CN117230043B

CN117230043B - Cas13 protein and application thereof

Info

Publication number: CN117230043B
Application number: CN202311508484.6A
Authority: CN
Inventors: 梁峻彬; 梁兴祥; 陈重建; 孙阳; 潘伟业; 徐辉; 司凯威; 李秋婷; 周琳; 张海辉
Original assignee: Zhejiang Xunzhi Biotechnology Co ltd; Guangzhou Ruifeng Biotechnology Co ltd
Current assignee: Zhejiang Xunzhi Biotechnology Co ltd; Guangzhou Ruifeng Biotechnology Co ltd
Priority date: 2023-11-14
Filing date: 2023-11-14
Publication date: 2024-04-12
Anticipated expiration: 2043-11-14
Also published as: CN117230043A

Abstract

The invention relates to an isolated Cas13 protein and application thereof, belonging to the technical field of genetic engineering. The amino acid sequence of the Cas13 protein is shown in any one of SEQ ID NO 1-3. The Cas13 protein has better editing efficiency and can be widely applied to various fields such as gene editing, detection and the like.

Description

Cas13 protein and application thereof

Technical Field

The invention relates to the technical field of genetic engineering, in particular to a Cas13 protein and application thereof.

Background

CRISPR-Cas13 is an RNA targeting and editing system based on the bacterial immune system that protects bacteria from viruses. The CRISPR-Cas13 system is similar to the CRISPR-Cas9 system, but unlike the Cas9 protein that targets DNA, the Cas13 protein targets RNA.

CRISPR-Cas13 belongs to the Type VI CRISPR-Cas13 system, which comprises one single effector protein Cas13. Currently, CRISPR-Cas13 can be divided into multiple subtypes (e.g., cas13a, cas13b, cas13c, and Cas13 d) according to phylogenetic development. However, there is still an urgent need to find new Cas13 systems that are compact in size (e.g., suitable for AAV delivery), high in editing efficiency in mammalian cells (e.g., RNA targeting/cleavage activity), and/or low in cytotoxicity (e.g., cell dormancy and apoptosis due to paracorporeal RNA degradation, or off-target).

Disclosure of Invention

In one aspect, the invention relates to an isolated Cas13 protein having an amino acid sequence that has at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to any one of SEQ ID NOs 1-3.

In some embodiments, the Cas13 protein is capable of forming a CRISPR complex with a guide polynucleotide comprising a cognate repeat sequence linked to a guide sequence engineered to hybridize to a target RNA. Where "engineering" refers to a process designed based on the target sequence to achieve the intended purpose, such as targeted hybridization to the target RNA, etc.

In some embodiments, the Cas13 protein is capable of forming a CRISPR complex with a guide polynucleotide, the CRISPR complex being capable of specifically binding to a target RNA sequence.

In some embodiments, the Cas13 protein is capable of forming a CRISPR complex with a guide polynucleotide, the CRISPR complex being capable of sequence-specifically binding and cleaving a target RNA.

In some embodiments, the Cas13 protein is capable of forming a CRISPR complex with a guide polynucleotide comprising a cognate repeat sequence linked to a guide sequence engineered to direct sequence specific binding of the CRISPR complex to a target RNA.

In some embodiments, the Cas13 protein is capable of forming a CRISPR complex with a guide polynucleotide comprising a cognate repeat sequence linked to a guide sequence engineered to direct the CRISPR complex to sequence specifically bind and cleave a target RNA.

In some embodiments, the amino acid sequence of the Cas13 protein has at least 80% sequence identity to any one of SEQ ID NOs 1-3. In some embodiments, the amino acid sequence of the Cas13 protein has at least 90% sequence identity to any one of SEQ ID NOs 1-3. In some embodiments, the amino acid sequence of the Cas13 protein has at least 95% sequence identity to any one of SEQ ID NOs 1-3. In some embodiments, the amino acid sequence of the Cas13 protein has at least 96% sequence identity to any one of SEQ ID NOs 1-3. In some embodiments, the amino acid sequence of the Cas13 protein has at least 97% sequence identity to any one of SEQ ID NOs 1-3. In some embodiments, the amino acid sequence of the Cas13 protein has at least 98% sequence identity to any one of SEQ ID NOs 1-3. In some embodiments, the amino acid sequence of the Cas13 protein has at least 99% sequence identity to any one of SEQ ID NOs 1-3. In some embodiments, the amino acid sequence of the Cas13 protein has at least 99.5% sequence identity to any one of SEQ ID NOs 1-3.

In some embodiments, the amino acid sequence of the Cas13 protein is as set forth in any one of SEQ ID NOs 1-3.

The Cas13 protein with the sequence of any one of SEQ ID NO 1-3 is identified based on bioinformatics analysis of procaryotic genome and metagenome in NCBI and CNGB databases and subsequent activity verification.

In some embodiments, the Cas13 proteins of the invention are from:

species (species) comprising a genome having an Average Nucleotide Identity (ANI) of 95% or more to the genome represented by GCA_016648615.1 in the NCBI database; or (b)

Species (specie) comprising a genome having an Average Nucleotide Identity (ANI) of 95% or more with the genome represented by CNA0007821 in the CNGB database.

Average nucleotide identity (average nucleotide identity, ANI) is an indicator of the similarity of all orthologous protein-encoding genes between two genomes at the nucleic acid level, and the threshold ANI=95% is generally used as a basis for determining whether bacteria/archaea are of the same species (Richter M, rossell, ra R. Shifting the genomic gold standard for the prokaryotic species definition. Proc Natl Acad Sci U S A. 2009 Nov 10;106 (45): 19126-31), therefore, the invention is defined by the above threshold, and species with ANI value of 95% or more with reference genome are considered to be of the same species, wherein the Cas13 protein has homology with the protein claimed by the invention and is functionally similar, and belongs to the scope of the invention. The ANI analysis tool includes procedures such as FastANI, JSpecies.

In some embodiments, the amino acid sequence of the Cas13 protein comprises 1, 2, 3, 4, 5, 6, 7, or more mutations, such as a single amino acid insertion, a single amino acid deletion, a single amino acid substitution, or a combination thereof, as compared to any of SEQ ID NOs 1-3.

In some embodiments, the Cas13 protein comprises one or more mutations in the catalytic domain and has reduced RNA cleavage activity. In some embodiments, the Cas13 protein comprises one mutation in the catalytic domain and has reduced RNA cleavage activity. In some embodiments, the Cas13 protein comprises one or more mutations in one or both HEPN domains and substantially lacks RNA cleavage activity. In some embodiments, the Cas13 protein comprises one mutation in any one HEPN domain and substantially lacks RNA cleavage activity.

The "substantially lack of RNA cleavage activity" refers to retention of only +.50%, +.40%, +.30%, +.20%, +.10%, +.5% or+.1% RNA cleavage activity, or no detectable RNA cleavage activity compared to the wild-type Cas13 protein.

In some embodiments, the rxxxxx H motif (x represents any amino acid, rxxxxH may also be denoted as Rx4H or R4 xH) in the HEPN domain of the Cas13 protein comprises one or more mutations and substantially lacks RNA cleavage activity.

In another aspect, the invention relates to a fusion protein comprising a Cas13 protein or a functional fragment thereof as described herein.

In some embodiments, the fusion protein comprises a Cas13 protein described herein.

In some embodiments, the fusion protein comprises an amino acid sequence as set forth in any one of SEQ ID NOs 1-3.

In some embodiments, the fusion protein comprises a functional fragment of a Cas13 protein described herein.

In some embodiments, the fusion protein comprises a protein functional domain.

In some embodiments, the protein functional domain is at least one of a protein domain and a polypeptide tag.

In some embodiments, the Cas13 protein is fused to a nuclear localization signal or a nuclear export signal.

In some embodiments, the protein functional domain is selected from any one or two or more of the following: functional protein domain: cytosine deaminase domain, adenine deaminase domain, translation activation domain, translation repression domain, RNA methylation domain, RNA demethylation domain, nuclease domain, splicing factor domain, reporter domain, affinity domain, subcellular localization signal, reporter tag and affinity tag.

In some embodiments, the fusion protein comprises a Cas13 protein or a functional fragment thereof as described herein, and a protein functional domain.

In some embodiments, the fusion protein comprises a Cas13 protein or a functional fragment thereof as described herein, and any one or two or more of the following fused to the Cas13 protein or functional fragment thereof: cytosine deaminase domain, adenine deaminase domain, translation activation domain, translation repression domain, RNA methylation domain, RNA demethylation domain, nuclease domain, splicing factor domain, subcellular localization signal, reporter tag and affinity tag.

In some embodiments, the fusion does not alter the original function of the Cas13 protein and/or the functional fragment thereof.

The original function of the Cas13 protein or functional fragment thereof is not altered, meaning that the fused protein still has the ability to recognize, bind and/or cleave target RNA when used in combination with gRNA. The Cas13 protein may have an increased or decreased ability to recognize, bind or cleave a target RNA when the fused protein is used in combination with a gRNA, but is in the "without changing the original function of the Cas13 protein" as long as the fused protein can effectively recognize, bind or cleave a target RNA when the fused protein is used in combination with a gRNA.

In some embodiments, the Cas13 protein or functional fragment thereof fuses a subcellular localization signal. In some embodiments, the subcellular localization signal is optionally derived from a Nuclear Localization Signal (NLS), a Nuclear Export Signal (NES), a chloroplast localization signal, or a mitochondrial localization signal.

In some embodiments, the Cas13 protein or functional fragment thereof is fused to a homologous or heterologous Nuclear Localization Signal (NLS). In some embodiments, the Cas13 protein or functional fragment thereof is fused to a homologous or heterologous Nuclear Export Signal (NES).

In some embodiments, the Cas13 protein or functional fragment thereof is covalently linked to a protein domain and/or a polypeptide tag. In some embodiments, the Cas13 protein or functional fragment thereof is directly covalently linked to a protein domain and/or polypeptide tag. In some embodiments, the Cas13 protein or functional fragment thereof is covalently linked to a protein domain and/or polypeptide tag through a linker sequence; further, in some embodiments, the linking sequence is an amino acid sequence.

In some embodiments, the Cas13 protein or functional fragment thereof of the fusion protein is linked to a homologous or heterologous protein domain and/or polypeptide tag by a rigid linking peptide sequence. In some embodiments, the Cas13 protein portion of the fusion protein is linked to a homologous or heterologous protein domain and/or polypeptide tag by a flexible linker peptide sequence.

In some embodiments, the fusion protein is capable of forming a CRISPR complex with a guide polynucleotide, the CRISPR complex capable of specifically binding to a target RNA sequence.

In some embodiments, the fusion protein comprises a Cas13 protein described herein fused to a homologous or heterologous protein domain and/or polypeptide tag, which fusion protein is capable of forming a CRISPR complex with a guide polynucleotide, which CRISPR complex is capable of specifically binding to a target RNA sequence.

In some embodiments, the fusion protein comprises a functional fragment of a Cas13 protein described herein fused to a homologous or heterologous protein domain and/or polypeptide tag, which fusion protein is capable of forming a CRISPR complex with a guide polynucleotide, which CRISPR complex is capable of specifically binding to a target RNA sequence.

In some embodiments, the fusion protein is capable of forming a CRISPR complex with a guide polynucleotide comprising a cognate repeat sequence linked to a guide sequence engineered to direct sequence specific binding of the CRISPR complex to a target RNA.

In some embodiments, the fusion protein comprises a Cas13 protein described herein fused to a homologous or heterologous protein domain and/or polypeptide tag, which fusion protein is capable of forming a CRISPR complex with a guide polynucleotide comprising a cognate repeat sequence linked to a guide sequence engineered to direct sequence-specific binding of the CRISPR complex to a target RNA.

In some embodiments, the fusion protein comprises a functional fragment of a Cas13 protein described herein fused to a homologous or heterologous protein domain and/or polypeptide tag, the fusion protein is capable of forming a CRISPR complex with a guide polynucleotide comprising a cognate repeat sequence linked to a guide sequence engineered to direct sequence-specific binding of the CRISPR complex to a target RNA.

In some embodiments, the fusion protein is capable of forming a CRISPR complex with a guide polynucleotide, the CRISPR complex capable of sequence-specifically binding and cleaving a target RNA.

In another aspect, the invention relates to a guide polynucleotide comprising (i) a cognate repeat having at least 50% sequence identity to any one of SEQ ID NOs 4-6 linked to (ii) a homologous or heterologous guide sequence engineered to hybridize to a target RNA, said guide polynucleotide being capable of forming a CRISPR complex with a Cas13 protein and guiding the sequence-specific binding of said CRISPR complex to said target RNA.

In some embodiments, the Cas13 protein is Cas13a, cas13b, cas13c, or Cas13d.

In some embodiments, the amino acid sequence of the Cas13 protein has at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100%, at least 95%, at least 98% or at least 99% sequence identity to any of SEQ ID NOs 1-3.

In some embodiments, the orthostatic repeat has at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100%, at least 95% or 100% sequence identity to any of SEQ ID NOs 4-6.

In some embodiments, the orthostatic repeat sequence has at least 80% sequence identity to any one of SEQ ID NOs 4-6. In some embodiments, the orthostatic repeat sequence has at least 85% sequence identity to any one of SEQ ID NOs 4-6. In some embodiments, the orthostatic repeat has at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to any one of SEQ ID NOs 4-6.

In some embodiments, the orthostatic repeat has 100% sequence identity to any one of SEQ ID NOs 4-6.

In some embodiments, the guide sequence is located 3' to the cognate repeat. In some embodiments, the guide sequence is located 5' to the cognate repeat.

