 2 (STMN2) gene, comprising: (i) a Cas12i polypeptide or a first nucleic acid encoding the Cas12i polypeptide; and (ii) A gene editing system comprising an RNA guide or a second nucleic acid encoding the RNA guide, wherein the RNA guide comprises a spacer sequence specific for a target sequence within exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, or an intron of the STMN2 gene, wherein the target sequence is adjacent to a protospacer adjacent motif (PAM), wherein the PAM comprises a 5'-TTN-3' motif, and the 5'-TTN-3' motif is located 5' to the target sequence.

17. The gene editing system described in Claim 16, wherein the Cas12i polypeptide is a Cas12i2 polypeptide.

18. 17. The gene editing system of Claim 16, wherein the RNA guide comprises the sequence of any one of SEQ ID NOs: 4505, 4506, 4507, 4508, 4509, 4510, 4511, 4512, 4513, 4514, 4515, 4554, 4555, 4556, 4557, 4558, 4559, 4560, 4561, and 4562.

19. 17. The gene editing system of Claim 16, comprising the first nucleic acid encoding the Cas12i polypeptide, wherein the first nucleic acid is messenger RNA (mRNA) or is contained within a viral vector.

20. A gene editing system according to any one of claims 1 to 19, wherein the system comprises one or more lipid nanoparticles (LNPs).

21. A pharmaceutical composition comprising the gene editing system of any one of claims 1 to 19.

22. The pharmaceutical composition described in claim 21, wherein the gene editing system comprises one or more lipid nanoparticles (LNPs).

23. A kit comprising elements (i) and (ii) of the gene editing system of any one of claims 1 to 19.

24. 20. An in vitro method for editing the stathmin 2 (STMN2) gene in a cell, the method comprising contacting a host cell with a gene editing system for editing the STMN2 gene described in any one of claims 1 to 19 to genetically edit the STMN2 gene in the host cell, wherein the host cell is cultured in vitro.

25. A pharmaceutical composition for treating a neurodegenerative disease in a subject, the pharmaceutical composition comprising a gene editing system for editing the stathmin 2 (STMN2) gene described in any one of claims 1 to 19.

26. The pharmaceutical composition described in claim 25, wherein the gene editing system comprises one or more lipid nanoparticles (LNPs).

27. The pharmaceutical composition described in claim 25, wherein the neurodegenerative disease is amyotrophic lateral sclerosis (ALS) or frontotemporal dementia (FTD).

28. 1. An RNA guide comprising: (i) a spacer sequence specific for a target sequence in a stathmin 2 (STMN2) gene, wherein the target sequence is flanked by protospacer adjacent motifs (PAMs), the PAMs comprising a 5'-TTN-3' motif, and the 5'-TTN-3' motif is located 5' to the target sequence; and (ii) a direct repeat sequence.

29. 29. The RNA guide of claim 28, wherein the target sequence is within exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, or an intron of the STMN2 gene.

30. 30. The RNA guide of claim 29, wherein the RNA guide comprises the sequence of any one of SEQ ID NOs: 4505, 4506, 4507, 4508, 4509, 4510, 4511, 4512, 4513, 4514, 4515, 4554, 4555, 4556, 4557, 4558, 4559, 4560, 4561, and 4562.

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