Gene editing system for treating retinitis pigmentosa and application thereof

Abstract

The invention discloses a gene editing system for treating retinitis pigmentosa and application thereof. The gene editing system disclosed by the invention comprises a V-type CRISPR-Cas12 polypeptide, and a guide RNA (Ribonucleic Acid) for guiding the V-type CRISPR-Cas12 polypeptide to bind to a target nucleic acid of the RHO gene. Specifically disclosed herein are the gene editing systems, nucleic acids, vectors, delivery systems, kits, and compositions.

Description

Technical Field

[0001] The present invention relates to the field of nucleic acid editing, in particular to the field of clustered regularly interspaced short palindromic repeats (CRISPR). Specifically, the present invention relates to a gene editing system for treating retinitis pigmentosa, comprising nucleic acid molecules, compositions or kits encoding them. Background Art

[0002] Retinitis pigmentosa (PR) is a group of progressive, blinding hereditary retinal diseases characterized by degeneration of retinal photoreceptors (cones and rods) and degeneration of the retinal pigment epithelium, with a worldwide incidence of about 1 / 3500. RP is divided into nonsyndromic RP (NSRP) and syndromic RP (SRP) based on whether there are accompanying symptoms. Most of the currently known NSRPs are inherited by a single gene, and there are four main modes of inheritance: autosomal dominant RP (ADRP), autosomal recessive RP (ARRP), X-linked RP (XLRP) and mitochondrial linked RP. ADRP accounts for about 30% of all cases of retinitis pigmentosa, of which 25% are caused by mutations in the rhodopsin (RHO) gene.

[0003] CRISPR-Cas gene editing technology has great application potential in basic research and clinical practice, and has developed into a potential option for the treatment of RP. Currently, the CRISPR-Cas system is used to perform site-specific editing of the mutated RHO gene, thereby directly knocking out the RHO gene, interfering with transcription factor binding sites, interfering with RHO gene translation, and interfering with RHO gene mRNA splicing, ultimately treating the disease from the root.

[0004] Therefore, it is of great significance to develop a novel CRISPR / Cas system that is easy to deliver and has good properties in many aspects for the treatment of retinitis pigmentosa. Summary of the invention

[0005] One aspect of the present invention provides a gene editing system for treating retinitis pigmentosa, comprising: (a) a V-type CRISPR-Cas12 polypeptide; and (b) a guide RNA (gRNA), wherein the guide RNA is complexed with the V-type CRISPR-Cas12 polypeptide to guide the V-type CRISPR-Cas12 polypeptide to bind to a RHO gene target nucleic acid; wherein the V-type CRISPR-Cas12 polypeptide is selected from a V-type CRISPR-Cas12i polypeptide or a V-type CRISPR-Cas12f polypeptide; the V-type CRISPR-Cas12i polypeptide comprises an amino acid sequence having at least 95% sequence identity compared to the amino acid sequence shown in any one of SEQ ID NO.1 to 6; the V-type CRISPR-Cas12f polypeptide comprises an amino acid sequence having at least 95% sequence identity compared to the amino acid sequence shown in SEQ ID NO.8; wherein the guide RNA comprises a guide segment hybridizing with the target nucleic acid and a repeat segment binding to the V-type CRISPR-Cas12 polypeptide, and the guide segment of the guide RNA comprises SEQ ID NO. The nucleotide sequence shown in any one of 19 to 34, 37 to 54, or a nucleotide sequence having 1 to 10 nucleotide substitutions, deletions and / or insertions compared to the nucleotide sequence shown in any one of SEQ ID NO. 19 to 34, 37 to 54; preferably, the guide segment of the guide RNA is the nucleotide sequence shown in any one of SEQ ID NO. 19 to 34, 37 to 54.

[0006] In some embodiments, the V-type CRISPR-Cas12i polypeptide comprises or is an amino acid sequence having at least 95% sequence identity compared to the amino acid sequence shown in any one of SEQ ID NO. 1 to 6. For example, the V-type CRISPR-Cas12i polypeptide comprises or is an amino acid sequence having at least 95%, 96%, 97%, 98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or 99.9% sequence identity to the amino acid sequence shown in SEQ ID NO.1, and has an amino acid substitution at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 positions relative to the amino acid sequence shown in SEQ ID NO.1. For example, the V-type CRISPR-Cas12i polypeptide comprises or is an amino acid sequence having at least 95%, 96%, 97%, 98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or 99.9% sequence identity to the amino acid sequence shown in SEQ ID NO.2, and has an amino acid substitution at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 positions relative to the amino acid sequence shown in SEQ ID NO.2. For example, the V-type CRISPR-Cas12i polypeptide comprises or is an amino acid sequence having at least 95%, 96%, 97%, 98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or 99.9% sequence identity to the amino acid sequence shown in SEQ ID NO.3, and has an amino acid substitution at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 positions relative to the amino acid sequence shown in SEQ ID NO.3.For example, the V-type CRISPR-Cas12i polypeptide comprises or is an amino acid sequence having at least 95%, 96%, 97%, 98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or 99.9% sequence identity to the amino acid sequence shown in SEQ ID NO.4, and has an amino acid substitution at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 positions relative to the amino acid sequence shown in SEQ ID NO.4. For example, the V-type CRISPR-Cas12i polypeptide comprises or is an amino acid sequence having at least 95%, 96%, 97%, 98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or 99.9% sequence identity to the amino acid sequence shown in SEQ ID NO.5, and has an amino acid substitution at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 positions relative to the amino acid sequence shown in SEQ ID NO.5. For example, the V-type CRISPR-Cas12i polypeptide comprises or is an amino acid sequence having at least 95%, 96%, 97%, 98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or 99.9% sequence identity to the amino acid sequence shown in SEQ ID NO.6, and has an amino acid substitution at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 positions relative to the amino acid sequence shown in SEQ ID NO.6.

[0007] In some embodiments, the V-type CRISPR-Cas12f polypeptide comprises or is an amino acid sequence having at least 95% sequence identity compared to the amino acid sequence shown in SEQ ID NO.8. For example, the V-type CRISPR-Cas12f polypeptide comprises or is an amino acid sequence having at least 95%, 96%, 97%, 98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or 99.9% sequence identity compared to the amino acid sequence shown in SEQ ID NO.8, and has an amino acid substitution at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 positions relative to the amino acid sequence shown in SEQ ID NO.8.

[0008] In other embodiments, the V-type CRISPR-Cas12i polypeptide is mutated to have the characteristic of enhanced nucleic acid cleavage activity; wherein the V-type CRISPR-Cas12i polypeptide, according to the sequence numbering shown in any one of SEQ ID NO.1 to 6, has an amino acid substitution at position N229, preferably substituted with lysine, arginine or histidine, more preferably substituted with arginine. Specifically, the amino acid sequence of the mutant polypeptide (enCas12i-001-N229R mutant) of the V-type CRISPR-Cas12i is shown in SEQ ID NO.7.

[0009] In other embodiments, the V-type CRISPR-Cas12f polypeptide is mutated to have the characteristic of enhanced nucleic acid cleavage activity; the V-type CRISPR-Cas12 polypeptide is the V-type CRISPR-Cas12f polypeptide, according to the sequence number shown in SEQ ID NO.8, the V-type CRISPR-Cas12f polypeptide has one or more amino acid substitutions at positions A38R, S114K, S128R, N129D, Q130H, E142R, C171K, G194K, N203K, A216R, N223R, I241A, E261K, L271R, E341K, Y371K, V399K and S428K; preferably, according to SEQ ID According to the sequence numbering shown in SEQ ID NO.8, the V-type CRISPR-Cas12f polypeptide has amino acid substitutions at Q130H, N203K, A216R and I241A positions; preferably, according to the sequence numbering shown in SEQ ID NO.8, the V-type CRISPR-Cas12f polypeptide has amino acid substitutions at A38R, S128R and N129D positions; preferably, according to the sequence numbering shown in SEQ ID NO.8, the V-type CRISPR-Cas12f polypeptide has amino acid substitutions at S114K, S128R and N129D positions; preferably, according to the sequence numbering shown in SEQ ID NO.8, the V-type CRISPR-Cas12f polypeptide has amino acid substitutions at S128R, N129D and G194K positions; preferably, according to the sequence numbering shown in SEQ ID NO.8, the V-type CRISPR-Cas12f polypeptide has amino acid substitutions at S128R, N129D and Y371K positions; preferably, according to the sequence numbering shown in SEQ ID NO.8, the V-type CRISPR-Cas12f polypeptide has amino acid substitutions at positions A38R, S128R, N129D and Y371K; preferably, according to the sequence numbering shown in SEQ ID NO.8, the V-type CRISPR-Cas12f polypeptide has amino acid substitutions at positions S114K, S128R, N129D and Y371K; preferably, according to the sequence numbering shown in SEQ ID NO.8, the V-type CRISPR-Cas12f polypeptide has amino acid substitutions at positions S128R, N129D, G194K and Y371K; preferably, according to the sequence numbering shown in SEQ ID NO.8, the V-type CRISPR-Cas12f polypeptide has amino acid substitutions at positions A38R, S114K, S128R, N129D and Y371K; preferably, according to the sequence numbering shown in SEQ ID NO.8, the V-type CRISPR-Cas12f polypeptide has amino acid substitutions at positions A38R, S114K, S128R, N129D and Y371K; preferably, according to SEQ ID NO.8, the V-type CRISPR-Cas12f polypeptide has amino acid substitutions at positions A38R, S128R, N129D, G194K and Y371K. Specifically, the amino acid sequence of the mutant polypeptide of the V-type CRISPR-Cas12f is shown in any one of the amino acid sequences of SEQ ID NO.9 to 18. .