In some embodiments, the guide sequence comprises 15-35 nucleotides. In some embodiments, the guide sequence hybridizes to the target RNA with no more than one nucleotide mismatch. In some embodiments, the orthostatic sequence comprises 25 to 40 nucleotides.

In some embodiments, the guide polynucleotide further comprises an aptamer sequence. In some embodiments, the aptamer sequence is inserted into a loop (loop) of the guide polynucleotide. In some embodiments, the aptamer sequence comprises an MS2 aptamer sequence, a PP7 aptamer sequence, or a qβ aptamer sequence.

In some embodiments, the guide polynucleotide comprises modified nucleotides. In some embodiments, the modification comprises 2' -O-methyl, 2' -O-methyl-3 ' -thiophosphate, or 2' -O-methyl-3 ' -thioppace.

In some embodiments, the target RNA of the guide polynucleotide is located in the nucleus of a eukaryotic cell.

In some embodiments, the target RNA is optionally selected from TTR RNA, SOD1 RNA, PCSK9 RNA, VEGFA RNA, VEGFR1 RNA, PTBP1 RNA, AQp1 RNA, or ANGPTL3 RNA. Alternatively, in some embodiments, the target RNA is optionally selected from VEGFA RNA, PTBP1 RNA, AQp1 RNA, or ANGPTL3 RNA.

In some embodiments, the Cas13 protein is Cas13a, cas13b, cas13c, or Cas13d. In some embodiments, the Cas13 protein is Cas13b. In some embodiments, the Cas13 protein is Cas13d. In some embodiments, the Cas13 protein has at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to any one of SEQ ID NOs 1-3.

In another aspect, the invention relates to a CRISPR-Cas13 system comprising: a Cas13 protein or fusion protein as described herein, or a nucleic acid encoding the Cas13 protein or fusion protein; and, a guide polynucleotide or a nucleic acid encoding the guide polynucleotide; the guide polynucleotide comprises a direct repeat sequence linked to a guide sequence engineered to hybridize to a target RNA; the guide polynucleotide is capable of forming a CRISPR complex with the Cas13 protein or fusion protein and directing sequence-specific binding of the CRISPR complex to a target RNA.

In some embodiments, the fusion protein comprises a Cas13 protein or a functional fragment thereof described herein fused to a homologous or heterologous protein domain and/or polypeptide tag.

In some embodiments, the Cas13 protein or functional fragment portion thereof of the fusion protein is fused to a homologous or heterologous Nuclear Localization Signal (NLS). In some embodiments, the Cas13 protein or functional fragment portion thereof of the fusion protein is fused to a homologous or heterologous Nuclear Export Signal (NES).

In some embodiments, the Cas13 protein comprises a mutation in the catalytic domain and has reduced RNA cleavage activity. In some embodiments, the Cas13 protein comprises mutations in one or both HEPN domains and substantially lacks RNA cleavage activity. In some embodiments, the "substantially lack of RNA cleavage activity" refers to retention of only +.50%, +.40%, +.30%, +.20%, +.10%, +.5% or+.1% RNA cleavage activity, or no detectable RNA cleavage activity compared to the wild-type Cas13 protein.

In some embodiments, the Cas13 protein or functional fragment portion thereof of the fusion protein is directly covalently linked to the homologous or heterologous protein domain and/or polypeptide tag. In some embodiments, the Cas13 protein portion of the fusion protein is linked to a homologous or heterologous protein domain and/or a polypeptide tag by a peptide sequence.

In some embodiments, the protein domain comprises a cytosine deaminase domain, an adenine deaminase domain, a translation activation domain, a translation repression domain, an RNA methylation domain, an RNA demethylation domain, a nuclease domain, or a splicing factor domain. In some embodiments, the Cas13 protein is covalently linked to an affinity tag or a reporter tag.

In some embodiments, the Cas13 protein has at least 95% sequence identity to any one of SEQ ID NOs 1-3. In some embodiments, the Cas13 protein has at least 97% sequence identity to any one of SEQ ID NOs 1-3. In some embodiments, the Cas13 protein has at least 98% sequence identity to any one of SEQ ID NOs 1-3. In some embodiments, the Cas13 protein has at least 99% sequence identity to any one of SEQ ID NOs 1-3. In some embodiments, the Cas13 protein has at least 99.5% sequence identity to any one of SEQ ID NOs 1-3. In some embodiments, the Cas13 protein has 100% sequence identity to any one of SEQ ID NOs 1-3. In some embodiments, the Cas13 protein comprises the sequence set forth in any one of SEQ ID NOs 1-3.

In some embodiments, the Cas13 protein is from:

In some embodiments, the Cas13 protein is used for RNA cleavage without the requirement for a pro-spacer flanking sequence (PFS).

In some embodiments, the guide polynucleotide comprises a linked guide sequence and a cognate repeat.

In some embodiments, the guide sequence comprises 15-35 nucleotides. In some embodiments, the guide sequence hybridizes to the target RNA with no more than one nucleotide mismatch.

In some embodiments, the orthostatic sequence comprises 25 to 40 nucleotides.

In some embodiments, the orthostatic repeat sequence has at least 80% sequence identity to any one of SEQ ID NOs 4-6. In some embodiments, the orthostatic repeat sequence has at least 90% sequence identity to any one of SEQ ID NOs 4-6. In some embodiments, the orthostatic repeat sequence has at least 95% sequence identity to any one of SEQ ID NOs 4-6. In some embodiments, the orthostatic repeat has 100% sequence identity to any one of SEQ ID NOs 4-6.

In some embodiments, the orthostatic repeat sequence is any one selected from SEQ ID NOS.4-6.

In some embodiments, the guide polynucleotide further comprises an aptamer sequence. In some embodiments, the aptamer sequence is inserted into a loop of a guide polynucleotide. In some embodiments, the aptamer sequence comprises an MS2 aptamer sequence, a PP7 aptamer sequence, or a qβ aptamer sequence.

In some embodiments, the CRISPR-Cas13 system comprises a fusion protein comprising a linker protein (adapter protein) capable of binding to the aptamer sequence and a homologous or heterologous protein domain, or a nucleic acid encoding the fusion protein.

In some embodiments, the linker protein comprises an MS2 bacteriophage coat protein, a PP7 bacteriophage coat protein, or a qβ bacteriophage coat protein.

In some embodiments, the protein domain comprises a cytosine deaminase domain, an adenine deaminase domain, a translation activation domain, a translation repression domain, an RNA methylation domain, an RNA demethylation domain, a nuclease domain, a splicing factor domain, a reporter domain, an affinity domain, a reporter tag, and an affinity tag.

In some embodiments, the guide polynucleotide comprises modified nucleotides.

In some embodiments, the modified nucleotide comprises 2' -O-methyl, 2' -O-methyl-3 ' -phosphorothioate, or 2' -O-methyl-3 ' -phosphorothioate.

In some embodiments, the Cas13 protein or fusion protein and the guide polynucleotide are not naturally co-occurring.

The present invention relates to a vector system comprising a CRISPR-Cas13 system as described herein, said vector system comprising one or more vectors comprising a polynucleotide sequence encoding a Cas13 protein or fusion protein as described herein and a polynucleotide sequence encoding a guide polynucleotide.

In another aspect, the invention relates to an adeno-associated virus (AAV) vector comprising a CRISPR-Cas13 system described herein, wherein the AAV vector comprises a DNA sequence encoding a Cas13 protein or fusion protein described herein and the guide polynucleotide.

In another aspect, the invention relates to a lipid nanoparticle comprising a CRISPR-Cas13 system described herein, the lipid nanoparticle comprising a guide polynucleotide described herein and an mRNA encoding a Cas13 protein or fusion protein described herein.

In another aspect, the invention relates to a lentiviral vector comprising a CRISPR-Cas13 system described herein, the lentiviral vector comprising a guide polynucleotide described herein and an mRNA encoding a Cas13 protein or fusion protein described herein. In some embodiments, the lentiviral vector is pseudotyped with a homologous or heterologous envelope protein, such as VSV-G. In some embodiments, the mRNA encoding the Cas13 protein or fusion protein is linked to an aptamer sequence.

In another aspect, the invention relates to a ribonucleoprotein complex comprising a CRISPR-Cas13 system described herein, wherein the ribonucleoprotein complex is formed from a guide polynucleotide described herein and a Cas13 protein or fusion protein described herein.

In another aspect, the invention relates to a virus-like particle comprising a CRISPR-Cas13 system described herein, the virus-like particle comprising a ribonucleoprotein complex formed from a guide polynucleotide described herein and a Cas13 protein or fusion protein described herein. In some embodiments, the Cas13 protein or fusion protein is fused to a gag protein.

In another aspect, the invention relates to a eukaryotic cell comprising a CRISPR-Cas13 system described herein. In some embodiments, the eukaryotic cell is a mammalian cell. In some embodiments, the eukaryotic cell is a human cell.

In another aspect, the invention relates to a pharmaceutical composition comprising a CRISPR-Cas13 system described herein, a Cas13 protein described herein, a fusion protein described herein, a guide polynucleotide described herein, a nucleic acid described herein, a vector system described herein, a lipid nanoparticle described herein, a lentiviral vector described herein, a ribonucleoprotein complex described herein, a virus-like particle described herein, or a eukaryotic cell described herein.

In some embodiments, the pharmaceutical composition comprises a CRISPR-Cas13 system described herein.

In another aspect, the invention relates to an in vitro composition comprising a CRISPR-Cas13 system described herein, and a labeled detector RNA that is incapable of hybridizing to or being targeted by a guide polynucleotide described herein.

In another aspect, the invention relates to an isolated nucleic acid encoding a Cas13 protein or fusion protein described herein.

In another aspect, the invention relates to an isolated nucleic acid encoding a guide polynucleotide described herein.

In another aspect, the invention relates to the use of a CRISPR-Cas13 system described herein for detecting a target RNA in a nucleic acid sample suspected to comprise the target RNA or for preparing a reagent for detecting a target RNA in a nucleic acid sample suspected to comprise the target RNA.

In another aspect, the invention relates to the use of a CRISPR-Cas13 system described herein for detecting a target RNA in a nucleic acid sample comprising the target RNA or for preparing a reagent for detecting the target RNA in a nucleic acid sample comprising the target RNA.

Another aspect of the invention relates to the use of a polypeptide comprising a CRISPR-Cas13 system described herein, a Cas13 protein described herein, a fusion protein described herein, a guide polynucleotide described herein, a nucleic acid described herein, a vector system described herein, a lipid nanoparticle described herein, a lentiviral vector described herein, a ribonucleoprotein complex described herein, a virus-like particle described herein or a eukaryotic cell described herein in a method or preparation of any one of the following to achieve any one of the following:

cleavage or nicking (nicking) of one or more target RNA molecules, activating or upregulating one or more target RNA molecules, activating or inhibiting translation of one or more target RNA molecules, inactivating one or more target RNA molecules, visualizing, labeling or detecting one or more target RNA molecules, binding one or more target RNA molecules, transporting one or more target RNA molecules, and masking one or more target RNA molecules.

In another aspect, the invention relates to the use of a CRISPR-Cas13 system described herein, a Cas13 protein described herein, a fusion protein described herein, a guide polynucleotide described herein, a nucleic acid described herein, a vector system described herein, a lipid nanoparticle described herein, a lentiviral vector described herein, a ribonucleoprotein complex described herein, a virus-like particle described herein or a eukaryotic cell described herein for cleaving one or more target RNA molecules or for the preparation of a reagent for cleaving one or more target RNA molecules.

In another aspect, the invention relates to the use of a CRISPR-Cas13 system described herein, a Cas13 protein described herein, a fusion protein described herein, a guide polynucleotide described herein, a nucleic acid described herein, a vector system described herein, a lipid nanoparticle described herein, a lentiviral vector described herein, a ribonucleoprotein complex described herein, a virus-like particle described herein or a eukaryotic cell described herein in binding to one or more target RNA molecules.

In another aspect, the invention relates to the use of a CRISPR-Cas13 system described herein in the preparation of an agent that binds or cleaves one or more target RNA molecules.

In another aspect, another aspect of the invention relates to the use of a CRISPR-Cas13 system described herein, a Cas13 protein described herein, a fusion protein described herein, a guide polynucleotide described herein, a nucleic acid described herein, a vector system described herein, a lipid nanoparticle described herein, a lentiviral vector described herein, a ribonucleoprotein complex described herein, a virus-like particle described herein, or a eukaryotic cell described herein, in cutting or editing a target RNA of a mammalian cell, the editing being base editing.

In another aspect, the invention relates to the use of a CRISPR-Cas13 system described herein for the preparation of an agent for cleaving or editing a target RNA of a mammalian cell, said editing being base editing.

In another aspect, the invention relates to the use of a CRISPR-Cas13 system described herein, a Cas13 protein described herein, a fusion protein described herein, a guide polynucleotide described herein, a nucleic acid described herein, a vector system described herein, a lipid nanoparticle described herein, a lentiviral vector described herein, a ribonucleoprotein complex described herein, a virus-like particle described herein or a eukaryotic cell described herein for activating or upregulating one or more target RNA molecules or for the preparation of an agent for activating or upregulating one or more target RNA molecules.

In another aspect, the invention relates to the use of a CRISPR-Cas13 system described herein, a Cas13 protein described herein, a fusion protein described herein, a guide polynucleotide described herein, a nucleic acid described herein, a vector system described herein, a lipid nanoparticle described herein, a lentiviral vector described herein, a ribonucleoprotein complex described herein, a virus-like particle described herein or a eukaryotic cell described herein for inhibiting the translation of one or more target RNA molecules or for the preparation of an agent that inhibits the translation of one or more target RNA molecules.

