WO2024240053A1 - Gene editing protein, corresponding gene editing system thereof, and use thereof - Google Patents

Abstract

Provided are a gene editing protein, a corresponding gene editing system thereof, and a use thereof. Specifically, the gene editing protein shows gene editing activity in vitro, can effectively edit or cut a target gene, and can effectively treat disorders or diseases of a subject in need.

Description

A gene editing protein, its corresponding gene editing system and application Technical Field

The present invention relates to the field of gene editing, and in particular, to a gene editing protein, a corresponding gene editing system and applications.

Background Art

Clustered regularly interspaced short palindromic repeats (CRISPR) systems are formed by bacteria and archaea to defend against invading phage DNA. CRISPR systems include two families, type 1 systems are further divided into types I, III, and IV; type 2 systems are divided into types II, V, and VI. These six system types are further divided into 19 subtypes. Many prokaryotes contain multiple CRISPR-Cas systems, suggesting that they are compatible and may share components.

The most common type II is the CRISPR/Cas9 system. The Cas9 protein can process pre-crRNA into mature crRNA that binds to tracrRNA with the assistance of trans-encoded small RNA (tracrRNA). Later, people found that by artificially constructing a single-stranded chimeric guide RNA (guide RNA, gRNA) that simulates the crRNA-tracrRNA complex, the Cas9 protein can effectively mediate the recognition and cutting of the target. The three bases adjacent to the 3′ end of the target must be in the form of 5′-NGG-3′, thus forming the PAM (protospacer adjacent motif) structure required for the Cas/crRNA complex to recognize the target.

Currently known CRISPR/Cas have their own advantages and disadvantages. For example, Cas9 requires two RNAs as guide RNAs.

There is an urgent need for alternative and robust editing systems and editing technologies targeting nucleic acids or polynucleotides with broad application value.

Therefore, the current development of biotechnology still requires the development of new CRISPR/Cas systems with diverse characteristics.

Summary of the invention

The main purpose of the present invention is to provide a new CRISPR/Cas system with diverse characteristics.

Another object of the present invention is to discover new CRISPR-Cas systems, provide alternative and robust systems and techniques for targeting nucleic acids or polynucleotides, and address the shortcomings of currently known CRISPR-Cas systems.

The first aspect of the present invention provides a gene editing protein, wherein the protein is selected from the following group:

(a) a polypeptide having an amino acid sequence as shown in SEQ ID NO: 1;

(b) a polypeptide having ≥80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5% homology (or identity) with the amino acid sequence of SEQ ID NO:1, and the polypeptide has the biological function of SEQ ID NO:1;

(c) A derivative polypeptide formed by replacing, deleting or adding one or more (preferably 1-20, more preferably 1-10, and more preferably 1-5) amino acid residues of any amino acid sequence shown in SEQ ID NO:1, and retaining the biological function of SEQ ID NO:1.

In another preferred embodiment, the gene editing protein is an effector protein in the CRISPR/Cas system.

The second aspect of the present invention provides a fusion protein comprising the gene editing protein described in the first aspect of the present invention; and one or more functional domains.

In another preferred embodiment, the functional domain is selected from a localization signal, a reporter protein, a Cas protein targeting portion, a DNA binding domain, an epitope tag, a transcription activation domain, a transcription repression domain, a nuclease, a deamination domain, a methylase, a demethylase, a transcription release factor, an HDAC, a cleavage active polypeptide, a ligase, an integrase, a transposase, a recombinase, a polymerase, and a base excision repair inhibitor (such as a uracil-DNA glycosylase inhibitor (UGI)).

In another preferred example, the functional domain includes one or more of the following enzymatic activities on the target sequence: methylase activity, demethylase activity, acetyltransferase activity, deacetylase activity, kinase activity, phosphatase activity, ubiquitin ligase activity, deubiquitinating activity, adenylation activity, deadenylation activity, SUMOylation activity, deSUMOylation activity, ribosylation activity, deribosylation activity, myristoylation activity, demyristoylation activity, glycosylation activity (e.g., from O-GlcNAc transferase) and deglycosylation activity.

In another preferred embodiment, the functional domain is selected from adenosine deaminase catalytic domain or cytidine deaminase catalytic domain.

In another preferred example, the adenosine deaminase catalytic domain or the cytidine deaminase catalytic domain includes one or more of ADAR1, ADAR2, APOBEC, AID or TAD.

In another preferred embodiment, the functional domain is the full length or functional fragment of TadA8e.

In another preferred example, the localization signal includes a nuclear localization signal (NLS) and/or a nuclear export signal (NES).