[0010] Specifically, the amino acid sequence of the mutant polypeptide (RaCas12f1-HKRA) of the V-type CRISPR-Cas12f is shown in SEQ ID NO.9; the amino acid sequence of the mutant polypeptide (RaCas12f1-V3.1) of the V-type CRISPR-Cas12f is shown in SEQ ID NO.10; the amino acid sequence of the mutant polypeptide (RaCas12f1-V3.2) of the V-type CRISPR-Cas12f is shown in SEQ ID NO.11; the amino acid sequence of the mutant polypeptide (RaCas12f1-V3.3) of the V-type CRISPR-Cas12f is shown in SEQ ID NO.12; the amino acid sequence of the mutant polypeptide (RaCas12f1-V3.4) of the V-type CRISPR-Cas12f is shown in SEQ ID NO.13; the amino acid sequence of the mutant polypeptide (RaCas12f1-V4.1) of the V-type CRISPR-Cas12f is shown in SEQ ID NO. IDNO.14; the amino acid sequence of the mutant polypeptide (RaCas12f1-V4.2) of the V-type CRISPR-Cas12f is shown in SEQ ID NO.15; the amino acid sequence of the mutant polypeptide (RaCas12f1-V4.3) of the V-type CRISPR-Cas12f is shown in SEQ ID NO.16; the amino acid sequence of the mutant polypeptide (RaCas12f1-V5.1) of the V-type CRISPR-Cas12f is shown in SEQ ID NO.17; the amino acid sequence of the mutant polypeptide (RaCas12f1-V5.2) of the V-type CRISPR-Cas12f is shown in SEQ ID NO.18.

[0011] In some embodiments, the mutation results in an enhancement of the nucleic acid cleavage activity of the V-type CRISPR-Cas12 polypeptide, for example, compared to the parent V-type CRISPR-Cas12 polypeptide (SEQ ID NO. 1 to 6), the nucleic acid cleavage activity is enhanced by at least 10%, for example, 10% to 500%, 10% to 100%, 10% to 200%, 10% to 300%, 10% to 50%, 10% to 30%, 10% to 20%, 50% to 100%, 50% to 200%, 50% to 300%, 100% to 200%, or 200% to 300%.

[0012] Specifically, the name of the mutant polypeptide in which the V-type CRISPR-Cas12 polypeptide is replaced by a specific amino acid at a specific position is named according to the mutation naming rules of the Human Genome Variation Society (HGVS), for example: "The V-type CRISPR-Cas12i polypeptide has an amino acid substitution at position N229" "N229R" means that the amino acid at position 229 of any one of SEQ ID NO.1 to 6 is replaced by R instead of the original N.

[0013] In a preferred embodiment, the V-type CRISPR-Cas12i polypeptide is selected from those Cas12i polypeptides disclosed in CN117431235A, and their disclosed contents are fully incorporated herein by reference; the V-type CRISPR-Cas12f polypeptide is selected from those Cas12f polypeptides disclosed in CN118127011A, and their disclosed contents are fully incorporated herein by reference.

[0014] In some embodiments, the V-type CRISPR-Cas12 polypeptide is a V-type CRISPR-Cas12i polypeptide, wherein the guide RNA does not contain and does not bind to tracrRNA; the repeat segment of the guide RNA contains the nucleotide sequence shown in any one of SEQ ID NO.55 to 62, or a nucleotide sequence having 1 to 10 nucleotide substitutions, deletions and / or insertions compared to the nucleotide sequence shown in any one of SEQ ID NO.55 to 62; preferably, the repeat segment of the guide RNA is the nucleotide sequence shown in any one of SEQ ID NO.55 to 62.

[0015] In some embodiments, the V-type CRISPR-Cas12 polypeptide is a V-type CRISPR-Cas12f polypeptide; the repeat segment of the guide RNA comprises the nucleotide sequence shown in SEQ ID NO.35 or 36 or a nucleotide sequence having 1 to 10 nucleotide substitutions, deletions and / or insertions compared to the nucleotide sequence shown in SEQ ID NO.35 or 36; preferably, the repeat segment of the guide RNA is the nucleotide sequence shown in SEQ ID NO.35 or 36.

[0016] In a preferred embodiment, the repeat segment suitable for the V-type CRISPR-Cas12i polypeptide is selected from the repeat segments of those guide RNAs disclosed in CN117431235A, and their disclosed contents are fully incorporated herein by reference; the repeat segment suitable for the V-type CRISPR-Cas12f polypeptide is selected from the repeat segments of those guide RNAs disclosed in CN118127011A, and their disclosed contents are fully incorporated herein by reference.

[0017] In some embodiments, the complementarity percentage between the guide segment of the guide RNA and the target site of the RHO gene target nucleic acid is 60% or more (e.g., 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100%). In some embodiments, the complementarity percentage between the guide segment of the guide RNA and the target site of the RHO gene target nucleic acid is 80% or more (e.g., 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100%). In some embodiments, the complementarity percentage between the guide segment of the guide RNA and the target site of the RHO gene target nucleic acid is 90% or more (e.g., 95% or more, 97% or more, 98% or more, 99% or more, or 100%). In some embodiments, the complementarity percentage between the guide segment of the guide RNA and the target site of the RHO gene target nucleic acid is 100%.

[0018] In some embodiments, the guide sequence of the guide RNA has a length of 15 to 50 nucleotides (e.g., 15 nucleotides (nt) to 20nt, 20nt to 25nt, 25nt to 30nt, 30nt to 35nt, 35nt to 40nt, 40nt to 45nt, or 45nt to 50nt). In some embodiments, the guide sequence of the guide RNA has a length in the range of 17-30 nucleotides (nt) (e.g., 17-25, 17-22, 17-20, 19-30, 19-25, 19-22, 19-20, 20-30, 20-25, or 20-22 nt). In some embodiments, the guide sequence of the guide RNA has a length in the range of 17-25 nucleotides (nt) (e.g., 17-22, 17-20, 19-25, 19-22, 19-20, 20-25, or 20-22 nt). In some embodiments, the guide sequence of the guide RNA has a length of 17 or more nt (e.g., 18 or more, 19 or more, 20 or more, 21 or more, or 22 or more nt; 19 nt, 20 nt, 21 nt, 22 nt, 23 nt, 24 nt, 25 nt, etc.). In some embodiments, the guide sequence of the guide RNA has a length of 19 or more nt (e.g., 20 or more, 21 or more, or 22 or more nt; 19 nt, 20 nt, 21 nt, 22 nt, 23 nt, 24 nt, 25 nt, etc.). In some embodiments, the guide sequence of the guide RNA has a length of 17 nt. In some embodiments, the guide sequence of the guide RNA has a length of 18 nt. In some embodiments, the guide sequence of the guide RNA has a length of 19 nt. In some embodiments, the guide sequence of the guide RNA has a length of 20 nt. In some embodiments, the guide sequence of the guide RNA has a length of 21 nt. In some embodiments, the guide sequence of the guide RNA has a length of 22 nt. In some embodiments, the guide sequence of the guide RNA has a length of 23 nt.

[0019] In some embodiments, the V-type CRISPR-Cas12 polypeptide is also fused with one or more heterologous polypeptides; wherein the one or more heterologous polypeptides are independently epitope tags, nuclear localization signals, reporter gene sequences, domains capable of binding to DNA molecules or intracellular molecules, 5'-3' exonuclease functional domains, enzymes that can detect signals, subcellular localization and protein transduction domains; preferably, the one or more heterologous polypeptides are fused to the N-terminus and / or C-terminus of the V-type CRISPR-Cas12 polypeptide.

[0020] In a preferred embodiment, the 5'-3' exonuclease domain is from T5 bacteriophage; the T5 exonuclease domain is fused to the N-terminus and / or C-terminus of the V-type CRISPR-Cas12 polypeptide.