In another aspect, the invention relates to the use of a CRISPR-Cas13 system described herein, a Cas13 protein described herein, a fusion protein described herein, a guide polynucleotide described herein, a nucleic acid described herein, a vector system described herein, a lipid nanoparticle described herein, a lentiviral vector described herein, a ribonucleoprotein complex described herein, a virus-like particle described herein or a eukaryotic cell described herein for inactivating one or more target RNA molecules or for the preparation of an agent for inactivating one or more target RNA molecules.

In another aspect, the invention relates to the use of a CRISPR-Cas13 system described herein, a Cas13 protein described herein, a fusion protein described herein, a guide polynucleotide described herein, a nucleic acid described herein, a vector system described herein, a lipid nanoparticle described herein, a lentiviral vector described herein, a ribonucleoprotein complex described herein, a virus-like particle described herein or a eukaryotic cell described herein for visualizing, labeling or detecting one or more target RNA molecules or for the preparation of a reagent for visualizing, labeling or detecting one or more target RNA molecules.

In another aspect, the invention relates to the use of a CRISPR-Cas13 system described herein, a Cas13 protein described herein, a fusion protein described herein, a guide polynucleotide described herein, a nucleic acid described herein, a vector system described herein, a lipid nanoparticle described herein, a lentiviral vector described herein, a ribonucleoprotein complex described herein, a virus-like particle described herein or a eukaryotic cell described herein for transporting one or more target RNA molecules or for the preparation of a reagent for transporting one or more target RNA molecules.

In another aspect, the invention relates to the use of a CRISPR-Cas13 system described herein, a Cas13 protein described herein, a fusion protein described herein, a guide polynucleotide described herein, a nucleic acid described herein, a vector system described herein, a lipid nanoparticle described herein, a lentiviral vector described herein, a ribonucleoprotein complex described herein, a virus-like particle described herein or a eukaryotic cell described herein for masking one or more target RNA molecules or for the preparation of a reagent for masking one or more target RNA molecules.

In another aspect, the invention relates to the use of a CRISPR-Cas13 system described herein, a Cas13 protein described herein, a fusion protein described herein, a guide polynucleotide described herein, a nucleic acid described herein, a vector system described herein, a lipid nanoparticle described herein, a lentiviral vector described herein, a ribonucleoprotein complex described herein, a virus-like particle described herein, or a eukaryotic cell described herein in the diagnosis, treatment, or prevention of a disease or disorder associated with a target RNA.

In another aspect, the invention relates to the use of a CRISPR-Cas13 system described herein in the diagnosis, treatment or prevention of a disease or disorder associated with a target RNA.

In another aspect, the invention relates to a method of diagnosing, treating or preventing a disease or disorder associated with a target RNA, the method comprising: administering to a sample of a subject in need thereof or to a subject in need thereof a Cas13 protein as described herein, a fusion protein as described herein, a guide polynucleotide as described herein, a CRISPR-Cas13 system as described herein, or an isolated nucleic acid as described herein.

In another aspect, the invention relates to the use of a CRISPR-Cas13 system described herein, a Cas13 protein described herein, a fusion protein described herein, a guide polynucleotide described herein, a nucleic acid described herein, a vector system described herein, a lipid nanoparticle described herein, a lentiviral vector described herein, a ribonucleoprotein complex described herein, a virus-like particle described herein or a eukaryotic cell described herein in the manufacture of a medicament for the diagnosis, treatment or prevention of a disease or disorder associated with a target RNA.

In another aspect, the invention relates to the use of a CRISPR-Cas13 system described herein in the manufacture of a medicament for the diagnosis, treatment or prevention of a disease or disorder associated with a target RNA.

The above preferred conditions can be arbitrarily combined on the basis of not deviating from the common knowledge in the art, and thus, each preferred embodiment of the present invention can be obtained.

Reagents and materials for the present disclosure are commercially available.

Compared with the prior art, the invention has the following beneficial effects:

the separated Cas13 protein has better editing efficiency and can be widely applied to various fields such as gene editing, detection and the like.

Drawings

FIGS. 1-3 show the secondary structure of the corresponding orthostatic repeat of C13-52/C13-55/C13-88, respectively, using RNA fold prediction.

Fig. 4 shows the results of a test targeting knockdown Aqp1 RNA.

Detailed Description

The present disclosure is further illustrated by way of examples below, but is not thereby limited to the scope of the examples described. The experimental methods, in which specific conditions are not noted in the following examples, were selected according to conventional methods and conditions, or according to the commercial specifications.

As used herein, the term "sequence identity" (identity or percentage identity) is used to refer to the match of sequences between two polypeptides or between two nucleic acids. When a position in both compared sequences is occupied by the same base or amino acid monomer subunit (e.g., a position in each of two DNA molecules is occupied by adenine, or a position in each of two polypeptides is occupied by lysine), then the molecules are identical at that position. "percent sequence identity" (percent identity) between two sequences is a function of the number of matched positions shared by the two sequences divided by the number of positions to be compared x 100%. For example, if 6 out of 10 positions of two sequences match, then the two sequences have 60% sequence identity. Typically, the comparison is made when two sequences are aligned to produce maximum sequence identity. Such alignment may be by using published and commercially available alignment algorithms and procedures such as, but not limited to, clustal omega, MAFFT, probcons, T-Coffee, probalign, BLAST, which one of ordinary skill in the art would have a reasonable choice to use. One skilled in the art can determine suitable parameters for aligning sequences, including, for example, any algorithm required to achieve a superior alignment or optimal alignment for the full length of the compared sequences, and any algorithm required to achieve a superior alignment or optimal alignment for the parts of the compared sequences.

As used herein, the term "guide polynucleotide" is used to refer to a molecule in a CRISPR-Cas system that forms a CRISPR complex with a Cas protein and directs the CRISPR complex to a target sequence. Typically, the guide polynucleotide comprises a backbone sequence linked to a guide sequence, which can hybridize to the target sequence. The backbone sequence typically comprises a homeotropic sequence and sometimes also a tracrRNA sequence. When the backbone sequence does not comprise a tracrRNA sequence, the guide polynucleotide comprises a guide sequence and a homeotic repeat sequence, in which case the guide polynucleotide may also be referred to as crRNA.

CRISPR-Cas13 system:

class 2 CRISPR-Cas systems confer multiple adaptive immune mechanisms to microorganisms. Provided herein are analyses of prokaryotic and metagenomes to identify previously uncharacterized RNA guided, RNA-targetable CRISPR-Cas13 systems comprising C13-52, C13-55 and C13-88, which are classified as type VI systems. The engineered CRISPR-Cas13 system based on C13-52, C13-55 and C13-88 has better activity in human cells. The results herein demonstrate that C13-52, C13-55 and C13-88 are useful as a programmable RNA binding module for the efficient targeting of cellular RNA, thereby providing a versatile platform for transcriptome engineering as well as therapeutic and diagnostic methods.

The engineered CRISPR-Cas13 system described herein can effectively knock down endogenous target RNAs in human cells, paving the way for RNA targeting applications as part of a transcriptome engineering kit. In some embodiments, C13-52, C13-55, and C13-88 mediated knockdown across multiple endogenous transcripts can achieve greater efficiency and/or specificity than PspCas13b, cas13X.1, and/or Cas13Y.1 mediated knockdown.

In some embodiments, the polynucleotide sequence encoding a Cas13 protein or fusion protein and/or the polynucleotide sequence encoding a guide polynucleotide are operably linked to regulatory sequences. In some embodiments, the polynucleotide sequence encoding a Cas13 protein or fusion protein is operably linked to a regulatory control sequence. In some embodiments, the polynucleotide sequence encoding the guide polynucleotide is operably linked to a regulatory sequence. In some embodiments, the regulatory sequence of the polynucleotide sequence encoding the Cas13 protein or fusion protein is the same as or different from the regulatory sequence of the polynucleotide sequence encoding the guide polynucleotide.

In some embodiments, a Cas13 protein or fusion protein described herein has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% sequence identity to any one of SEQ ID NOs 1-3. When the CRISPR-Cas13 system comprises a fusion protein comprising the Cas13 protein and a protein domain and/or a polypeptide tag, the percent sequence identity between the Cas13 portion of the fusion protein and a reference sequence is calculated.

In some embodiments, a Cas13 protein or fusion protein described herein comprises one or more (e.g., 1 or 2) native HEPN domains, each native HEPN domain comprising an RX4H amino acid motif (where X represents any amino acid, and the subscript "4" represents 4 consecutive amino acids). In some embodiments, the Cas13 proteins or fusion proteins described herein comprise one or more mutated HEPN domains. In some embodiments, the mutant Cas13 protein or fusion protein can process its guide polynucleotide, but is unable to cleave the target RNA.

In some embodiments, the Cas13 proteins or fusion proteins described herein are used for RNA cleavage without the requirement of a protospacer flanking sequence (Protospacer Flanking Sequence, PFS).

The CRISPR-Cas13 system described herein can be introduced into cells (or cell-free systems) in a variety of non-limiting ways: (i) as Cas13 mRNA or fusion protein mRNA and guide polynucleotide, (ii) as part of a single vector or plasmid, or split into multiple vectors or plasmids, (iii) as a separate Cas13 protein or fusion protein, and guide polynucleotide, or (iv) as a RNP complex of Cas13 protein or fusion protein, and guide polynucleotide.

In some embodiments, the CRISPR-Cas13 system, composition or kit comprises a nucleic acid molecule encoding the Cas13 protein or fusion protein, wherein the coding sequence is codon optimized for expression in a eukaryotic cell. In some embodiments, the CRISPR-Cas13 system, composition or kit comprises a nucleic acid molecule encoding the Cas13 protein or fusion protein, wherein the coding sequence is codon optimized for expression in a mammalian cell. In some embodiments, the CRISPR-Cas13 system, composition or kit comprises a nucleic acid molecule encoding the Cas13 protein or fusion protein, wherein the coding sequence is codon optimized for expression in a human cell.

In some embodiments, the nucleic acid molecule encoding the Cas13 protein or fusion protein is a plasmid. In some embodiments, the nucleic acid molecule encoding the Cas13 protein or fusion protein is part of a viral vector genome, e.g., the DNA genome of an AAV vector flanked by ITRs. In some embodiments, the nucleic acid molecule encoding the Cas13 protein or fusion protein is mRNA.

Guide polynucleotide:

in some embodiments, the guide polynucleotide of the CRISPR-Cas13 system is a guide RNA. In some embodiments, the guide polynucleotide is a chemically modified guide polynucleotide. In some embodiments, the guide polynucleotide comprises at least one chemically modified nucleotide. In some embodiments, the guide polynucleotide is hybrid RNA-DNA guide. In some embodiments, the guide polynucleotide is a hybrid RNA-LNA (locked nucleic acid) guide.

In some embodiments, the guide polynucleotide comprises at least one guide sequence (also referred to as a spacer sequence) linked to at least one Direct Repeat (DR). In some embodiments, the guide sequence is located 3' to the orthostatic repeat sequence. In some embodiments, the guide sequence is located 5' to the orthostatic repeat sequence.

In some embodiments, the guide sequence comprises at least 15 nucleotides, at least 16 nucleotides, at least 17 nucleotides, at least 18 nucleotides, at least 19 nucleotides, at least 20 nucleotides, at least 21 nucleotides, at least 22 nucleotides, at least 23 nucleotides, at least 24 nucleotides, at least 25 nucleotides, at least 26 nucleotides, at least 27 nucleotides, at least 28 nucleotides, at least 29 nucleotides, or at least 30 nucleotides. In some embodiments, the guide sequence comprises no more than 60 nucleotides, no more than 55 nucleotides, no more than 50 nucleotides, no more than 45 nucleotides, no more than 40 nucleotides, no more than 35 nucleotides, or no more than 30 nucleotides. In some embodiments, the guide sequence comprises 15-20 nucleotides, 20-25 nucleotides, 25-30 nucleotides, 30-35 nucleotides, or 35-40 nucleotides.

In some embodiments, the guide sequence has sufficient complementarity to the target RNA sequence to hybridize to the target RNA and direct sequence-specific binding of the CRISPR-Cas13 complex to the target RNA. In some embodiments, the guide sequence has 100% complementarity to the target RNA (or region of RNA to be targeted), but the guide sequence may have less than 100% complementarity to the target RNA, such as at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% complementarity.

In some embodiments, the guide sequence is engineered to hybridize to the target RNA with no more than two nucleotides mismatched. In some embodiments, the guide sequence is engineered to hybridize to the target RNA with no more than one nucleotide mismatches. In some embodiments, the guide sequence is engineered to hybridize to the target RNA with or without mismatches.

In some embodiments, the orthostatic sequence comprises at least 20 nucleotides, at least 21 nucleotides, at least 22 nucleotides, at least 23 nucleotides, at least 24 nucleotides, at least 25 nucleotides, at least 26 nucleotides, at least 27 nucleotides, at least 28 nucleotides, at least 29 nucleotides, at least 30 nucleotides, at least 31 nucleotides, at least 32 nucleotides, at least 33 nucleotides, at least 34 nucleotides, at least 35 nucleotides, or at least 36 nucleotides. In some embodiments, the orthostatic sequence comprises no more than 60 nucleotides, no more than 55 nucleotides, no more than 50 nucleotides, no more than 45 nucleotides, no more than 40 nucleotides, or no more than 35 nucleotides. In some embodiments, the orthostatic sequence comprises 20-25 nucleotides, 25-30 nucleotides, 30-35 nucleotides, or 35-40 nucleotides.

In some embodiments, the orthostatic sequence is modified to alter the nucleotide sequence in the stem loop. In some embodiments, an aptamer (aptamer) sequence is inserted into or appended to the end of the orthostatic sequence. In some embodiments, the orthostatic repeat sequence has at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity compared to any of SEQ ID NOs 4-6.