[0021] In some embodiments, the heterologous polypeptide is selected from an epitope tag. Such epitope tags are conventional tags, including but not limited to His, V5, FLAG, HA, Myc, VSV-G, Trx, etc., and those skilled in the art know how to select a suitable epitope tag according to the desired purpose (e.g., purification, detection or tracing).

[0022] In some embodiments, the heterologous polypeptide is selected from a reporter gene sequence. Such reporter genes are well known to those skilled in the art, and examples thereof include but are not limited to GST, HRP, CAT, GFP, HcRed, DsRed, CFP, YFP, BFP, etc.

[0023] In some embodiments, the heterologous polypeptide is selected from a domain capable of binding to a DNA molecule or an intracellular molecule, such as maltose binding protein (MBP), the DNA binding domain (DBD) of LexA, the DBD of GAL4, and the like.

[0024] In some embodiments, the heterologous polypeptide can also be an enzyme, a radioisotope, a member of a specific binding pair, a fluorophore, a fluorescent protein, a quantum dot, etc. that can detect a signal.

[0025] In some embodiments, the heterologous polypeptide provides subcellular localization, i.e., the heterologous polypeptide contains a subcellular localization sequence (e.g., a nuclear localization signal (NLS) for targeting to the nucleus, a sequence for retaining the fusion protein outside the nucleus (e.g., a nuclear export sequence (NES)), a sequence for retaining the fusion protein in the cytoplasm, a mitochondrial localization signal for targeting to mitochondria, a chloroplast localization signal for targeting to chloroplasts, an ER retention signal, etc.).

[0026] In some embodiments, the V-type CRISPR-Cas12 polypeptide provided by the present invention is (fused with) a nuclear localization signal (NLS) (for example, in some embodiments, 2 or more, 3 or more, 4 or more, or 5 or more NLS). In some embodiments, one or more NLSs are located at the N-terminus of the V-type CRISPR-Cas12 polypeptide, and one or more NLSs are located at the C-terminus of the V-type CRISPR-Cas12 polypeptide. Specifically, the nuclear localization signal (NLS) connection sequence can be: NH2-[V-type CRISPR-Cas12]-[NLS]-COOH; NH2-[NLS]-[V-type CRISPR-Cas12]-COOH; NH2-[NLS]-[V-type CRISPR-Cas12]-[NLS]-COOH; wherein]-[ represents an optionally present connecting peptide as defined below (the same below).

[0027] In some embodiments, the fusion polypeptide provided by the present invention comprises (is fused with) 1 to 10 NLS (e.g., 1-9, 1-8, 1-7, 1-6, 1-5, 2-10, 2-9, 2-8, 2-7, 2-6 or 2-5 NLS). In some embodiments, the Cas12i fusion polypeptide comprises (is fused with) 2 to 5 NLS (e.g., 2-4 or 2-3 NLS).

[0028] Non-limiting examples of NLS include, but are not limited to, the NLS of SV40 virus large T antigen, nucleoplasmin bipartite NLS, c-mycNLS, hRNPA1 M9 NLS, the IBB domain of importin-α, myoma T protein, human p53, mouse c-abl IV, influenza virus NS1, hepatitis virus delta antigen, mouse Mx1 protein, human poly (ADP-ribose) polymerase, steroid hormone receptor (human) glucocorticoid and other commonly used NLSs.

[0029] In some embodiments, a "protein transduction domain" or PTD (also known as CPP-cell penetrating peptide) refers to a polypeptide, polynucleotide, carbohydrate or organic or inorganic compound that facilitates traversal of a lipid bilayer, a micelle, a cell membrane, an organelle membrane or a vesicle membrane. The PTD attached to another molecule (the molecule may range from a small polar molecule to a large macromolecular and / or nanoparticle) facilitates the molecule to traverse the membrane, for example, from the extracellular space into the intracellular space or from the cytosol into the organelle. In some embodiments, the PTD is covalently linked to the amino terminus of the V-type CRISPR-Cas12 polypeptide to generate a fusion protein. In some embodiments, the PTD is covalently linked to the carboxyl terminus of the V-type CRISPR-Cas12 polypeptide to generate a fusion protein. In some embodiments, the PTD is inserted into the fusion polypeptide at a suitable insertion site (ie, not at the N-terminus or C-terminus of the Cas12i fusion polypeptide). In some embodiments, the fusion polypeptide comprises (conjugated to, fused to) one or more PTDs (e.g., two or more, three or more, four or more PTDs). In some embodiments, a PTD comprises a nuclear localization signal (NLS) (eg, in some embodiments, 2 or more, 3 or more, 4 or more, or 5 or more NLS).

[0030] In some embodiments, the V-type CRISPR-Cas12 polypeptide can be fused with a heterologous polypeptide via one or more linker polypeptides (or linker peptides, linkers). Specifically, the linker polypeptide (or linker peptide, linker) may have any of a variety of amino acid sequences. Proteins can be connected by spacer peptides, which are generally flexible, but other chemical bonds are not excluded. Suitable linkers include polypeptides with a length of between 4 and 40 amino acids or a length of between 4 and 25 amino acids. These linkers can be produced by using synthetic oligonucleotides encoding linkers to couple proteins, or can be encoded by a nucleic acid sequence encoding a fusion protein. Peptide linkers with a certain degree of flexibility can be used. The connecting peptide can actually have any amino acid sequence, and it should be remembered that the preferred linker will have a sequence that produces a generally flexible peptide. The use of small amino acids (such as glycine and alanine) is used to produce flexible peptides. It is conventional for those skilled in the art to produce such sequences. A variety of different linkers are commercially available and are considered suitable for use.

[0031] Specifically, examples of linker polypeptides include glycine polymers ((G)n, wherein n is selected from 1, 2, 3, 4, 5 or 6), glycine-serine polymers ((GGGGS)n, (GGGS)n, (GGS)n, (GS)n or (G)n, wherein n is selected from 1, 2, 3, 4, 5 or 6), glycine-alanine polymers, alanine-serine polymers and α-helical linkers ((EAAAK)n, wherein n is selected from 1, 2, 3, 4, 5 or 6). The connecting peptide can also be various XTEN linkers, the length of XTEN linker is about 16-80 amino acids, and the XTEN linker can be XTEN16 linker, XTEN18 linker, XTEN32 linker, XTEN80 linker. Those skilled in the art will recognize that the design of the peptide conjugated to any desired element may include a fully or partially flexible linker, so that the linker may include a flexible linker and one or more portions that impart a less flexible structure.

[0032] Another aspect of the present invention provides a complex comprising any V-type CRISPR-Cas12 polypeptide provided by the present invention and a guide RNA, wherein the guide RNA is complexed with the fusion polypeptide to guide the fusion polypeptide to bind to the RHO gene target nucleic acid. In a preferred embodiment, in the complex, the guide RNA comprises a guide segment hybridized with the target nucleic acid and a repeat segment bound to the V-type CRISPR-Cas12 polypeptide. In a preferred embodiment, in the complex, the guide segment of the guide RNA comprises a nucleotide sequence shown in any one of SEQ ID NO.19 to 34, 37 to 54, or a nucleotide sequence having 1 to 10 nucleotide substitutions, deletions and / or insertions compared to the nucleotide sequence shown in any one of SEQ ID NO.19 to 34, 37 to 54; preferably, the guide segment of the guide RNA is a nucleotide sequence shown in any one of SEQ ID NO.19 to 34, 37 to 54; more preferably, the guide segment of the guide RNA is a nucleotide sequence shown in any one of SEQ ID NO.19, 31 or 47.

[0033] Another aspect of the present invention provides a nucleic acid comprising a polynucleotide encoding a type V CRISPR-Cas12 polypeptide of any gene editing system provided by the present invention. In a preferred embodiment, the polynucleotide is codon optimized for expression in prokaryotic or eukaryotic cells.

[0034] Another aspect of the present invention provides a nucleic acid comprising a guide RNA or a nucleotide sequence encoding the guide RNA, wherein the guide segment of the guide RNA comprises a nucleotide sequence as shown in any one of SEQ ID NOs. 19 to 34, 37 to 54, or a nucleotide sequence having 1 to 10 nucleotide substitutions, deletions and / or insertions compared with the nucleotide sequence as shown in any one of SEQ ID NOs. 19 to 34, 37 to 54; preferably, the guide segment of the guide RNA is a nucleotide sequence as shown in any one of SEQ ID NOs. 19 to 34, 37 to 54; the V-type CRISPR-Cas12 polypeptide is a V-type CRISPR-Cas12i polypeptide, and the repeat segment of the guide RNA comprises a nucleotide sequence as shown in any one of SEQ ID NOs. 55 to 62, or a nucleotide sequence having 1 to 10 nucleotide substitutions, deletions and / or insertions compared with the nucleotide sequence as shown in any one of SEQ ID NOs. 55 to 62; preferably, the repeat segment of the guide RNA is SEQ ID NO.55 to 62; the V-type CRISPR-Cas12 polypeptide is a V-type CRISPR-Cas12f polypeptide; the repeat segment of the guide RNA comprises the nucleotide sequence shown in SEQ ID NO.35 or 36 or a nucleotide sequence having 1 to 10 nucleotide substitutions, deletions and / or insertions compared with the nucleotide sequence shown in SEQ ID NO.35 or 36; preferably, the repeat segment of the guide RNA is the nucleotide sequence shown in SEQ ID NO.35 or 36; preferably, the nucleic acid is DNA or mRNA.