In some embodiments, the CRISPR-Cas13 system, composition, or kit comprises at least 2, at least 3, at least 4, at least 5, at least 10, or at least 20 different guide polynucleotides. In some embodiments, the guide polynucleotide targets at least 2, at least 3, at least 4, at least 5, at least 10, or at least 20 different target RNA molecules, or targets at least 2, at least 3, at least 4, at least 5, at least 10, or at least 20 different regions of one or more target RNA molecules.

In some embodiments, the guide polynucleotide comprises a constant, homologous repeat sequence upstream of the variable guide sequence. In some embodiments, the plurality of guide polynucleotides is part of an array (which may be part of a vector, such as a viral vector or plasmid). For example, a guide array comprising the sequence DR-spacer-DR may comprise three unique unprocessed guide polynucleotides. Once introduced into a cell or cell-free system, the array is processed by the Cas13 protein into three separate maturation-guiding polynucleotides. This allows multiplexing, for example, delivery of multiple guide polynucleotides to a cell or system to target multiple target RNAs or multiple regions within a single target RNA.

The ability of the guide polynucleotide to direct sequence-specific binding of the CRISPR complex to the target RNA can be assessed by any suitable assay. For example, components of the CRISPR system sufficient to form a CRISPR complex, including the guide polynucleotide to be tested, can be provided to a host cell having a corresponding target RNA molecule, e.g., by transfection of a vector encoding the components of the CRISPR complex, and then preferential cleavage within the target sequence is assessed. Similarly, cleavage of a target RNA sequence can be assessed in a test tube by providing components of the target RNA, CRISPR complex, including a guide polynucleotide to be tested and a control guide polynucleotide different from the test guide polynucleotide, and comparing the ability to bind to or the rate of cleavage of the target RNA between the test and control guide polynucleotides.

Cas13 mutant:

in some embodiments, the Cas13 proteins provided herein comprise one or more mutations, such as a single amino acid insertion, a single amino acid deletion, a single amino acid substitution, or a combination thereof, as compared to the wild-type Cas13 protein (any one of SEQ ID NOs: 1-3). In some examples, the Cas13 protein comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, or 90 amino acid changes (e.g., insertions, deletions, or substitutions) as compared to the wild-type Cas13 protein (any of SEQ ID NO: 1-3), but retains the ability of the complementary sequence to bind to the guide RNA to the guide sequence of the polynucleotide to guide the RNA to retain the RNA to process the target molecule. In some examples, the Cas13 protein comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, or 90 amino acid changes (e.g., insertions, deletions, or substitutions) as compared to the wild-type Cas13 protein (any of SEQ ID NO: 1-3), but retains the ability to bind to the complementary target sequence of the polynucleotide to guide the RNA. In some examples, the Cas13 protein comprises 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, or 30 amino acid changes (e.g., insertions, deletions, or substitutions) as compared to the wild-type Cas13 protein (any of SEQ ID NOs: 1-3), but retains the ability to bind to a target RNA molecule complementary to a guide sequence of a guide polynucleotide, and/or retains the ability to process a guide array RNA transcript into a guide polynucleotide molecule.

In some embodiments, the Cas13 protein comprises one or more mutations in the catalytic domain and has reduced RNA cleavage activity. In some embodiments, the Cas13 protein comprises one mutation in the catalytic domain and has reduced RNA cleavage activity. In some embodiments, the Cas13 protein comprises one or more mutations in one or both HEPN domains and substantially lacks RNA cleavage activity. In some embodiments, the Cas13 protein comprises mutations in one or both HEPN domains and substantially lacks RNA cleavage activity. In some embodiments, the "substantially lack of RNA cleavage activity" refers to retention of only +.50%, +.40%, +.30%, +.20%, +.10%, +.5% or+.1% RNA cleavage activity, or no detectable RNA cleavage activity compared to the wild-type Cas13 protein.

In some embodiments, the Cas13 protein has 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 at least 99% sequence identity to any of SEQ ID NOs 1-3. When the CRISPR-Cas 13 system comprises a fusion protein of the Cas13 with a protein domain and/or a polypeptide tag, the percent sequence identity between the Cas13 portion of the fusion protein and a reference sequence is calculated.

One type of modification or mutation includes substitution of amino acid residues with similar biochemical properties, i.e., conservative substitutions (e.g., conservative substitutions of 1-4, 1-8, 1-10, or 1-20 amino acids). In general, conservative substitutions have little or no effect on the activity of the resulting protein or peptide. For example, conservative substitutions are amino acid substitutions in the Cas13 protein that do not substantially affect the binding of the Cas13 protein to a target RNA molecule that is complementary to a gRNA molecule guide sequence, and/or the process of processing the guide array RNA transcript into a gRNA molecule. Alanine scanning can be used to identify which amino acid residues in the Cas13 protein can tolerate amino acid substitutions. In one example, the ability of a variant Cas13 protein to modify gene expression in a CRISPR-Cas system does not change by more than 25%, such as by not more than 20%, such as by not more than 10% when alanine or other conserved amino acids are substituted with 1-4, 1-8, 1-10, or 1-20 natural amino acids. Examples of amino acids that may be substituted and are considered conservatively substituted include: replacement of Ala with Ser; substitution of Lys for Arg; substitution of Asn with Gln or His; substitution of Glu for Asp; replacement of Cys with Ser; substitution of Asn for Gln; substitution of Asp for Glu; substitution of Pro for Gly; substitution of Asn or Gln for His; substitution of le with Leu or Val; substitution of Ile or Val for Leu; substitution of Arg or Gln for Lys; substitution of Leu or Ile for Met; substitution of Met, leu or Tyr for Phe; substitution of Thr for Ser; substitution of Ser for Thr; replacement of Trp with Tyr; replacement of Tyr with Trp or Phe; substitution of Ile or Leu for Val.

More substantial changes may be made by using less conservative substitutions, for example, selecting residues that differ more in maintaining the following effects: (a) Substitution occurs in the region of the polypeptide backbone structure, for example, as a helix or folded conformation; (b) Charge or hydrophobicity of the region that interacts with the target site; or (c) the volume of the side chain. Substitutions that are generally expected to produce the greatest change in polypeptide function are (a): substitution between a hydrophilic residue (e.g., serine or threonine) and a hydrophobic residue (e.g., leucine, isoleucine, phenylalanine, valine, or alanine); (b) Substitution between cysteine or proline and any other residue; (c) Substitution between a positively charged side chain residue (e.g., lysine, arginine, or histidine) and a negatively charged residue (e.g., glutamic acid or aspartic acid); or (d) substitution between a residue having a bulky side chain (e.g., phenylalanine) and a residue having no side chain (e.g., glycine).

In some embodiments, the RxxxxH motif (x represents any amino acid, rxxxxH may also be denoted as Rx4H or R4 xH) of the Cas13 protein comprises one or more mutations and substantially lacks RNA cleavage activity.

Subcellular localization signal (or localization signal):

in some embodiments, the Cas13 protein or functional fragment thereof is fused to at least one homologous or heterologous subcellular localization signal. Exemplary subcellular localization signals include organelle localization signals such as Nuclear Localization Signals (NLS), nuclear Export Signals (NES), or mitochondrial localization signals.

In some embodiments, the Cas13 protein or functional fragment thereof is fused to at least 1 homologous or heterologous NLS. In some embodiments, the Cas13 protein or functional fragment thereof is fused to at least 2 NLS. In some embodiments, the Cas13 protein or functional fragment thereof is fused to at least 3 NLS. In some embodiments, the Cas13 protein or functional fragment thereof is fused to at least 1N-terminal NLS and at least 1C-terminal NLS. In some embodiments, the Cas13 protein or functional fragment thereof is fused to at least 2C-terminal NLS. In some embodiments, the Cas13 protein or functional fragment thereof is fused to at least 2N-terminal NLS.

In some embodiments, the NLS is independently selected from SPKKKRKVEAS (SEQ ID NO: 41), GPKKKRKVAAA (SEQ ID NO: 42), PKKKKKKKKKV (SEQ ID NO: 43), KRPAATKKA GQA KKKK (SEQ ID NO: 44), PAAKRVKLD (SEQ ID NO: 45), RQRRNELKRSP (SEQ ID NO: 46), NQSSNFGPMKGGNFGGRSSGPYGGGGQYFAKPRNQGGY (SEQ ID NO: 47), RMRIZFKGKDTARTYVERRVERRVERSKKKKKKDEQILKRRV (SEQ ID NO: 48), VSRKRPRP (SEQ ID NO: 49), PPKKARED (SEQ ID NO: 50), POPKKKPL (SEQ ID NO: 51), SALIKKKKKMAP (SEQ ID NO: 52), DRLRR (SEQ ID NO: 53), PKQKKK (SEQ ID NO: 54), RKLKKKIKKL (SEQ ID NO: 55), REKKKFLKRR (SEQ ID NO: 56), KRKGDEVDGVDEVAKKKSKK (SEQ ID NO: 57), RKCLQAGMNLEARKTKK (SEQ ID NO: 58), and PAAKKKKLD (SEQ ID NO: 59).

In some embodiments, the Cas13 protein or functional fragment thereof is fused to a homologous or heterologous NES. In some embodiments, the Cas13 protein or functional fragment thereof is fused to at least two NES. In some embodiments, the Cas13 protein or functional fragment thereof is fused to at least three NES. In some embodiments, the Cas13 protein or functional fragment thereof is fused to at least one N-terminal NES and at least one C-terminal NES. In some embodiments, the Cas13 protein or functional fragment thereof is fused to at least two C-terminal NES. In some embodiments, the Cas13 protein or functional fragment thereof is fused to at least two N-terminal NES.

In some embodiments, the NES is independently selected from adenovirus type 5E 1B NES, HIV Rev NES, MAPK NES, and PTK2 NES.

In some embodiments, the Cas13 protein or functional fragment thereof is fused to a homologous or heterologous NLS and NES, with a cleavable linker present between the NLS and the NES. In some embodiments, the NES facilitates production of a delivery particle (e.g., a virus-like particle) comprising the Cas13 protein or a functional fragment thereof in a production cell line. In some embodiments, cleavage of the linker in the target cell can expose the NLS and promote nuclear localization of the Cas13 protein or functional fragment thereof in the target cell.

Protein domain and polypeptide tag:

in some embodiments of the invention, the fusion protein comprises a Cas13 protein or a functional fragment thereof as described herein, as well as a homologous or heterologous protein domain and/or a polypeptide tag.

In some embodiments, the Cas13 protein or functional fragment thereof is covalently linked or fused to a homologous or heterologous protein domain and/or a polypeptide tag. In some embodiments, the Cas13 protein or functional fragment thereof is fused to a homologous or heterologous protein domain and/or a polypeptide tag.

In some embodiments, the protein domain and polypeptide tag are selected from the group consisting of: cytosine deaminase domain, adenine deaminase domain, translation activation domain, translation repression domain, RNA methylation domain, RNA demethylation domain, nuclease domain, splicing factor domain, reporter domain, affinity domain, subcellular localization signal, reporter tag and affinity tag.

In some embodiments, the protein domain comprises a cytosine deaminase domain, an adenine deaminase domain, a translation activation domain, a translation repression domain, an RNA methylation domain, an RNA demethylation domain, a ribonuclease domain, a splicing factor domain, a reporter domain, and an affinity domain. In some embodiments, the polypeptide tag comprises a reporter tag and an affinity tag.

In some embodiments, the amino acid sequence of the protein domain is no less than 40 amino acids, no less than 50 amino acids, no less than 60 amino acids, no less than 70 amino acids, no less than 80 amino acids, no less than 90 amino acids, no less than 100 amino acids, no less than 150 amino acids, no less than 200 amino acids, no less than 250 amino acids, no less than 300 amino acids, no less than 350 amino acids, or no less than 400 amino acids in length.

Exemplary protein domains include domains that can cleave RNA (e.g., PIN endonuclease domains, NYN domains, SMR domains from SOT1, or RNase domains from staphylococcal nucleases), domains that can affect RNA stability (e.g., tristetraprolin (TTP) or domains from UPF1, EXOSC5, and STAU 1), domains that can edit nucleotides or ribonucleotides (e.g., cytidine deaminase, PPR proteins, adenine deaminase, ADAR family proteins, or APOBEC family proteins), domains that can activate translation (e.g., eIF4E and other translation initiation factors, domains of yeast poly (A) binding proteins or GLD 2), domains that can inhibit translation (e.g., pumilo or FBF PUF proteins, deadenosine, CAF1, argonaute protein), a domain that can methylate RNA (e.g., a domain from an m6A methyltransferase factor (e.g., METTL14, METTL3, or WTAP), a domain that can demethylate RNA (e.g., human alkylated repair homolog 5), a domain that can affect splicing (e.g., RS-rich domain of SRSF1, gly-rich domain of hnRNP A1, alanine-rich motif of RBM4, or proline-rich motif of DAZAP 1), a domain that can achieve affinity purification or immunoprecipitation, and a domain that can achieve proximity-dependent (proximity-based) protein labeling and recognition (e.g., biotin ligase (e.g., birA) or peroxidase (e.g., APEX 2) such that a target DNA interacting protein is biotinylated).

In some embodiments, the protein domain comprises an adenine deaminase domain. In some embodiments, the Cas13 protein, the catalytically inactivated Cas13 protein, or the functional fragment thereof with a mutant HEPN domain is covalently linked or fused to an adenine deaminase domain to direct a-to-I deaminase activity of RNA transcripts in mammalian cells. Adenine deaminase domains for targeting a-to-I RNA editing based on ADAR2 engineering are described in Science 358 (6366): 1019-1027 (2017), which is incorporated herein by reference in its entirety. In other embodiments, the adenine deaminase domain is covalently linked or fused to a linker protein that is capable of binding to an aptamer sequence inserted or appended within the guide polynucleotide, allowing non-covalent linkage of the adenine deaminase domain to a Cas13 protein or functional fragment thereof complexed with the guide polynucleotide.