[0035] Another aspect of the present invention provides a vector comprising any one of the nucleic acids provided by the present invention. In a preferred embodiment, the vector is a plasmid or a viral vector. In a preferred embodiment, the viral vector is an adeno-associated viral vector, an adenoviral vector, a retroviral vector, a lentiviral vector or a herpes simplex viral vector. In a preferred embodiment, a V-type CRISPR-Cas12 polypeptide expression frame and a gRNA expression frame are provided between the two ITRs of the adeno-associated viral vector; preferably, the gRNA expression frame is arranged forward or reversely at the N-terminus or C-terminus of the V-type CRISPR-Cas12 polypeptide expression frame; preferably, one or two NLSs are connected to both ends of the V-type CRISPR-Cas12 polypeptide.

[0036] In some preferred embodiments, the adeno-associated viral vector includes a V-type CRISPR-Cas12 polypeptide expression frame and a forward-set gRNA expression frame in sequence from the 5' end to the 3' end between the two ITRs; preferably, the adeno-associated viral vector includes a V-type CRISPR-Cas12 polypeptide expression frame and a reverse-set gRNA expression frame in sequence from the 5' end to the 3' end between the two ITRs; preferably, the adeno-associated viral vector includes a forward-set gRNA expression frame and a V-type CRISPR-Cas12 polypeptide expression frame in sequence from the 5' end to the 3' end between the two ITRs; preferably, the adeno-associated viral vector includes a reverse-set gRNA expression frame and a V-type CRISPR-Cas12 polypeptide expression frame in sequence from the 5' end to the 3' end between the two ITRs.

[0037] In some preferred embodiments, the promoter of the V-type CRISPR-Cas12 polypeptide expression frame is a promoter such as a CAG promoter and a CMV promoter; the V-type CRISPR-Cas12 polypeptide expression frame comprises polyA; the promoter of the gRNA expression frame is a promoter of an RNA polymerase, such as a U6 promoter, a T7 promoter, etc.; the gRNA expression frame comprises a PolIII terminator.

[0038] In some preferred embodiments, in the V-type CRISPR-Cas12 polypeptide expression frame, the N-terminus or / and C-terminus of the V-type CRISPR-Cas12 nucleotide are connected to NLS or 2×NLS. Preferably, the V-type CRISPR-Cas12 polypeptide expression frame is: CAG promoter / CMV promoter, NLS, V-type CRISPR-Cas12 nucleotide and polyA in sequence; preferably, the V-type CRISPR-Cas12 polypeptide expression frame is: CAG promoter / CMV promoter, 2×NLS, V-type CRISPR-Cas12 nucleotide and polyA in sequence; preferably, the V-type CRISPR-Cas12 polypeptide expression frame is: CAG promoter / CMV promoter, V-type CRISPR-Cas12 nucleotide, NLS and polyA in sequence; preferably, the V-type CRISPR-Cas12 polypeptide expression frame is: CAG promoter / CMV promoter, V-type CRISPR-Cas12 nucleotide, 2×NLS and polyA in sequence.

[0039] In some preferred embodiments, the gRNA expression frame is: RNA polymerase promoter, gRNA and PolIII terminator.

[0040] In some preferred embodiments, the gRNA expression frame set in the forward direction and the gRNA expression frame set in the reverse direction are both conventional expression frame settings, and the promoter, gRNA and PolIII terminator of the RNA polymerase in the gRNA expression frame set in the forward direction and the gRNA expression frame set in the reverse direction are placed in opposite orders.

[0041] Specifically, in some preferred embodiments, the two ITRs of the AAV vector are, from the 5' end to the 3' end, sequentially: L-ITR, V-type CRISPR-Cas12 polypeptide expression frame, forward-set gRNA expression frame and R-ITR; preferably, the two ITRs of the AAV vector are, from the 5' end to the 3' end, sequentially: L-ITR, V-type CRISPR-Cas12 polypeptide expression frame, reverse-set gRNA expression frame and R-ITR; preferably, the two ITRs of the AAV vector are, from the 5' end to the 3' end, sequentially: L-ITR, forward-set gRNA expression frame, V-type CRISPR-Cas12 polypeptide expression frame and R-ITR; preferably, the two ITRs of the AAV vector are, from the 5' end to the 3' end, sequentially: L-ITR, reverse-set gRNA expression frame, V-type CRISPR-Cas12 polypeptide expression frame and R-ITR.wherein the L-ITR is the nucleotide sequence shown in SEQ ID NO.63; the R-ITR is the nucleotide sequence shown in SEQ ID NO.64; the CAG promoter is the nucleotide sequence shown in SEQ ID NO.65; the CMV promoter is the nucleotide sequence shown in SEQ ID NO.66; the NLS may be SV40NLS, and the SV40 NLS is the nucleotide sequence shown in SEQ ID NO.67; the 2×NLS may be 2×SV40NLS, and the 2×SV40 NLS is the nucleotide sequence shown in SEQ ID NO.68; the V-type CRISPR-Cas12 nucleotide may be a nucleotide of a eukaryotic codon-optimized RaCas12f1-HKRA protein (SEQ ID NO.9) or a RaCas12f1-V5.1 protein (SEQ ID NO.17), preferably, the V-type CRISPR-Cas12 nucleotide is the nucleotide sequence shown in SEQ ID NO.72; the polyA may be bGHpolyA, and the bGHpolyA is the nucleotide sequence shown in SEQ ID NO.69; the U6 promoter is SEQ IDNO.70; the PolIII terminator is the nucleotide sequence shown in SEQ ID NO.71; from the 5' end to the 3' end between the two ITRs, the gRNA is Scaffold [Rasgmax-1 (SEQ ID NO.35) or Rasgmax-2 (SEQ ID NO.36)] and RHO target gRNA in sequence, preferably, the RHO target gRNA comprises the nucleotide sequence shown in any one of SEQ ID NOs.19 to 34, 37 to 54, or a nucleotide sequence having 1 to 10 nucleotide substitutions, deletions and / or insertions compared with the nucleotide sequence shown in any one of SEQ ID NOs.19 to 34, 37 to 54; preferably, the RHO target gRNA is the nucleotide sequence shown in any one of SEQ ID NOs.19 to 34, 37 to 54; more preferably, the RHO target gRNA is the nucleotide sequence shown in any one of SEQ ID NOs.19 or 31.

[0042] Another aspect of the present invention provides a vector system, comprising a first vector and a second vector different from the first vector, wherein the first vector comprises a polynucleotide encoding a V-type CRISPR-Cas12 polypeptide of any gene editing system provided by the present invention; the second vector comprises a guide RNA or a nucleotide sequence encoding the guide RNA. In a preferred embodiment, the first vector and the second vector are independently plasmids or viral vectors. In a preferred embodiment, the viral vector is an adeno-associated viral vector, an adenoviral vector, a retroviral vector, a lentiviral vector, or a herpes simplex viral vector.

[0043] Another aspect of the present invention provides a delivery system, comprising any gene editing system for treating retinitis pigmentosa provided by the present invention, any complex provided by the present invention, any nucleic acid provided by the present invention, any vector provided by the present invention, or any vector system provided by the present invention. In a preferred embodiment, the delivery system comprises liposomes, nanoparticles or exosomes.

[0044] Another aspect of the present invention provides a cell comprising any gene editing system for treating retinitis pigmentosa provided by the present invention, any complex provided by the present invention, any nucleic acid provided by the present invention, any vector provided by the present invention, any vector system provided by the present invention, or any delivery system provided by the present invention. In a preferred embodiment, the cell is a eukaryotic cell. In a preferred embodiment, the cell is a human cell.

[0045] Another aspect of the present invention provides a composition or kit, which comprises any gene editing system for treating retinitis pigmentosa provided by the present invention, any complex provided by the present invention, any nucleic acid provided by the present invention, any vector provided by the present invention, any vector system provided by the present invention, any delivery system provided by the present invention, or any cell provided by the present invention; and a pharmaceutically acceptable carrier. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 The structural diagrams of the RaCas12f1-HKRA cutting vector, RaCas12f1-1 target vector and enCas12i-001-N229R-crRNA-RHO cutting vector of the present invention are shown.

[0047] Figure 2 The editing efficiency of the RaCas12f1-HKRA protein of the present invention from the RHO-1 target to the RHO-15 target is demonstrated.

[0048] Figure 3 The editing efficiency of the enCas12i-001-N229R protein of the present invention from the RHO-1 target site to the RHO-15 target site is demonstrated.

[0049] Figure 4 The editing efficiency of the RaCas12f1-HKRA protein of the present invention at the RHO-0 target, RHO-3 target, RHO-7 target, RHO-11 target, RHO-12 target and RHO-13 target is demonstrated.

[0050] Figure 5 The editing efficiency of the enCas12i-001-N229R protein of the present invention is shown at the RHO-1 target, RHO-2 target, RHO-4 target, RHO-5 target, RHO-6 target, RHO-7 target, RHO-8 target, RHO-9 target, RHO-10 target, RHO-11 target, RHO-12 target, RHO-14 target and RHO-15 target.

[0051] Figure 6 The long-term editing properties of the RaCas12f1-HKRA protein of the present invention at the RHO-0 target and RHO-7 target and the enCas12i-001-N229R protein at the RHO-7 target and RHO-10 target are demonstrated.

[0052] Figure 7 The vector structure diagram shows the ssAAV-CMV-RaCas12f1-HKRA cutting vector, ssAAV-CMV-RaCas12f1-V5.1 cutting vector, RaCas12f1-HKRA-RHO-0 target vector and RaCas12f1-V5.1-RHO-12 target vector of the present invention.

[0053] Figure 8 The editing efficiency of the AAV vectors of the RaCas12f1-HKRA protein and the RaCas12f1-V5.1 protein of the present invention at the RHO-0 target and the RHO-12 target is demonstrated.

[0054] Fig. 9 The TIDE results show the editing efficiency of the AAV vectors of the RaCas12f1-HKRA protein and RaCas12f1-V5.1 protein of the present invention at the RHO-0 target and the RHO-12 target.

[0055] Fig.10 Structural diagram showing different single AAV vectors constructed with the RaCas12f1-V5.1 protein and RHO-0 / RHO-12 targets of the present invention.

[0056] Fig.11 The editing efficiency of vectors A to H based on the RaCas12f1-V5.1 protein of the present invention at the RHO-0 target site and the RHO-12 target site is demonstrated.

[0057] Fig.12 The TIDE results show the editing efficiency of vectors A to H based on the RaCas12f1-V5.1 protein of the present invention at the RHO-0 target and the RHO-12 target.

[0058] Fig.13 The editing efficiencies of vectors A, I, O, E, J, and P based on the RaCas12f1-V5.1 protein of the present invention at the RHO-0 target and the RHO-12 target are demonstrated.

[0059] Fig.14 The editing efficiency of vector A, vector I, vector O, vector E, vector J and vector P based on the RaCas12f1-V5.1 protein of the present invention at the RHO-0 target and the RHO-12 target was demonstrated by TIDE results.

[0060] Fig.15 The editing efficiency of vector A, vector K, vector L, vector E, vector M and vector N based on the RaCas12f1-V5.1 protein of the present invention at the RHO-0 target and the RHO-12 target is demonstrated.

[0061] Fig.16 The editing efficiency of vector A, vector K, vector L, vector E, vector M and vector N based on the RaCas12f1-V5.1 protein of the present invention at the RHO-0 target and the RHO-12 target was demonstrated by TIDE results.

[0062] Fig.17 A structural diagram showing the ssAAV-CAG-RaCas12f1-V5.1 cutting vector and the Rasgmax-2 target vector of the present invention.

[0063] Fig.18 The editing efficiency of the ssAAV-CAG-RaCas12f1-V5.1 cutting vector of the present invention from the RHO-16 target site to the RHO-33 target site is demonstrated. DETAILED DESCRIPTION

[0064] Sequence Listing Example

[0065] Example 1. Screening of target nucleic acid sequences that can effectively knock out the RHO gene in human cell lines based on RaCas12f1-HKRA protein

[0066] In this experiment, Sanger sequencing and Tracking of Indels by Decomposition (TIDE) analysis were used to screen the target nucleic acid sequence suitable for knocking out the RHO gene by RaCas12f1-HKRA protein in 293T cells. Figure 1 The RaCas12f1-HKRA cutting vector was constructed as shown in Figure 1 a) and RaCas12f1 target vector ( Figure 1 b), RaCas12f1-HKRA cutting vector construction method is: RaCas12f1-HKRA (SEQ ID NO.9) is constructed in the AAV eukaryotic expression vector through eukaryotic codon-optimized nucleotides, the RaCas12f1-HKRA protein is expressed by the CAG promoter, and EGFP (for cell sorting) is connected by T2A downstream of RaCas12f1-HKRA. The RaCas12f1 target vector construction method is: gRNA sequences are constructed separately downstream of the U6 promoter of the commercial vector pUC19, and the gRNA sequences are repeat segments and targeting segments from the 5' end to the 3' end, and the repeat segment is Rasgmax-1 (SEQ ID NO.35), and the targeting segment is RHO target gRNA (SEQ ID NO.20 to SEQ ID NO.34, respectively), and the targeting segment of the gRNA (RHO target gRNA) is targeted to Exon1 of the RHO gene, and the RaCas12f1 target vector (RHO-1 to RHO-15 targets) is obtained.

[0067] Then, the above RaCas12f1-HKRA cutting vector and the above corresponding RaCas12f1 target vector (RHO-1 to RHO-15 target) were co-transfected into human 293T cells, and then cultured at 37°C and 5% carbon dioxide concentration. After 3 days of culture, the cell genome was extracted without cell sorting, and then Sanger sequencing and TIDE analysis were performed, and a blank control was set. The results are as follows: Figure 2 The electroporation efficiency was >90%, and the overall efficiency of RaCas12f1-HKRA protein on the RHO Exon1 target was low. The cutting efficiency of RHO-12 (SEQ ID NO.31) was the highest, greater than 20%, followed by RHO-3 (SEQ ID NO.22) and RHO-7 (SEQ ID NO.26). The cutting efficiency of the remaining RHO target gRNAs was low, which may be due to the low adaptability of RaCas12f1-HKRA to high GC genes.

[0068] Construct a new RaCas12f1 target vector, and construct a gRNA sequence and a polIII terminator (SEQ ID NO.71) downstream of the U6 promoter (SEQ ID NO.70) of the commercial vector pUC19 according to the above method. The gRNA sequence is followed by a repeat segment and a targeting segment from the 5' end to the 3' end, and its repeat segment is Rasgmax-1 (SEQ ID NO.35), and its targeting segment is RHO targetgRNA (SEQ ID NO.19, respectively), and the targeting segment (RHO-0) of the gRNA is targeted to the promoter region of the RHO gene. Then, select 5 RHO target gRNAs (RHO-3, RHO-7, RHO-11, RHO-12, and RHO-13) with higher efficiency in the above experiment to repeat the experiment. The above RaCas12f1-HKRA cutting vector and the corresponding RaCas12f1 target vector (RHO-0, RHO-3, RHO-7, RHO-11, RHO-12 and RHO-13 targets) were co-transfected into 293T cells, and the cells were sorted after 48 h of culture (EGFP-positive cells were collected by flow cytometry FACS), and the cells were cultured for 72 h after sorting. The genome of the sorted cells was then extracted, and Sanger sequencing and TIDE analysis were performed. A blank control was also set up. The results are shown in Figure 2. Figure 4 The results showed that the target efficiency of RHOtarget gRNA changed little before and after sorting, and the overall editing efficiency was not high. The better RHO target gRNA was the promoter region RHO-0 (SEQ ID NO.19), with an editing efficiency of about 50%, followed by the exon 1 region RHO-12 (SEQ ID NO.31), with an editing efficiency of about 30%.

[0069] Example 2. Screening of target nucleic acid sequences for knocking out the RHO gene in human cell lines based on enCas12i-001-N229R mutant protein

[0070] The target nucleic acid sequence for knocking out the RHO gene suitable for the enCas12i-001-N229R mutant protein was screened according to the method of Example 1. Figure 1 The enCas12i-001-N229R-crRNA-RHO cutting single vector was constructed as shown ( Figure 1c), the method for constructing the cutting single vector is as follows: the enCas12i-001-N229R mutant (SEQ ID NO.7) is constructed into a eukaryotic expression vector AAV through a eukaryotic codon-optimized nucleotide, the enCas12i-001-N229R mutant is expressed by a CAG promoter, EGFP (for cell sorting) is connected to the downstream of the enCas12i-001-N229R mutant through T2A, and a RHO gRNA expression cassette is constructed in the vector at the same time, the RHO gRNA expression cassette includes a U6 promoter (SEQ ID NO.70), a gRNA sequence and a polIII terminator (SEQ ID NO.71) in sequence, the gRNA sequence is a repeat segment and a targeting segment from the 5' end to the 3' end, the repeat segment is crRNA-1 (SEQ ID NO.37), the targeting segment is RHO target gRNA (SEQ ID NO.20 to SEQ ID NO.34, respectively), and the targeting segment of the gRNA (RHO targetgRNA) targets Exon1 of the RHO gene to obtain enCas12i-001-crRNA-RHO cutting single vector (RHO-1 to RHO-15 targets).