In some embodiments, the protein domain comprises a cytosine deaminase domain. In some embodiments, the Cas13 protein, the catalytically inactivated Cas13 protein, or the functional fragment thereof with a mutant HEPN domain is covalently linked or fused to a cytosine deaminase domain to direct the C-to-U deaminase activity of an RNA transcript in a mammalian cell. Cytosine deaminase domains evolved from ADAR2 for targeting C-to-U RNA editing are described in Abudayyeh et al, science 365 (6451): 382-386 (2019), incorporated herein by reference in its entirety. In other embodiments, the cytosine deaminase domain is covalently linked or fused to a linker protein that is capable of binding to an aptamer sequence inserted or attached to the guide polynucleotide, thereby allowing the cytosine deaminase domain to be non-covalently linked to the Cas13 protein or functional fragment thereof complexed with the guide polynucleotide.

In some embodiments, the protein domain comprises a splicing factor domain. In some embodiments, the Cas13 protein, the catalytically inactivated Cas13 protein, or the functional fragment thereof with a mutant HEPN domain is covalently linked or fused to a splicing factor domain to direct alternative splicing of a target RNA in a mammalian cell. Splice factor domains for targeting alternative splicing are described in Konermann et al, cell 173 (3): 665-676 (2018), which is incorporated herein by reference in its entirety. Non-limiting examples of splicing factor domains include the RS-rich domain of SRSF1, the Gly-rich domain of hnRNPA1, the alanine-rich motif of RBM4, or the proline-rich motif of DAZAP 1. In other embodiments, the splicing factor domain is covalently linked or fused to a linker protein capable of binding to an aptamer sequence inserted or attached to the guide polynucleotide, thereby allowing non-covalent attachment of the splicing factor domain to the Cas13 protein or functional fragment thereof complexed with the guide polynucleotide.

In some embodiments, the protein domain comprises a translation activation domain. In some embodiments, the Cas13 protein, the catalytically inactivated Cas13 protein, or the functional fragment thereof with a mutant HEPN domain is covalently linked or fused to a translation activation domain to activate or increase expression of a target RNA. Non-limiting examples of translation activation domains include the domains of eIF4E and other translation initiation factors, yeast poly (a) binding proteins, or GLD 2. In other embodiments, the translational activation domain is covalently linked or fused to a linker protein capable of binding to an aptamer sequence inserted or attached to the guide polynucleotide, thereby allowing non-covalent linkage of the translational activation domain to the Cas13 protein or functional fragment thereof complexed with the guide polynucleotide.

In some embodiments, the protein domain comprises a translational inhibition domain. In some embodiments, the Cas13 protein, the catalytically inactivated Cas13 protein, or the functional fragment thereof with a mutant HEPN domain is covalently linked or fused to a translation suppression domain to inhibit or reduce expression of a target RNA. Non-limiting examples of translational inhibition domains include the Pumilio or FBF PUF proteins, deadenase, CAF1, argonaute proteins. In other embodiments, the translational inhibition domain is covalently linked or fused to a linker protein capable of binding to an aptamer sequence inserted or appended within the guide polynucleotide, thereby allowing non-covalent linking of the translational inhibition domain to the Cas13 protein or functional fragment thereof complexed with the guide polynucleotide.

In some embodiments, the protein domain comprises an RNA methylation domain. In some embodiments, the Cas13 protein, the catalytically inactivated Cas13 protein, or the functional fragment thereof with a mutant HEPN domain is covalently linked or fused to an RNA methylation domain for methylation of a target RNA. Non-limiting examples of RNA methylation domains include m6A domains, such as METTL14, METTL3, or WTAP. In other embodiments, the RNA methylation domain is covalently linked or fused to a linker protein capable of binding to an aptamer sequence inserted or attached to the guide polynucleotide, thereby allowing non-covalent linkage of the RNA methylation domain to the Cas13 protein or functional fragment thereof complexed with the guide polynucleotide.

In some embodiments, the protein domain comprises an RNA demethylation domain. In some embodiments, the Cas13 protein, catalytically inactivated Cas13 protein, or functional fragment thereof with a mutant HEPN domain is covalently linked or fused to an RNA demethylation domain for demethylation of a target RNA. Non-limiting examples of RNA demethylation domains include the human alkylated repair homolog 5 or alk bh5. In other embodiments, the RNA demethylating domain is covalently linked or fused to a linker protein capable of binding to an aptamer sequence inserted or attached to the guide polynucleotide, thereby allowing non-covalent attachment of the RNA demethylating domain to the Cas13 protein or functional fragment thereof complexed with the guide polynucleotide.

In some embodiments, the protein domain comprises a ribonuclease domain. In some embodiments, the Cas13 protein, the catalytically inactivated Cas13 protein, or the functional fragment thereof with a mutant HEPN domain is covalently linked or fused to a ribonuclease domain to cleave a target RNA. Non-limiting examples of ribonuclease domains include a PIN endonuclease domain, a NYN domain, an SMR domain from SOT1, or an RNase domain from staphylococcal nuclease.

In some embodiments, the protein domain comprises an affinity domain (affinity domain) and/or a reporting domain (reporting domain). In some embodiments, the Cas13 protein or functional fragment thereof is covalently linked or fused to a reporter domain, such as a fluorescent protein. Non-limiting examples of reporting fields include GST, HRP, CAT, GFP, hcRed, dsRed, CFP, YFP, BFP.

In some embodiments, the Cas13 protein is covalently linked or fused to a polypeptide tag. In some embodiments, an example of the polypeptide tag is a small polypeptide sequence. In some embodiments, the amino acid sequence of the polypeptide tag is less than or equal to 50 amino acids, less than or equal to 40 amino acids, less than or equal to 30 amino acids, less than or equal to 25 amino acids, less than or equal to 20 amino acids, less than or equal to 15 amino acids, less than or equal to 10 amino acids, or less than or equal to 5 amino acids in length. In some embodiments, the Cas13 protein is covalently linked or fused to an affinity tag, such as a purification tag. Non-limiting examples of affinity tags include HA-tags, his-tags (e.g., 6-His), myc-tags, E-tags, S-tags, calmodulin tags, FLAG-tags, GST-tags, MBP-tags, halo tags, or biotin.

In some embodiments, the inactivated mutant of Cas13 is fused to a protein domain and/or a polypeptide tag.

In some embodiments, the Cas13 protein is fused to an ADAR.

In some embodiments, the inactivated mutant of Cas13 is fused to a cytosine deaminase or an adenine deaminase.

In some embodiments, the inactivated mutant of Cas13 is directly covalently linked to a cytosine deaminase or an adenine deaminase, linked by a rigid linking peptide sequence, or linked by a flexible linking peptide sequence.

Aptamer Sequence (Aptamer Sequence):

in some embodiments, the guide polynucleotide further comprises an aptamer (aptamer) sequence. In some embodiments, the aptamer sequence is inserted into a loop (loop) of a guide polynucleotide. In some embodiments, the aptamer sequence is inserted into a tetra loop of a guide polynucleotide. In some embodiments, the aptamer sequence is attached to the end of the guide polynucleotide.

The insertion of an aptamer sequence onto the guide polynucleotide of the CRISPR-Cas system is described in Konermann et al, nature 517:583-588 (2015), which is incorporated herein by reference in its entirety. In some embodiments, the aptamer sequence comprises an MS2 aptamer sequence, a PP7 aptamer sequence, or a qβ aptamer sequence.

Adaptor protein:

in some embodiments, the CRISPR-Cas13 system further comprises a fusion protein comprising a linker protein and a homologous or heterologous protein domain and/or polypeptide tag, or a nucleic acid encoding the fusion protein, wherein the linker protein is capable of binding to an aptamer sequence.

Fusion proteins of a linker protein and a protein domain are described in Konermann et al, nature 517:583-588 (2015), which is incorporated herein by reference in its entirety. In some embodiments, the linker protein comprises MS2 phage coat protein (MCP), PP7 Phage Coat Protein (PCP), or qβ phage coat protein (QCP). In some embodiments, the protein domain comprises a cytosine deaminase domain, an adenine deaminase domain, a translation activation domain, a translation repression domain, an RNA methylation domain, an RNA demethylation domain, a nuclease domain, a splicing factor domain, an affinity domain, or a reporter domain.

Modified guide polynucleotide:

in some embodiments, the guide polynucleotide comprises modified nucleotides. In some embodiments, the modified nucleotide comprises 2' -O-methyl, 2' -O-methyl-3 ' -phosphorothioate, or 2' -O-methyl-3 ' -phosphorothioate. In some embodiments, the guide polynucleotide is a chemically modified guide polynucleotide. Chemically modified guide polynucleotides are described in Hendel et al, nat.Biotechnol.33 (9): 985-989 (2015), which is incorporated herein by reference in its entirety.

In some embodiments, the guide polynucleotide is a hybrid RNA-DNA guide, a hybrid RNA-LNA (locked nucleic acid) guide, a hybrid DNA-LNA guide, or a hybrid DNA-RNA-LNA guide. In some embodiments, the orthostatic sequence comprises one or more ribonucleotides that are substituted with a corresponding deoxyribonucleotide. In some embodiments, the guide sequence comprises one or more ribonucleotides that were substituted with a corresponding deoxyribonucleotide. Hybrid RNA-DNA guide polynucleotides are described in WO2016/123230, which is incorporated herein by reference in its entirety.

Carrier system:

another aspect of the invention relates to a vector system comprising a CRISPR-Cas13 system described herein, said vector system comprising one or more vectors comprising a polynucleotide sequence encoding said Cas13 protein or fusion protein and a polynucleotide sequence encoding said guide polynucleotide.

In some embodiments, the vector system comprises at least one plasmid or viral vector (e.g., retrovirus, lentivirus, adenovirus, adeno-associated virus, or herpes simplex virus). In some embodiments, the polynucleotide sequence encoding the Cas13 protein or fusion protein and the polynucleotide sequence encoding the guide polynucleotide are on the same vector. In some embodiments, the polynucleotide sequence encoding the Cas13 protein or fusion protein and the polynucleotide sequence encoding the guide polynucleotide are on multiple vectors.

In some embodiments, the polynucleotide sequence encoding a Cas13 protein or fusion protein and/or the polynucleotide sequence encoding a guide polynucleotide are operably linked to regulatory sequences. In some embodiments, the polynucleotide sequence encoding a Cas13 protein or fusion protein is operably linked to a regulatory control sequence. In some embodiments, the polynucleotide sequence encoding the guide polynucleotide is operably linked to a regulatory sequence. In some embodiments, the regulatory sequence of the polynucleotide sequence encoding the Cas13 protein or fusion protein is the same as or different from the regulatory sequence of the polynucleotide sequence encoding the guide polynucleotide. In some embodiments, the regulatory sequences are optionally derived from promoters, enhancers, internal Ribosome Entry Sites (IRES), and other expression control elements (e.g., transcription termination signals, such as polyadenylation signals and poly-U sequences). In some embodiments, the regulatory sequences include regulatory sequences that allow constitutive expression of a nucleotide sequence in many types of host cells, as well as regulatory sequences that allow expression of the nucleotide sequence in only certain host cells (e.g., tissue-specific regulatory sequences). Tissue-specific promoters may be expressed primarily directly in the desired tissue of interest, such as muscle, neurons, bone, skin, blood, specific organs (e.g., liver, pancreas), or specific cell types (e.g., lymphocytes). Regulatory sequences may also direct expression in a time-dependent manner, for example in a cell cycle-dependent or developmental stage-dependent manner, which may or may not also be tissue or cell type specific. In some embodiments, the regulatory sequence is an enhancer element, such as WPRE, CMV enhancer, R-U5 segment in LTR of HTLV-1, SV40 enhancer, or an intron sequence between rabbit β -globin exons 2 and 3.

In some embodiments, the vector comprises a pol III promoter (e.g., U6 and H1 promoters), a pol II promoter (e.g., a retroviral Rous Sarcoma Virus (RSV) LTR promoter (optionally with an RSV enhancer), a Cytomegalovirus (CMV) promoter (optionally with a CMV enhancer), an SV40 promoter, a dihydrofolate reductase promoter, a β -actin promoter, a phosphoglycerate kinase (PGK) promoter, or an EF1 a promoter), or a pol III promoter and a pol II promoter.

In some embodiments, the promoter is a constitutive promoter that is continuously active and not regulated by external signals or molecules. Suitable constitutive promoters include, but are not limited to, CMV, RSV, SV, EF1 alpha, CAG, and beta-actin promoters. In some embodiments, the promoter is an inducible promoter regulated by an external signal or molecule (e.g., a transcription factor).

In some embodiments, the promoter is a tissue-specific promoter that can be used to drive tissue-specific expression of the Cas13 protein or fusion protein. Suitable muscle-specific promoters include, but are not limited to, CK8, MHCK7, myoglobin promoter (Mb), desmin promoter (Desmin), muscle creatine kinase promoter (MCK) and variants thereof, and SPc5-12 synthetic promoters. Suitable immune cell specific promoters include, but are not limited to, the B29 promoter (B cells), the CD14 promoter (monocytes), the CD43 promoter (leukocytes and platelets), the CD68 (macrophages) and the SV40/CD43 promoter (leukocytes and platelets). Suitable blood cell specific promoters include, but are not limited to, the CD43 promoter (white blood cells and platelets), the CD45 promoter (hematopoietic cells), INF- β (hematopoietic cells), the WASP promoter (hematopoietic cells), the SV40/CD43 promoter (white blood cells and platelets), and the SV40/CD45 promoter (hematopoietic cells). Suitable pancreatic specific promoters include, but are not limited to, elastase-1 promoters. Suitable endothelial cell specific promoters include, but are not limited to, the Fit-1 promoter and the ICAM-2 promoter. Suitable neuronal tissue/cell specific promoters include, but are not limited to, the GFAP promoter (astrocytes), the SYN1 promoter (neurons), and NSE/RU5' (mature neurons). Suitable kidney-specific promoters include, but are not limited to, the NphsI promoter (podocyte). Suitable bone-specific promoters include, but are not limited to, the OG-2 promoter (osteoblasts, odontoblasts). Suitable lung specific promoters include, but are not limited to, the SP-B promoter (lung). Suitable liver-specific promoters include, but are not limited to, the SV40/Alb promoter. Suitable heart-specific promoters include, but are not limited to, alpha-MHC.

AAV vector:

another aspect of the invention relates to an adeno-associated virus (AAV) vector comprising a CRISPR-Cas13 system described herein, wherein the adeno-associated virus (AAV) vector comprises DNA encoding a Cas13 protein or fusion protein described herein, and a guide polynucleotide.

Delivery of CRISPR-Cas systems by AAV vectors is described in Maeder et al, nature Medicine 25:229-233 (2019), which is incorporated herein by reference in its entirety. In some embodiments, the AAV vector comprises a ssDNA genome comprising a coding sequence for a Cas13 protein or fusion protein, and a guide polynucleotide flanking the ITR.

In some embodiments, a CRISPR-Cas13 system described herein is packaged in an AAV vector, such as AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, and AAVrh74. In some embodiments, a CRISPR-Cas13 system described herein is packaged in an AAV vector comprising an engineered capsid having tissue tropism, e.g., an engineered muscle tropism capsid. Engineering AAV capsids with tissue tropism by directed evolution is described in Tabebordbar et al, cell 184:4919-4938 (2021), which is incorporated herein by reference in its entirety.

Lipid nanoparticles:

another aspect of the invention relates to a Lipid Nanoparticle (LNP) comprising a CRISPR-Cas13 system described herein, wherein the LNP comprises a guide polynucleotide described herein, and an mRNA encoding a Cas13 protein or fusion protein described herein.

LNP delivery of the CRISPR-Cas system is described in Gillmore et al, N.Engl.J.Med., 385:493-502 (2021), which is incorporated herein by reference in its entirety. In some embodiments, the Lipid Nanoparticle (LNP) comprises four components in addition to the RNA payload (Cas 13 mRNA and guide polynucleotide): cationic or ionizable lipids, cholesterol, helper lipids, and PEG-lipids. In some embodiments, the cationic or ionizable lipids include cKK-E12, C12-200, ALC-0315, DLin-MC3-DMA, DLin-KC2-DMA, FTT5, moderna SM-102, and Intellia LP01. In some embodiments, the PEG-lipid comprises PEG-2000-C-DMG, PEG-2000-DMG, or ALC-0159. In some embodiments, the helper lipid comprises DSPC. The components of LNP are described in Paunovska et al, nature Reviews Genetics 23:265-280 (2022), incorporated herein by reference in its entirety.

Lentiviral vector:

another aspect of the invention relates to a lentiviral vector comprising a CRISPR-Cas13 system described herein, wherein the lentiviral vector comprises a guide polynucleotide described herein, and an mRNA encoding a Cas13 protein or fusion protein described herein. In some embodiments, the lentiviral vector is pseudotyped with a homologous or heterologous envelope protein, such as VSV-G. In some embodiments, the mRNA encoding the Cas13 protein or fusion protein is linked to an aptamer sequence.

RNP complex:

another aspect of the invention relates to ribonucleoprotein complexes comprising a CRISPR-Cas13 system described herein, wherein the ribonucleoprotein complexes are formed from a guide polynucleotide described herein and a Cas13 protein or fusion protein. In some embodiments, the ribonucleoprotein complex can be delivered to eukaryotic, mammalian, or human cells by microinjection or electroporation. In some embodiments, the ribonucleoprotein complex can be packaged in a virus-like particle and delivered to a mammalian or human subject in vivo.

Virus-like particles:

another aspect of the invention relates to a virus-like particle (VLP) comprising a CRISPR-Cas13 system described herein, wherein said virus-like particle comprises: a guide polynucleotide as described herein, and a Cas13 protein or fusion protein; or ribonucleoprotein complexes consisting of the guide polynucleotide, cas13 protein, or fusion protein.

Engineered VLPs are described in Banskota et al, cell 185 (2): 250-265 (2022), mangeot et al, nature Communications (1): 1-15 (2019), campbell, et al, molecular Therapy 27:151-163 (2019), campbell, et al, molecular Therapy,27 (2019): 151-163, and Mangeot et al Molecular Therapy, 19 (9): 1656-1666 (2011), the entire disclosures of which are incorporated herein by reference. In some embodiments, the engineered virus-like particle (VLP) is pseudotyped with a homologous or heterologous envelope protein, such as VSV-G. In some embodiments, the Cas13 protein is fused to a gag protein (e.g., MLVgag) via a cleavable linker, wherein cleavage of the linker in the target cell exposes the NLS located between the linker and the Cas13 protein. In some embodiments, the fusion protein comprises (e.g., from 5 'to 3') a gag protein (e.g., MLVgag), one or more NES, a cleavable linker, one or more NLS, and Cas13, as described in Banskota et al, cell 185 (2): 250-265 (2022). In some embodiments, the Cas13 protein is fused to a first dimerization domain capable of dimerizing or heterodimerizing with a second dimerization domain fused to a membrane protein, wherein the presence of a ligand promotes the dimerization and enriches the Cas13 protein or fusion protein into VLPs as described in Campbell, et al, molecular Therapy 27:151-163 (2019).

And (3) cells:

another aspect of the invention relates to a cell comprising a CRISPR-Cas13 system described herein. The cells (e.g., which may be used to produce a cell-free system) may be eukaryotic or prokaryotic. Examples of such cells include, but are not limited to, bacteria, archaebacteria, plants, fungi, yeasts, insect and mammalian cells, such as lactobacillus, lactococcus, bacillus (e.g., bacillus subtilis), escherichia (e.g., escherichia), clostridium, saccharomyces or pichia (e.g., saccharomyces cerevisiae or pichia pastoris), kluyveromyces lactis, salmonella typhimurium, drosophila cells, caenorhabditis elegans cells, xenopus cells, SF9 cells, C129 cells, 293 cells, neurospora and immortalized mammalian cell lines (e.g., hela cells, bone marrow cell lines and lymphoid cell lines).

In some embodiments, the cell is a prokaryotic cell, such as a bacterial cell, e.g., e. In some embodiments, the cell is a eukaryotic cell, such as a mammalian cell or a human cell. In some embodiments, the cells are primary eukaryotic cells, stem cells, tumor/cancer cells, circulating Tumor Cells (CTCs), blood cells (e.g., T cells, B cells, NK cells, tregs, etc.), hematopoietic stem cells, specialized immune cells (e.g., tumor-infiltrating lymphocytes or tumor-suppressing lymphocytes), stromal cells in the tumor microenvironment (e.g., cancer-associated fibroblasts, etc.). In some embodiments, the cell is a brain or neuronal cell of the central or peripheral nervous system (e.g., neuron, astrocyte, microglial cell, retinal ganglion cell, rod/cone cell, etc.).

Target RNA molecule:

the CRISPR-Cas13 systems, compositions, or kits described herein can be used to target one or more target RNA molecules, for example, target RNA molecules present in a biological sample, environmental sample (e.g., soil, air, or water sample), or the like. In some embodiments, the target RNA is a coding RNA, such as pre-mRNA or mature mRNA. In some embodiments, the target RNA is nuclear RNA. In some embodiments, the target RNA is an RNA transcript located in a eukaryotic cell nucleus. In some embodiments, the target RNA is a non-coding RNA, such as functional RNA, siRNA, microRNA, snRNA, snoRNA, piRNA, scaRNA, tRNA, rRNA, lncRNA or lincRNA.

In some embodiments, in addition to targeting a target RNA molecule, the CRISPR-Cas13 system, composition, or kit described herein performs one or more of the following functions on the target RNA: cleaving or nicking (nicking) one or more target RNA molecules, activating or upregulating one or more target RNA molecules, activating or inhibiting translation of one or more target RNA molecules, inactivating one or more target RNA molecules, visualizing, labeling or detecting one or more target RNA molecules, binding one or more target RNA molecules, editing one or more target RNA molecules, transporting one or more target RNA molecules, and masking one or more target RNA molecules. In some examples, a CRISPR-Cas13 system, composition, or kit described herein modifies one or more target RNA molecules, including one or more of the following: RNA base substitution, RNA base deletion, RNA base insertion, cleavage of target RNA, RNA methylation, and RNA demethylation. In some embodiments, a CRISPR-Cas13 system, composition, or kit described herein can target one or more target RNA molecules. In some embodiments, a CRISPR-Cas13 system, composition, or kit described herein can bind to one or more target RNA molecules. In some embodiments, a CRISPR-Cas13 system, composition, or kit described herein can cleave one or more target RNA molecules. In some embodiments, a CRISPR-Cas13 system, composition, or kit described herein can activate translation of one or more target RNA molecules. In some embodiments, a CRISPR-Cas13 system, composition, or kit described herein can inhibit translation of one or more target RNA molecules. In some embodiments, a CRISPR-Cas13 system, composition, or kit described herein can detect one or more target RNA molecules. In some embodiments, a CRISPR-Cas13 system, composition, or kit described herein can edit one or more target RNA molecules.

In some embodiments, the target RNA is AQp1 RNA. Knocking down Aqp1 RNA levels using the CRISPR-Cas13 system described herein can reduce aqueous humor production and lower ocular tension, and can be used to treat glaucoma and other diseases. In some embodiments, the target RNA is AQP1 RNA and the guide polynucleotide has a guide sequence of SEQ ID NO. 8.

In some embodiments, the target RNA is PTBP1 RNA. Knocking down the level of PTBP1 RNA using the CRISPR-Cas13 system described herein may promote brain astrocyte transdifferentiation into neurons, which may be useful for treating diseases such as parkinson's disease. In some embodiments, the target RNA is PTBP1 RNA.

In some embodiments, the target RNA is VEGFA RNA. Reducing the level of VEGFA RNA using the CRISPR-Cas13 system described herein can prevent choroidal neovascularization, which can be used to treat diseases such as age-related macular degeneration.

In some embodiments, the target RNA is ANGPTL3 RNA. Knocking down ANGPTL3 RNA levels using the CRISPR-Cas13 system described herein can reduce blood lipids such as low density lipoprotein cholesterol (LDL-C), and can be used to treat atherosclerotic cardiovascular diseases such as hyperlipidemia, familial hypercholesterolemia, and the like.

Therapeutic application:

another aspect of the invention relates to a pharmaceutical composition comprising a CRISPR-Cas13 system described herein, a Cas13 protein described herein, a fusion protein described herein, a guide polynucleotide described herein, a nucleic acid described herein, a vector system described herein, a lipid nanoparticle described herein, a lentiviral vector described herein, a ribonucleoprotein complex described herein, a virus-like particle described herein, or a eukaryotic cell described herein. The pharmaceutical composition can comprise, for example, an AAV vector encoding a Cas13 protein or fusion protein or guide polynucleotide as described herein. The pharmaceutical composition can comprise, for example, a lipid nanoparticle comprising a guide polynucleotide described herein and an mRNA encoding the Cas13 protein or fusion protein. The pharmaceutical composition can comprise, for example, a lentiviral vector comprising a guide polynucleotide as described herein and an mRNA encoding the Cas13 protein or fusion protein. The pharmaceutical composition may comprise, for example: a virus-like particle comprising a guide polynucleotide as described herein, a Cas13 protein or a fusion protein; or ribonucleoprotein complexes formed from the guide polynucleotide, cas13 protein, or fusion protein.

Another aspect of the invention relates to the use of a CRISPR-Cas13 system described herein, a Cas13 protein described herein, a fusion protein described herein, a guide polynucleotide described herein, a nucleic acid described herein, a vector system described herein, a lipid nanoparticle described herein, a lentiviral vector described herein, a ribonucleoprotein complex described herein, a virus-like particle described herein, or a eukaryotic cell described herein for cleaving or editing a target RNA in a mammalian cell.

Another aspect of the invention relates to the use of a CRISPR-Cas13 system described herein, a Cas13 protein described herein, a fusion protein described herein, a guide polynucleotide described herein, a nucleic acid described herein, a vector system described herein, a lipid nanoparticle described herein, a lentiviral vector described herein, a ribonucleoprotein complex described herein, a virus-like particle described herein, or a eukaryotic cell described herein in any of the following: cleavage or nicking (nicking) of one or more target RNA molecules, activating or upregulating one or more target RNA molecules, activating or inhibiting translation of one or more target RNA molecules, inactivating one or more target RNA molecules, visualizing, labeling or detecting one or more target RNA molecules, binding one or more target RNA molecules, transporting one or more target RNA molecules, and masking one or more target RNA molecules.

Another aspect of the invention relates to the use of a CRISPR-Cas13 system described herein, a Cas13 protein described herein, a fusion protein described herein, a guide polynucleotide described herein, a nucleic acid described herein, a vector system described herein, a lipid nanoparticle described herein, a lentiviral vector described herein, a ribonucleoprotein complex described herein, a virus-like particle described herein, or a eukaryotic cell described herein to modify one or more target RNA molecules in a mammalian cell, the modification of one or more target RNA molecules comprising one or more of: RNA base substitution, RNA base deletion, RNA base insertion, cleavage of target RNA, RNA methylation, and RNA demethylation.