[0071] Then, the above enCas12i-001-N229R-crRNA-RHO cleavage single vector (RHO-1 to RHO-15 target sites) was transfected into human 293T cells, and then cultured at 37°C and 5% carbon dioxide concentration. The cell genome was extracted 3 days after transfection, and then Sanger sequencing and TIDE analysis were performed. At the same time, a blank control was set. The results are as follows Figure 3 The electroporation efficiency was >90%, and the overall efficiency of the enCas12i-001-N229R mutant on the RHO target was relatively uniform. The excision efficiency of most RHO target gRNAs was between 20% and 30%, indicating that the editing efficiency of a single vector was generally higher than that of the dual vector in Example 1.

[0072] Next, the 13 RHO target gRNAs (RHO-1, RHO-2, RHO-4, RHO-5, RHO-6, RHO-7, RHO-8, RHO-9, RHO-10, RHO-11, RHO-12, RHO-14 and RHO-15 targets) with higher efficiency in the above experiment were selected for repeated experiments. The above enCas12i-001-N229R-crRNA-RHO cleavage single vector (RHO-1, RHO-2, RHO-4, RHO-5, RHO-6, RHO-7, RHO-8, RHO-9, RHO-10, RHO-11, RHO-12, RHO-14 and RHO-15 targets) was transfected into 293T cells, and the cells were sorted after 48 h of culture (EGFP-positive cells were collected by flow cytometry FACS), and the cells were cultured for 72 hours after sorting. The genome of the sorted cells was then extracted, and Sanger sequencing and TIDE analysis were performed. A blank control was also set up. The results are shown in the figure. Figure 5 The results showed that enCas12i-001-N229R had a high editing efficiency in the overall target site of RHO target gRNA, and the efficiency of these targets was significantly improved before and after sorting.

[0073] Example 3. Long-term detection of RaCas12f1-HKRA protein and enCas12i-001-N229R mutant at RHO target gRNA target site

[0074] The long-term cutting effect of RaCas12f1-HKRA protein and enCas12i-001-N229R mutant on RHOtarget gRNA target in mammalian cells was tested. Sanger sequencing and TIDE analysis were used for verification. RaCas12f1-HKRA protein was verified by RaCas12f1-HKRA cutting vector and RaCas12f1 target vector in the above experiment. The enCas12i-001-N229R mutant was verified by enCas12i-001-N229R-crRNA-RHO cutting single vector in the above experiment. The target vectors selected RaCas12f1 target vector (RHO-0 target), RaCas12f1 target vector (RHO-7 target), enCas12i-001-N229R-crRNA-RHO cutting single vector (RHO-7 target) and enCas12i-001-N229R-crRNA-RHO cutting single vector (RHO-10 target). Divided into four groups: ①RaCas12f1-HKRA cutting vector and RaCas12f1 target vector (RHO-0 target), ②RaCas12f1-HKRA cutting vector and RaCas12f1 target vector (RHO-7 target), ③enCas12i-001-N229R-crRNA-RHO cutting single vector (RHO-7 target) and ④enCas12i-001-N229R-crRNA-RHO cutting single vector (RHO-10 target), the above four groups of double plasmids were transfected into 293T cells, and cells were sorted after 48 hours of culture (EGFP-positive cells were collected by flow cytometry FACS), and cultured after sorting. The genomes of the sorted cells were extracted on the 2nd, 5th, 7th, 14th and 21st days, respectively, and Sanger sequencing and TIDE analysis were performed. The results are as follows Figure 6 . Figure 6 aThe results showed that RaCas12f1-HKRA protein maintained long-term stable editing efficiency at both RHO-0 (SEQ ID NO.19) and RHO-7 (SEQ ID NO.26) targets, with the RHO-0 (SEQ ID NO.19) target showing an upward trend from the 2nd day to the 5th day. Figure 6 b The results show that the enCas12i-001-N229R mutant protein maintained long-term stable editing efficiency at both RHO-7 (SEQ ID NO.26) and RHO-10 (SEQ ID NO.29) targets, and both showed an upward trend. Figure 6This indicates that the RaCas12f1-HKRA protein has long-acting and strong editing efficiency at the RHO-0 (SEQ ID NO.19) target site, and the enCas12i-001-N229R mutant has long-acting and strong editing efficiency at the RHO-10 (SEQ ID NO.29) target site.

[0075] Example 4. Detection of cleavage efficiency of different RaCas12f1 mutants at the RHO target gRNA target site

[0076] In order to improve the cutting efficiency of different RaCas12f1 mutants at the RHO target gRNA target site, the vector of Example 1 was transformed into an AAV expression vector (① without fusion of T2A-EGFP, ② replacing the CAG promoter of the cutting vector of Example 1 with a CMV promoter), and ssAAV-CMV-RaCas12f1-HKRA cutting vector and ssAAV-CMV-RaCas12f1-V5.1 cutting vector were obtained; wherein, the structure of the ssAAV-CMV-RaCas12f1-HKRA cutting vector is as follows Figure 7 a, the two ITRs of the vector are L-ITR (SEQ ID NO.63), CMV promoter (SEQ ID NO.66), SV40NLS (SEQ ID NO.67), RaCas12f1-HKRA protein (SEQ ID NO.9), SV40NLS (SEQ ID NO.67), bGHpolyA (SEQ ID NO.69) and R-ITR (SEQ ID NO.64) from 5' to 3' ends; the ssAAV-CMV-RaCas12f1-V5.1 cutting vector structure is as follows Figure 7 b, the two ITRs of the vector are L-ITR (SEQ ID NO.63), CMV promoter (SEQ ID NO.66), SV40NLS (SEQ ID NO.67), RaCas12f1-V5.1 protein (SEQ ID NO.17), SV40NLS (SEQ ID NO.67), bGH polyA (SEQ ID NO.69) and R-ITR (SEQ ID NO.64) from the 5' end to the 3' end. The target vector selects the RaCas12f1 target vector (RHO-0 target and RHO-12 target) of Example 1, and the RaCas12f1-HKRA-RHO-0 target vector structure and the RaCas12f1-V5.1-RHO-12 target vector structure used are as follows Figure 7c and 7d. Divided into four groups: ①ssAAV-CMV-RaCas12f1-HKRA cutting vector and RaCas12f1-HKRA-RHO-0 target vector, ②ssAAV-CMV-RaCas12f1-HKRA cutting vector and RaCas12f1-V5.1-RHO-12 target vector, ③ssAAV-CMV-RaCas12f1-V5.1 cutting vector and RaCas12f1-HKRA-RHO-0 target vector, ④ssAAV-CMV-RaCas12f1-V5.1 cutting vector and RaCas12f1-V5.1-RHO-12 target vector. The above four groups of double plasmids were co-transfected into 293T cells, respectively, and then cultured at 37°C and 5% carbon dioxide concentration. After 2 days of culture, the cell genome was extracted without cell sorting, and then Sanger sequencing and TIDE analysis were performed. The results are as follows Figure 8 and Fig. 9 . Figure 8 It shows that at the RHO-0 (SEQ ID NO.19) target and the RHO-12 (SEQ ID NO.31) target, the editing efficiency of the RaCas12f1-V5.1 protein is significantly improved compared with that of the RaCas12f1-HKRA protein; Fig. 9 a is the TIDE analysis result of the cutting efficiency of RaCas12f1-HKRA at the RHO-0 (SEQ ID NO.19) target, Fig. 9 b is the TIDE analysis result of the cutting efficiency of RaCas12f1-HKRA at the RHO-12 (SEQ ID NO.31) target, Fig. 9 c is the TIDE analysis result of the cutting efficiency of RaCas12f1-V5.1 at the RHO-0 (SEQ ID NO.19) target, Fig. 9 d is the TIDE analysis result of the cleavage efficiency of RaCas12f1-V5.1 at the RHO-12 (SEQ ID NO.31) target. Fig. 9 TIDE analysis showed that the editing frame of RaCas12f1-V5.1 at the RHO target site was wider than that of RaCas12f1-HKRA protein, and the AAV vector of RaCas12f1-V5.1 was subsequently optimized.