Another aspect of the invention relates to the use of a CRISPR-Cas13 system described herein, a Cas13 protein described herein, a fusion protein described herein, a guide polynucleotide described herein, a nucleic acid described herein, a vector system described herein, a lipid nanoparticle described herein, a lentiviral vector described herein, a ribonucleoprotein complex described herein, a virus-like particle described herein, or a eukaryotic cell described herein in the diagnosis, treatment or prevention of a disease or disorder associated with a target RNA. In some embodiments, the disease or disorder is parkinson's disease. In some embodiments, the disease or disorder is parkinson's disease and the target RNA is PTBP1 RNA. In some embodiments, the disease or disorder is glaucoma. In some embodiments, the disease or disorder is glaucoma and the target RNA is AQp1 RNA. In some embodiments, the disease or disorder is amyotrophic lateral sclerosis. In some embodiments, the disease or disorder is amyotrophic lateral sclerosis, and the target RNA is superoxide dismutase 1 (SOD 1) RNA. In some embodiments, the disease or disorder is age-related macular degeneration, and the target RNA is VEGFA RNA. In some embodiments, the disease or disorder is age-related macular degeneration, and the target RNA is VEGFA RNA or VEGFR1 RNA. In some embodiments, the disease or disorder is elevated plasma LDL cholesterol levels. In some embodiments, the disease or disorder is elevated plasma LDL cholesterol levels and the target RNA is PCSK9 RNA or ANGPTL3 RNA.

Another aspect of the invention relates to the use of a CRISPR-Cas13 system described herein, a Cas13 protein described herein, a fusion protein described herein, a guide polynucleotide described herein, a nucleic acid described herein, a vector system described herein, a lipid nanoparticle described herein, a lentiviral vector described herein, a ribonucleoprotein complex described herein, a virus-like particle described herein, or a eukaryotic cell described herein in the manufacture of a medicament for the diagnosis, treatment or prevention of a disease or disorder associated with a target RNA. In some embodiments, the disease or disorder is parkinson's disease. In some embodiments, the disease or disorder is glaucoma. In some embodiments, the disease or disorder is amyotrophic lateral sclerosis. In some embodiments, the disease or disorder is age-related macular degeneration. In some embodiments, the disease or disorder is elevated plasma LDL cholesterol levels. In some embodiments, the disease or disorder is parkinson's disease and the target RNA is PTBP1 RNA. In some embodiments, the disease or disorder is glaucoma and the target RNA is AQp1 RNA. In some embodiments, the disease or disorder is amyotrophic lateral sclerosis, and the target RNA is superoxide dismutase 1 (SOD 1) RNA. In some embodiments, the disease or disorder is age-related macular degeneration, and the target RNA is VEGFA RNA or VEGFR1 RNA. In some embodiments, the disease or disorder is elevated plasma LDL cholesterol levels and the target RNA is PCSK9 RNA or ANGPTL3 RNA.

In some embodiments, the pharmaceutical composition is delivered to a human subject in vivo. The pharmaceutical composition may be delivered by any effective route. Exemplary routes of administration include, but are not limited to, intravenous infusion, intravenous injection, intraperitoneal injection, intramuscular injection, intratumoral injection, subcutaneous injection, intradermal injection, intraventricular injection, intravascular injection, intracerebral injection, intraocular injection, subretinal injection, intravitreal injection, intracameral injection, intrathecal injection, intranasal administration, and inhalation.

In some embodiments, the method of targeting RNA results in editing the sequence of the target RNA. For example, by using Cas13 proteins or fusion proteins with non-mutated HEPN domains and guide polynucleotides comprising guide sequences specific for the target RNA, the target RNA can be cleaved at precise locations or nicked (nick, e.g., cleavage of either single strand when the target RNA is present as a double-stranded nucleic acid molecule). In some examples, this method is used to reduce expression of the target RNA, which will reduce translation of the corresponding protein. This method can be used in cells where increased RNA expression is not required. In one example, the RNA is associated with a disease such as cystic fibrosis, huntington's disease, tay-Sachs, fragile X syndrome, fragile X-related tremor/ataxia syndrome, muscular dystrophy, tonic muscular dystrophy, spinal muscular atrophy, spinocerebellar ataxia, age-related macular degeneration, or familial ALS. In another example, the RNA is associated with cancer (e.g., lung cancer, breast cancer, colon cancer, liver cancer, pancreatic cancer, prostate cancer, bone cancer, brain cancer, skin cancer (e.g., melanoma), or renal cancer). Examples of target RNAs include, but are not limited to, those associated with cancer (e.g., PD-L1, BCR-ABL, ras, raf, p, BRCA1, BRCA2, CXCR4, β -catenin, HER2, and CDK 4). Editing such target RNAs can produce therapeutic effects.

In some embodiments, the RNA is expressed in immune cells. For example, the target RNA may encode a protein that results in the inhibition of a desired immune response (e.g., tumor infiltration). Knocking down such RNAs may promote such desired immune responses (e.g., PD1, CTLA4, LAG3, TIM 3). In another example, the target RNA encodes a protein that results in activation of an undesired immune response, for example in the case of an autoimmune disease such as multiple sclerosis, crohn's disease, lupus, or rheumatoid arthritis.

Diagnostic application:

another aspect of the invention relates to an in vitro composition comprising a CRISPR-Cas13 system described herein and a labeled detector RNA that is not capable of hybridizing to a guide polynucleotide described herein.

Another aspect of the invention relates to the use of a CRISPR-Cas13 system described herein for detecting a target RNA in a nucleic acid sample suspected of containing the target RNA.

In some embodiments, the method of detecting a target RNA comprises a Cas13 protein or fusion protein fused to a fluorescent protein or other detectable label, and a guide polynucleotide comprising a guide sequence specific for the target RNA. Binding of Cas13 protein or fusion protein to target RNA can be visualized by microscopy or other imaging methods. In another example, RNA aptamer sequences may be appended to or inserted into guide polynucleotides, such as MS2, PP7, qβ, and other aptamers. The introduction of proteins that specifically bind to these aptamers, such as MS2 phage coat proteins fused to fluorescent proteins or other detectable labels, can be used to detect target RNAs, as Cas 13-guide-target RNA complexes will be labeled by aptamer interactions.

In some embodiments, the method of detecting a target RNA in a cell-free system results in the production of a detectable label or enzyme activity. For example, by using a Cas13 protein, a guide polynucleotide comprising a guide sequence specific for the target RNA, and a detectable label, the target RNA will be recognized by Cas 13. Binding of Cas13 to the target RNA triggers its RNase activity, which results in cleavage of the target RNA as well as the detectable label.

In some embodiments, the detectable label is an RNA linked to a fluorescent probe and a quencher. The complete detectable RNA is linked to a fluorescent probe and a quencher, which inhibits fluorescence. After cleavage of the detectable RNA by Cas13, the fluorescent probe is released from the quencher and exhibits fluorescent activity. Such methods can be used to determine whether a target RNA is present in a lysed cell sample, a lysed tissue sample, a blood sample, a saliva sample, an environmental sample (e.g., a water, soil, or air sample), or other lysed cell or cell-free sample. Such methods may also be used to detect pathogens, such as viruses or bacteria, or to diagnose disease states, such as cancer.

In some embodiments, detection of the target RNA is useful in diagnosing a disease and/or pathological condition, or the presence of a viral or bacterial infection. For example, cas 13-mediated detection of non-coding RNAs such as PCA3, if detected in patient urine, can be used to diagnose prostate cancer. In another example, cas 13-mediated detection of lncRNA-AA174084, a biomarker for gastric cancer, can be used to diagnose gastric cancer.

EXAMPLE 1 screening of Cas13 protein

1. CRISPR and annotation of genes

Microbial genomes from NCBI Gebank and CNGB (national gene bank) databases were predicted for whole genome proteins, and then analysis annotation was performed using software to predict CRISPR array on the genome.

2. Preliminary screening of proteins

Redundant proteins were removed by cluster analysis, while proteins with amino acid sequence lengths less than 800AA (amino acids) or greater than 1400AA were filtered out.

3. CRISPR related protein acquisition

Protein sequences within 10kb upstream and downstream of the CRISPR Array were aligned with known Cas13 proteins and proteins with values greater than 1*e-5 were filtered out. And then comparing with NR library of NCBI and EBI library, filtering out proteins with high similarity, and selecting to obtain candidate proteins.

The inventors further screened and tested a number of candidate proteins to finally obtain the C13-52 protein (SEQ ID NO: 1), the C13-55 protein (SEQ ID NO: 2) and the C13-88 protein (SEQ ID NO: 3). The C13-52 protein is also known as CasRfg.7. The C13-55 protein is also known as CasRfg.5. The C13-88 protein is also known as CasRfg.6. The genomic sequence sources of these proteins are shown in table 1.

Table 1. Source of genomic sequence of Cas13 protein.

The corresponding Direct Repeat (DR) sequence of C13-52 is: 5'-GTTGTGAAAGGCCACCGAAATGGTGGTTCAAGCAAC-3' (SEQ ID NO: 4).

The corresponding Direct Repeat (DR) sequence of C13-55 is: 5'-GTTGAGAAACACCGCTGATGGCGGCTGTTCTGAGAC-3' (SEQ ID NO: 5).

The corresponding Direct Repeat (DR) sequence of C13-88 is: 5'-GTTGTTTAGACCGCCATTTCGGCGACCCTTCGCAAC-3' (SEQ ID NO: 6).

RNA fold predictions were used to obtain the RNA secondary structure of the above-described orthostatic repeats as shown in FIGS. 1-3.

Example 2 editing efficiency validation of Cas13 protein

1. Construction of editing vector targeting AQP1 RNA

The test target nucleic acid selected for this experiment was AQp1 (Aquaporin 1) RNA using 293T cell lines that highly expressed AQp 1.

The construction method of the 293T cell line (293T-AQP 1 cell) for highly expressing AQP1 is as follows:

the vector Lv-AQP1-T2a-GFP (sequence shown as SEQ ID NO: 7) for over-expressing the AQP1 gene was constructed according to the conventional method. The vector is based on a lentiviral vector, and is inserted with an AQP1 gene and an EGFP gene, wherein the AQP1 and the EGFP are separated by using a 2A peptide (T2A). The Lv-AQP1-T2a-GFP plasmid is packaged into lentivirus and then transduced into 293T cells to form a cell line for stably over-expressing the AQP1 gene.

In the CRISPR-Cas13 system, the guide sequence of the gRNA targeting AQp1 is:

AGGGCAGAACCGATGCTGATGAAGAC（SEQ ID NO: 8）。

the C13-55-BsaI plasmid vector (with the sequence shown as SEQ ID NO: 9) and the C13-88-BsaI plasmid vector (with the sequence shown as SEQ ID NO: 10) with the general gRNA skeleton expression frame are respectively synthesized by outsourcing companies.

When selecting positive control proteins, taking Cas rx as the highest editing efficiency Cas13 protein currently disclosed in the art as a control, the following control vectors were prepared using conventional methods:

a negative control vector, casRx-blank (SEQ ID NO: 11), for expressing CasRx and gRNA that does not target any gene of eukaryotic cells;

a positive control vector, casRx-AQP1 (SEQ ID NO: 12), was used to express CasRx and gRNA targeting AQP1 RNA.

Primer annealing is used to obtain a fragment targeting the AQP1 target site, and the primers are as follows:

C13-55-AQP1 corresponding primer:

AGACagggcagaaccgatgctgatgaagac（SEQ ID NO: 13）

AAAAgtcttcatcagcatcggttctgccct（SEQ ID NO: 14）

C13-88-AQP1 corresponding primer:

CACCGagggcagaaccgatgctgatgaagac（SEQ ID NO: 15）

CAACgtcttcatcagcatcggttctgccctc（SEQ ID NO: 16）

the primer annealing reaction system is as follows:

Oligo-F（10 μM）2 μl

Oligo-R（10 μM）2 μl

2 μl of 10×endonuclease reaction buffer

Deionized water up to 20. Mu.l.

Incubation at 95℃for 5 minutes in a PCR apparatus followed by immediate removal and incubation on ice for 5 minutes allowed the primers to anneal to each other to form double stranded DNA containing cohesive ends.

The endoenzyme is used for synthesizing the C13-55-BsaI and the C13-88-BsaI plasmid BsaI) After enzyme digestion, the purified and recovered skeleton and the annealed product are connected by T4, and positive clones are selected and plasmids are extracted after escherichia coli is transformed. The C13-55-AQP1 and C13-88-AQP1 vectors are obtained, the carrier architecture is CMV-Cas13-U6-gRNA, and the vectors can express C13-55 and C13-88 proteins (the architecture is NLS-Cas13-SV40 NLS-nucleoplasmin NLS-HA) and gRNA containing corresponding DR sequences and targeting AQP 1.

2. Vectors to be validated transfect 293T-AQP1 cells

The C13-55-AQP1, C13-88-AQP1 and control plasmids were transfected into 293T-AQP1 cells in 24 well plates according to 500ng following Lipofectamine 2000 (Thermo) instructions. 293T-AQP1 cells not transfected with plasmid served as blank.

3. qPCR detection of RNA changes in target genes

Cells 72h after transfection were used to extract RNA using the SteadyPureUniversal RNA Extraction Kit AG21017 kit and the RNA concentration was detected using an ultra-micro spectrophotometer. The RNA product was reverse transcribed using the Evo M-MLVMix Kit with gDNA Clean for qPCR AG11728 reverse transcription kit and the reverse transcription product was detected using the SYBRGreen PremixPro TaqHS qPCR Kit (Low Rox Plus) AG11720 qPCR kit.