[0077] Example 5. Optimization of the AAV vector structure of RaCas12f1-V5.1

[0078] The above examples have verified that RaCas12f1-V5.1 delivered by AAV vector can efficiently and long-term perform cutting and editing at RHO-0 and RHO-12 target sites, and further adjust the order of each expression frame of the above AAV expression vector, change the promoter type, and adjust the number and position of NLS to optimize the AAV vector of RaCas12f1-V5.1. Fig.10 Different single vectors (containing RaCas12f1-V5.1 expression frame and gRNA expression frame) were constructed as shown. Fig.10 The vector construction method is as follows: the eukaryotic codon-optimized RaCas12f1-V5.1 protein (SEQ ID NO.17) nucleotide (SEQ ID NO.72) is constructed in an AAV eukaryotic expression vector, the two ITRs of the AAV vector are L-ITR (SEQ ID NO.63) and R-ITR (SEQ ID NO.64), and the RaCas12f1-V5.1 protein is expressed by a CAG promoter (composed of a CMV early enhancer, a chicken β-actin promoter and a chimeric intron, SEQ ID NO.65) or a CMV promoter (composed of a CMV enhancer and a CMV promoter, SEQ ID NO.66), and at the same time, a SV40NLS (SEQ ID NO.67) or 2×SV40 NLS (SEQID NO.68) is constructed at the N-terminus or / and C-terminus of the RaCas12f1-V5.1 protein, and a bovine growth hormone gene polyA (bGH polyA, SEQ ID NO.69) is constructed downstream of the RaCas12f1-V5.1 protein. NO.69), the gRNA expression frame includes U6 promoter (SEQ ID NO.70), gRNA sequence and PolIII terminator (SEQ ID NO.71), the gRNA sequence constructed downstream of U6 promoter is a repeat segment and a targeting segment from 5' end to 3' end, the repeat segment is Rasgmax-1 (SEQ ID NO.35) or Rasgmax-2 (SEQ ID NO.36), and the targeting segment is RHO-0 (SEQ ID NO.19) or RHO-12 (SEQ ID NO.31).

[0079] As shown in Table 1 and Fig.10 , multiple single AVV vectors were constructed to test the effects of different promoters (CAG promoter or CMV promoter), different repeat segments of gRNA, the relative positions of RaCas12f1-V5.1 expression frame and RHO gRNA expression frame on the editing efficiency of the editor. These single AVV vectors are marked as vector A to vector J.

[0080] Table 1

[0081] As shown in Table 2 and Fig.10 , multiple single AVV vectors were constructed to test the effect of 2×SV40NLS at the N-terminus or C-terminus of the RaCas12f1-V5.1 protein on the editing efficiency of the editor, and these single AVV vectors were labeled from vector K to vector N.

[0082] Table 2

[0083] As shown in Table 3 and Fig.10 Two single AVV vectors were constructed, and the expression frame of U6 promoter-Rasgmax-2-RHO targetgRNA-PolIII terminator was placed in the vector in reverse order to test the effect of the reverse setting of the RHO gRNA expression frame at the N-terminus of the RaCas12f1-V5.1 expression frame on the editing efficiency of the editor. These single AVV vectors were marked as vector O and vector P.

[0084] Table 3

[0085] Then, the above single plasmids (vector A to vector H) were transfected into 293T cells respectively, and then cultured at 37°C and 5% carbon dioxide concentration. After culturing for 2 days, the cell genome was extracted without cell sorting, and then Sanger sequencing and TIDE analysis were performed. The results are as follows: Fig.11 and Fig.12 . When the gRNA expression frame is located at the C-terminus of the RaCas12f1-V5.1 expression frame, the gene editing efficiency mediated by the CAG promoter (54.8%) is not much different from that of the CMV promoter (56.7%) on the RHO-0 target site. On the RHO-12 target site, the gene editing efficiency mediated by the CAG promoter (29.7%) is significantly better than that of the CMV promoter (19.4%); when the promoter (CAG promoter) is the same, on the RHO-0 target site, the gene editing efficiency mediated by the gRNA expression frame located at the C-terminus (54.8%) in the case of tandem in the same direction is significantly better than that at the N-terminus (37.1%). On the RHO-12 target site, the gene editing efficiency mediated by the gRNA expression frame located at the C-terminus (29.7%) in the case of tandem in the same direction is also significantly better than that at the N-terminus (20.0%). Therefore, the CAG promoter is used to initiate the expression of RaCas12f1-V5.1 in the future.

[0086] Next, the above single plasmids (vector A, vector E, vector K, vector L, vector M and vector N) were transfected into 293T cells respectively, and then cultured at 37°C and 5% carbon dioxide concentration. After culturing for 2 days, the cell genome was extracted without cell sorting, and then Sanger sequencing and TIDE analysis were performed. The analysis results are as follows: Fig.15 and Fig.16These results show that, from the plasmid level, 2×NLS set at the N-terminus or C-terminus of RaCas12f1-V5.1 protein is slightly better than a single NLS set at both ends of RaCas12f1-V5.1 protein; the editing frame of 2×NLS set at the N-terminus or C-terminus of RaCas12f1-V5.1 is similar to that of a single NLS set at both ends of RaCas12f1-V5.1, indicating that 2×NLS set at the N-terminus or C-terminus of RaCas12f1-V5.1 does not affect the editing frame size of the RHO target site.

[0087] Finally, the above single plasmids (vector A, vector E, vector I, vector J, vector O and vector P) were transfected into 293T cells respectively, and then cultured at 37°C and 5% carbon dioxide concentration. After culturing for 2 days, the cell genome was extracted without cell sorting, and then Sanger sequencing and TIDE analysis were performed. The analysis results are shown in Fig.13 and Fig.14 . These results show that for the same target (RHO-0 and RHO-12), the RHO gRNA expression frame mediated by Rasgmax-1 has a higher editing efficiency than the RHO gRNA expression frame mediated by Rasgmax-2; for the same target (RHO-0 and RHO-12), the U6 promoter-mediated RHO gRNA expression frame reversely set at the N-terminus of the RaCas12f1-V5.1 expression frame has a higher gene editing efficiency than its forward setting at the C-terminus. The results show that adjusting the gRNA expression frame to the N-terminus of the RaCas12f1-V5.1 expression frame and the reverse setting of the gRNA expression frame can improve the gene editing efficiency of the AAV vector to a certain extent.

[0088] Example 6. Screening for RHO targets suitable for Rasgmax-2

[0089] The target nucleic acid sequence suitable for knocking out the RHO gene of RaCas12f1-V5.1 and Rasgmax-2 was screened by referring to the method of Example 1, and the RHO target gRNA suitable for Rasgmax-2 was designed with 5'-NTC-3' (N = A, T, C or G) as PAM. Fig.17 The ssAAV-CAG-RaCas12f1-V5.1 cutting vector was constructed as shown ( Fig.17 a) and Rasgmax-2 target vector ( Fig.17b), the RaCas12f1-V5.1 cutting vector construction method is: the eukaryotic codon-optimized nucleotides of RaCas12f1-V5.1 (SEQ ID NO.17) are constructed in the AAV eukaryotic expression vector, and the RaCas12f1-V5.1 protein is expressed by the CAG promoter. The Rasgmax-2 target vector construction method is: gRNA sequences are constructed downstream of the U6 promoter of the commercial vector pUC19, and the gRNA sequences are repeating segments and targeting segments from the 5' end to the 3' end, and the repeating segment is Rasgmax-2 (SEQ ID NO.36), and the targeting segment is RHO target gRNA (SEQ ID NO.37 to SEQ ID NO.54, respectively), to obtain the Rasgmax-2 target vector (RHO-16 to RHO-33 targets).

[0090] Then, the above RaCas12f1-V5.1 cutting vector and the above corresponding Rasgmax-2 target vector (RHO-16 to RHO-33 target) were co-transfected into human 293T cells, and then cultured at 37°C and 5% carbon dioxide concentration. After 3 days of culture, the cell genome was extracted without cell sorting, and then Sanger sequencing and TIDE analysis were performed, and a blank control was set up. The results are as follows: Fig.18 The electroporation efficiency was >90%, and the overall efficiency of RaCas12f1-V5.1 protein on the RHO target was low. RHO-26 (SEQ ID NO.47) had the highest cutting efficiency, with a cutting efficiency greater than 20%, followed by RHO-33 (SEQ ID NO.54), RHO-29 (SEQ ID NO.50) and RHO-18 (SEQ ID NO.39), with a cutting efficiency greater than 10%, and then RHO-21 (SEQ ID NO.42) and RHO-30 (SEQ ID NO.51), with a cutting efficiency greater than 9%, and the cutting efficiency of the remaining RHO target gRNAs was low.

Claims

1. A gene editing system for treating retinitis pigmentosa, comprising: (a) Type V CRISPR-Cas12 polypeptide; and (b) a guide RNA (gRNA), which is complexed with the V-type CRISPR-Cas12 polypeptide to guide the V-type CRISPR-Cas12 polypeptide to bind to the RHO gene target nucleic acid; wherein the V-type CRISPR-Cas12 polypeptide is selected from a V-type CRISPR-Cas12i polypeptide or a V-type CRISPR-Cas12f polypeptide; the V-type CRISPR-Cas12i polypeptide comprises an amino acid sequence having at least 95% sequence identity compared to the amino acid sequence shown in any one of SEQ ID NO.1 to 6; the V-type CRISPR-Cas12f polypeptide comprises an amino acid sequence having at least 95% sequence identity compared to the amino acid sequence shown in SEQ ID NO.8; The guide RNA comprises a guide segment that hybridizes with the target nucleic acid and a repeat segment that binds to a V-type CRISPR-Cas12 polypeptide, and the guide segment of the guide RNA comprises a nucleotide sequence as shown in any one of SEQ ID NOs. 19 to 34, 37 to 54, or a nucleotide sequence having 1 to 10 nucleotide substitutions, deletions and / or insertions compared with the nucleotide sequence as shown in any one of SEQ ID NOs. 19 to 34, 37 to 54; preferably, the guide segment of the guide RNA is a nucleotide sequence as shown in any one of SEQ ID NOs. 19 to 34, 37 to 54.

2. The gene editing system according to claim 1, wherein the V-type CRISPR-Cas12i polypeptide is mutated to have enhanced nucleic acid cleavage activity; The V-type CRISPR-Cas12i polypeptide, according to the sequence numbering shown in any one of SEQ ID NO.1 to 6, has an amino acid substitution at position N229, preferably substituted with lysine, arginine or histidine, more preferably substituted with arginine.

3. The gene editing system according to claim 2, wherein the V-type CRISPR-Cas12 polypeptide is a V-type CRISPR-Cas12i polypeptide, and the guide RNA does not contain and does not bind to tracrRNA; the repeat segment of the guide RNA comprises the nucleotide sequence shown in any one of SEQ ID NOs.55 to 62, or a nucleotide sequence having 1 to 10 nucleotide substitutions, deletions and / or insertions compared to the nucleotide sequence shown in any one of SEQ ID NOs.55 to 62; preferably, the repeat segment of the guide RNA is the nucleotide sequence shown in any one of SEQ ID NOs.55 to 62.

4. The gene editing system according to claim 1, wherein the V-type CRISPR-Cas12f polypeptide is mutated to have enhanced nucleic acid cleavage activity; The V-type CRISPR-Cas12 polypeptide is the V-type CRISPR-Cas12f polypeptide, and according to the sequence numbering shown in SEQ ID NO.8, the V-type CRISPR-Cas12f polypeptide has one or more amino acid substitutions at positions A38R, S114K, S128R, N129D, Q130H, E142R, C171K, G194K, N203K, A216R, N223R, I241A, E261K, L271R, E341K, Y371K, V399K and S428K; Preferably, according to the sequence numbering shown in SEQ ID NO.8, the V-type CRISPR-Cas12f polypeptide has amino acid substitutions at positions Q130H, N203K, A216R and I241A; Preferably, according to the sequence numbering shown in SEQ ID NO.8, the V-type CRISPR-Cas12f polypeptide has amino acid substitutions at positions A38R, S128R and N129D; Preferably, according to the sequence numbering shown in SEQ ID NO.8, the V-type CRISPR-Cas12f polypeptide has amino acid substitutions at positions S114K, S128R and N129D; Preferably, according to the sequence numbering shown in SEQ ID NO.8, the V-type CRISPR-Cas12f polypeptide has amino acid substitutions at positions S128R, N129D and G194K; Preferably, according to the sequence numbering shown in SEQ ID NO.8, the V-type CRISPR-Cas12f polypeptide has amino acid substitutions at positions S128R, N129D and Y371K; Preferably, according to the sequence numbering shown in SEQ ID NO.8, the V-type CRISPR-Cas12f polypeptide has amino acid substitutions at positions A38R, S128R, N129D and Y371K; Preferably, according to the sequence numbering shown in SEQ ID NO.8, the V-type CRISPR-Cas12f polypeptide has amino acid substitutions at positions S114K, S128R, N129D and Y371K; Preferably, according to the sequence numbering shown in SEQ ID NO.8, the V-type CRISPR-Cas12f polypeptide has amino acid substitutions at positions S128R, N129D, G194K and Y371K; Preferably, according to the sequence numbering shown in SEQ ID NO.8, the V-type CRISPR-Cas12f polypeptide has amino acid substitutions at positions A38R, S114K, S128R, N129D and Y371K; Preferably, according to the sequence numbering shown in SEQ ID NO.8, the V-type CRISPR-Cas12f polypeptide has amino acid substitutions at positions A38R, S128R, N129D, G194K and Y371K.

5. The gene editing system according to claim 4, wherein the V-type CRISPR-Cas12 polypeptide is a V-type CRISPR-Cas12f polypeptide; the repeating segment of the guide RNA comprises the nucleotide sequence shown in SEQ ID NO.35 or 36, or a nucleotide sequence having 1 to 10 nucleotide substitutions, deletions and / or insertions compared to the nucleotide sequence shown in SEQ ID NO.35 or 36; preferably, the repeating segment of the guide RNA is the nucleotide sequence shown in SEQ ID NO.35 or 36.

6. The gene editing system according to claim 1, wherein the V-type CRISPR-Cas12 polypeptide is also fused with one or more heterologous polypeptides; wherein the one or more heterologous polypeptides are independently epitope tags, nuclear localization signals, reporter gene sequences, domains capable of binding to DNA molecules or intracellular molecules, 5'-3' exonuclease functional domains, enzymes that can detect signals, subcellular localization and protein transduction domains; preferably, the one or more heterologous polypeptides are fused to the N-terminus and / or C-terminus of the V-type CRISPR-Cas12 polypeptide.

7. A nucleic acid comprising: A polynucleotide encoding a type V CRISPR-Cas12 polypeptide of the gene editing system according to any one of claims 1 to 6; preferably, the polynucleotide is codon-optimized for expression in prokaryotic or eukaryotic cells; and a guide RNA or a nucleotide sequence encoding the guide RNA.

8. The nucleic acid according to claim 7, comprising a guide RNA or a nucleotide sequence encoding the guide RNA, wherein the guide segment of the guide RNA comprises a nucleotide sequence as shown in any one of SEQ ID NOs. 19 to 34, 37 to 54, or a nucleotide sequence having 1 to 10 nucleotide substitutions, deletions and / or insertions compared to the nucleotide sequence as shown in any one of SEQ ID NOs. 19 to 34, 37 to 54; preferably, the guide segment of the guide RNA is a nucleotide sequence as shown in any one of SEQ ID NOs. 19 to 34, 37 to 54; The V-type CRISPR-Cas12 polypeptide is a V-type CRISPR-Cas12i polypeptide, and the repeat segment of the guide RNA comprises a nucleotide sequence as shown in any one of SEQ ID NOs. 55 to 62, or a nucleotide sequence having 1 to 10 nucleotide substitutions, deletions and / or insertions compared to the nucleotide sequence as shown in any one of SEQ ID NOs. 55 to 62; preferably, the repeat segment of the guide RNA is a nucleotide sequence as shown in any one of SEQ ID NOs. 55 to 62; The V-type CRISPR-Cas12 polypeptide is a V-type CRISPR-Cas12f polypeptide; the repeating segment of the guide RNA comprises the nucleotide sequence shown in SEQ ID NO.35 or 36 or a nucleotide sequence having 1 to 10 nucleotide substitutions, deletions and / or insertions compared to the nucleotide sequence shown in SEQ ID NO.35 or 36; preferably, the repeating segment of the guide RNA is the nucleotide sequence shown in SEQ ID NO.35 or 36; preferably, the nucleic acid is DNA or mRNA.

9. A vector comprising the nucleic acid according to claim 7 or 8; preferably, the vector is a plasmid or a viral vector; preferably, the viral vector is an adeno-associated viral vector, an adenoviral vector, a retroviral vector, a lentiviral vector or a herpes simplex viral vector; a V-type CRISPR-Cas12 polypeptide expression frame and a gRNA expression frame are provided between the two ITRs of the adeno-associated viral vector; preferably, the gRNA expression frame is arranged forward or reversely at the N-terminus or C-terminus of the V-type CRISPR-Cas12 polypeptide expression frame; preferably, one or two NLSs are connected to both ends of the V-type CRISPR-Cas12 polypeptide.

10. A composition or kit comprising the gene editing system of any one of claims 1 to 6, the nucleic acid of claim 7 or 8, or the vector of claim 9; and a pharmaceutically acceptable carrier.

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