The primers used for qPCR are as follows:

Detection of AQp1: gctcttctggagggcagtgg (SEQ ID NO: 17)

cagtgtgacagccgggttgag（SEQ ID NO: 18）

Detecting an internal reference GAPDH: CCATGGGGAAGGTGAAGGTC (SEQ ID NO: 19)

GAAGGGGTCATTGATGGCAAC（SEQ ID NO: 20）

The reaction System was configured according to SYBR Green Premix Pro Taq HS qPCR Kit (Rox Plus) AG11718 instructions and tested using Quantum studio ™ 5 Real-Time PCR System.

The change in target RNA was calculated using the relative quantification method, the 2-DeltaCt method. The calculation mode is as follows:

△Ct= Ct (AQp1)-Ct (GAPDH)

delta Ct= [ Delta ] Ct (sample to be verified) - [ Delta ] Ct (CasRx-blank)

2-△△Ct=2^(-△△Ct)

The experiment was repeated 3 times independently and the result data averaged over 3 tests. The results are shown in Table 2 and FIG. 4.

TABLE 2 results of the knockout test of Cas13 on AQP1 RNA

The experimental results show that both C13-55 and C13-88 have obvious knockdown (P < 0.05) to AQP1 RNA, the editing efficiency is as high as 97% and 78%, and the editing efficiency of the C13-55 protein is close to that of CasRx.

Example 3 comparison with known Cas13 tools

1. Verification vector and control vector construction

When selecting positive control proteins, casRx is considered to be Cas13 protein with highest editing efficiency which is currently disclosed in the art, and Cas13 proteins such as PspCas13b, cas13x.1 and cas13y.1 which are reported more are set as controls.

Verification vectors for targeting PTBP1 and VEGFA shown in Table 3 were constructed to obtain CasRx, pspCas13b, cas13X.1, cas13Y.1, C13-52, C13-55, C13-88, C13-115. All the experimental examples verify that the vectors used the same plasmid backbone as in example 2, which was structured as CMV-Cas13-U6-crRNA, and the expressed Cas13 had a structure of 1 NLS at the N-terminus and 2 NLS at the C-terminus, for a total of 3×nls. crrnas consist of guide sequences and the corresponding DR sequences of each of the prior art disclosures. Exemplary, plasmid sequences are given for CasRx-EGFP (SEQ ID NO: 32), casRx-VEGFA (SEQ ID NO: 33), pspCas13b-VEGFA (SEQ ID NO: 34), cas13X.1-VEGFA (SEQ ID NO: 35), cas13Y.1-VEGFA (SEQ ID NO: 36), C13-52-VEGFA (SEQ ID NO: 37), C13-55-VEGFA (SEQ ID NO: 38), C13-88-VEGFA (SEQ ID NO: 39) and C13-115-VEGFA (SEQ ID NO: 40). The plasmid sequence targeting PTBP1 differs only in the gRNA coding sequence.

The amino acid sequence of C13-115 is shown as SEQ ID NO. 21, and the corresponding DR sequence is shown as SEQ ID NO. 22.

Because 293T cells do not contain EGFP sequences, casRx-targeted EGFP vectors were used as negative controls.

EGFP-targeting spacer is tgccgttcttctgcttgtcggccatgatat (SEQ ID NO: 29).

The spacer targeting PTBP1 was GTGGTTGGAGAACTGGATGTAGATGGGCTG (SEQ ID NO: 30).

The spacer targeting VEGFA was TGGGTGCAGCCTGGGACCACTTGGCATGG (SEQ ID NO: 31).

The constructed verification vector is shown below:

TABLE 3 verification Carrier and architecture

2. Vector to be verified transfects 293T cells

The verification vector and the control vector were transfected into 293T cells.

24-well plates were transfected according to Lipofectamine 2000 (Thermo) protocol. 293T cells not transfected with plasmid served as a blank.

3. qPCR detection of RNA changes in target genes

Cells 48h after transfection were used to extract RNA using the SteadyPureUniversal RNA Extraction Kit AG21017 kit and the RNA concentration was detected using an ultra-micro spectrophotometer. RNA products were reverse transcribed using Evo M-MLVMix Kit with gDNA Clean for qPCR AG11728 reverse transcription kit and reverse transcription products were detected using SYBRGreen PremixPro TaqHS qPCR Kit (Low Rox Plus) qPCR kit.

The primers used for qPCR are as follows:

detection of VEGFA: ACCTCCACCATGCCAAGTGG (SEQ ID NO: 23)

CAGGGTCTCGATTGGATGGC（SEQ ID NO：24）

Detection of PTBP1: ATTGTCCCAGATATAGCCGTTG (SEQ ID NO: 25)

GCTGTCATTTCCGTTTGCTG（SEQ ID NO：26）

Detecting an internal reference GAPDH: CCATGGGGAAGGTGAAGGTC (SEQ ID NO: 27)

GAAGGGGTCATTGATGGCAAC（SEQ ID NO：28）

The reaction System was configured according to SYBR Green Premix Pro Taq HS qPCR Kit (Rox Plus) instructions and tested using Quantum studio ™ Real-Time PCR System.

The present experiment uses a relative quantitative method, namely the 2- Δct method, to calculate the change in target RNA. The calculation mode is as follows:

Δct=ct (VEGFA or PTBP 1) -Ct (GAPDH)

DeltaCt= DeltaCt (sample to be verified) -DeltaCt (CasRx-EGFP)

2-△△Ct=2^(-△△Ct)

The 2- ΔΔct values for targeted VEGFA or PTBP1 were calculated as described above. The experiment was repeated 4 times independently and the result data averaged 4 tests. As shown in tables 4 and 5.

TABLE 4 knockdown results targeting VEGFA

In the test of targeting VEGFA RNA, the editing efficiency average was ordered as CasRx > C13-55 > PspCas13b > C13-52 > Cas13X.1 > C13-88 > Cas13Y.1 > C13-115. Compared with the CasRx-EGFP group, the VEGFA RNA can be significantly knocked down in the C13-55, C13-52 and C13-88 groups, and the statistical difference is present (P < 0.05).

TABLE 5 knock-down results targeting PTBP1

In the PTBP1 RNA targeting test, the editing efficiency average rank was CasRx > C13-55 > PspCas13b > Cas13X.1 > C13-52 > C13-88 > Cas13Y.1. Compared with the CasRx-EGFP group, the groups C13-55, C13-52 and C13-88 can significantly knock down PTBP1 RNA, and the statistical difference is present (P < 0.05).

4. Off-target analysis

RNAseq sequencing.

RNAseq sequencing (n=3 per group of samples) was performed on total RNA samples of VEGFA-targeted experimental and control groups, the library was a LncRNA strand-specific library, the amount of sequencing data was 16G, and the sequencing strategy was PE150.

RNAseq analysis

(1) Data were quality controlled using fastqc, multiqc and low quality reads were removed using fastp.

(2) Alignment reads to human rRNA sequences were removed and the remaining reads were aligned to the hg38 reference genome using Hisat2 alignment software.

(3) After alignment, the expression level of the gene was quantified using kalisto software, and then the difference analysis of the expression level was performed using sleuth software, and the genes of |b| >0.5, qval <0.05, mean_obs >2 were regarded as Differentially Expressed Genes (DEG).

(4) gRNA guide sequences were aligned to reference cDNA using embos water software, transcripts with aligned base number > =18, mismatched base number < =6, minimum consecutive paired base number > =8 were considered predicted to target transcripts (on target+off target), and the corresponding genes were considered to target genes (on target+off target).

(5) And (3) taking intersection of the differential expression gene with remarkably down-regulated expression and the target gene for predicting off-target, and removing the differential expression gene from the target gene to obtain an off-target gene set.

The results are shown in Table 6 below. The number of off-target genes CasRx > Cas13X.1 > C13-88 > C13-52 > C13-55. Wherein C13-55 has high editing efficiency and low off-target.

TABLE 6 comparison of off-target Gene numbers

In conclusion, the invention of the C13-52, C13-55, C13-88 and C13-115-gRNA systems discovered by the invention expands the range of Cas13 protein tools and further paves the way for the application of the CRISPR-Cas13 system in RNA targeting.

The technical features of the above-described embodiments may be arbitrarily combined, and all possible combinations of the technical features in the above-described embodiments are not described for brevity of description, however, as long as there is no contradiction between the combinations of the technical features, they should be considered as the scope of the description.

The above examples illustrate only a few embodiments of the invention, which are described in detail and are not to be construed as limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention. Accordingly, the scope of protection of the present invention is to be determined by the appended claims.

Claims

1. An isolated Cas13 protein having an amino acid sequence as set forth in any one of SEQ ID NOs 1-3.

2. A fusion protein comprising the Cas13 protein of claim 1 and a protein functional domain that is at least one of a protein domain and a polypeptide tag.

3. The fusion protein of claim 2, wherein the Cas13 protein is fused to a nuclear localization signal or a nuclear export signal.

4. The fusion protein of claim 2, wherein the Cas13 protein is fused to any one or two or more protein functional domains selected from the group consisting of: cytosine deaminase domain, adenine deaminase domain, translation activation domain, translation repression domain, RNA methylation domain, RNA demethylation domain, nuclease domain, splicing factor domain, reporter domain, affinity domain, subcellular localization signal, reporter tag and affinity tag.

5. A CRISPR-Cas13 system, characterized in that it comprises:

the Cas13 protein of claim 1 or the fusion protein of any one of claims 2-4, or the nucleic acid encoding the Cas13 protein of claim 1 or the fusion protein of any one of claims 2-4; and

A guide polynucleotide or a nucleic acid encoding the guide polynucleotide; the guide polynucleotide is capable of forming a CRISPR complex with the Cas13 protein or fusion protein and directing sequence-specific binding of the CRISPR complex to a target RNA.

6. The CRISPR-Cas13 system according to claim 5, wherein said guide polynucleotide comprises a linked guide sequence and a homeotropic repeat sequence of any of SEQ ID NOs 4 to 6.

7. The CRISPR-Cas13 system of claim 5, wherein the guide polynucleotide comprises a modified nucleotide.

8. The CRISPR-Cas13 system according to claim 7, wherein said modification is selected from 2' -O-methyl, 2' -O-methyl-3 ' -phosphorothioate or 2' -O-methyl-3 ' -phosphorothioate modification.

9. The CRISPR-Cas13 system according to claim 5, the target RNA is optionally selected from VEGFA RNA, PTBP1 RNA and AQp1 RNA.

10. A vector system comprising the CRISPR-Cas13 system of any one of claims 5-9, wherein the vector system comprises one or more vectors comprising a polynucleotide sequence encoding the Cas13 protein or fusion protein and a polynucleotide sequence encoding the guide polynucleotide.

11. An adeno-associated viral vector comprising the CRISPR-Cas13 system of any one of claims 5-9, wherein the adeno-associated viral vector comprises DNA encoding the Cas13 protein or fusion protein and a guide polynucleotide.

12. A lipid nanoparticle comprising the CRISPR-Cas13 system of any one of claims 5-9, wherein the lipid nanoparticle comprises the guide polynucleotide and an mRNA encoding the Cas13 protein or fusion protein.

13. A lentiviral vector comprising the CRISPR-Cas13 system of any one of claims 5-9, wherein the lentiviral vector comprises the guide polynucleotide and mRNA encoding the Cas13 protein or fusion protein.

14. The lentiviral vector of the CRISPR-Cas13 system of claim 13, wherein: the lentiviral vector is pseudotyped with an envelope protein; and/or mRNA encoding the Cas13 protein or fusion protein is linked to an aptamer sequence.

15. A ribonucleoprotein complex comprising the CRISPR-Cas13 system of any one of claims 5-9, wherein the ribonucleoprotein complex is formed from the guide polynucleotide and a Cas13 protein or fusion protein.

16. A virus-like particle comprising the CRISPR-Cas13 system of any one of claims 5-9, wherein the virus-like particle comprises a ribonucleoprotein complex formed from the guide polynucleotide and a Cas13 protein or fusion protein.

17. The virus-like particle of claim 16, wherein: the Cas13 protein or fusion protein is fused to a gag protein.

18. A eukaryotic cell comprising the Cas13 protein of claim 1, the fusion protein of any one of claims 2-4, or the CRISPR-Cas13 system of any one of claims 5-9.

19. A pharmaceutical composition comprising the Cas13 protein of claim 1, the fusion protein of any one of claims 2-4, or the CRISPR-Cas13 system of any one of claims 5-9.

20. An isolated nucleic acid encoding the Cas13 protein of claim 1 or the fusion protein of any one of claims 2-4.

21. Use of the Cas13 protein of claim 1, the fusion protein of any one of claims 2-4, the CRISPR-Cas13 system of any one of claims 5-9, or the isolated nucleic acid of claim 20 in the preparation of a reagent for detecting a target RNA in a nucleic acid sample comprising a target RNA or in a nucleic acid sample comprising a target RNA.

22. Use of the Cas13 protein of claim 1, the fusion protein of any one of claims 2-4, the CRISPR-Cas13 system of any one of claims 5-9, or the isolated nucleic acid of claim 20 in the preparation of a reagent that implements the method of any one of: cleavage or nicking of one or more target RNA molecules, activation or upregulation of one or more target RNA molecules, activation or inhibition of translation of one or more target RNA molecules, inactivation of one or more target RNA molecules, visualization, labeling or detection of one or more target RNA molecules, binding of one or more target RNA molecules, transportation of one or more target RNA molecules, and masking of one or more target RNA molecules.

23. Use of the Cas13 protein of claim 1, the fusion protein of any one of claims 2-4, the CRISPR-Cas13 system of any one of claims 5-9, or the isolated nucleic acid of claim 20 in the manufacture of a medicament for diagnosing, treating, or preventing a disease or disorder associated with a target RNA, including age-related macular degeneration, glaucoma, or parkinson's disease.