In another preferred embodiment, the sequence of the nuclear localization signal is located at, near or close to the end (eg, N-terminus or C-terminus) of the protein according to claim 1.

In another preferred embodiment, the nuclear export signal includes protein tyrosine kinase 2 (such as human protein tyrosine kinase 2).

In another preferred embodiment, the reporter protein includes glutathione-S-transferase (GST), horseradish peroxidase (HRP), chloramphenicol acetyltransferase (CAT), β-galactosidase, β-glucuronidase, and autofluorescent protein.

In another preferred embodiment, the autofluorescent protein includes green fluorescent protein (e.g., GFP, GFP-2, tagGFP, turboGFP, eGFP, CopGFP, AceGFP, etc.), HcRed, DsRed, cyan fluorescent protein (e.g., eCFP, Cerulean, CyPet, AmCyanl, etc.), yellow fluorescent protein (e.g., YFP, eYFP, Citrine, Venus, YPet, PhiYFP, etc.), blue fluorescent protein (e.g., eBFP, eBFP2, Azurite, mKalamal, GFPuv, Sapphire, T-sapphire).

In another preferred embodiment, the DNA binding domain includes methylation binding protein, LexADBD, and Gal4DBD.

In another preferred embodiment, the epitope tag includes a histidine tag, a V5 tag, a FLAG tag, an influenza virus hemagglutinin tag, a Myc tag, a VSV-G tag, a thioredoxin tag, or a streptavidin tag.

In another preferred embodiment, the transcriptional activation domain includes VP64 and/or VPR.

In another preferred embodiment, the transcriptional repression domain includes KRAB and/or SID.

In another preferred embodiment, the nuclease includes FokI.

In another preferred embodiment, the cleavage active polypeptide includes a polypeptide having single-stranded RNA cleavage activity, a polypeptide having double-stranded RNA cleavage activity, a polypeptide having single-stranded DNA cleavage activity or a polypeptide having double-stranded DNA cleavage activity.

In another preferred embodiment, the ligase includes DNA ligase and/or RNA ligase.

In another preferred embodiment, the functional domain is connected to the N-terminus and/or C-terminus of the gene editing protein.

In another preferred embodiment, the functional domain is inserted between the N-terminus and the C-terminus of the gene editing protein.

In another preferred embodiment, the one or more functional domains are optionally connected to the N-terminus and/or C-terminus of the gene editing protein via a linker.

In another preferred embodiment, the functional domain is inserted between the N-terminus and the C-terminus of the gene editing protein through a linker.

In another preferred embodiment, the fusion protein has the following structure from N-terminus to C-terminus:

Z1-Z2(I); or

Z2-Z1(II); or

Z3-Z1-Z4(III);

wherein Z1 is cytosine deaminase or adenosine deaminase;

Z2 is the gene editing protein described in the first aspect of the present invention;

Z3 is the N-terminal fragment of the gene editing protein described in the first aspect of the present invention;

Z4 is the C-terminal fragment of the gene editing protein described in the first aspect of the present invention;

Furthermore, each "-" is independently a bond or a linker.

The third aspect of the present invention provides an isolated polynucleotide, which encodes the gene editing protein described in the first aspect of the present invention or the fusion protein described in the second aspect of the present invention.

In another preferred embodiment, the polynucleotide is selected from the following group:

(a) a polynucleotide whose sequence is shown as SEQ ID NO.2;

(b) a polynucleotide having a nucleotide sequence homology ≥ 70% (preferably ≥ 80%, more preferably ≥ 90%, more preferably ≥ 95%, and most preferably ≥ 99%) to the sequence shown in SEQ ID NO.2 and encoding the polypeptide shown in SEQ ID NO.1;

(c) A polynucleotide complementary to the polynucleotide described in any one of (a) to (b).

In another preferred embodiment, the polynucleotide further contains auxiliary elements selected from the following groups on the flank of the ORF of the variant: a signal peptide, a secretory peptide, a tag sequence (such as 6His), or a combination thereof.

In another preferred embodiment, the polynucleotide is selected from the following group: genomic sequence, cDNA sequence, RNA sequence, or a combination thereof.

In another preferred example, the polynucleotide further comprises a promoter operably linked to the ORF sequence of the variant.

In another preferred embodiment, the promoter is selected from the following group: a constitutive promoter, a tissue-specific promoter, an inducible promoter, or a strong promoter.

In another preferred embodiment, the host cell includes a prokaryotic cell or a eukaryotic cell.

In another preferred embodiment, the host cell is a eukaryotic cell, such as a yeast cell, a plant cell or a mammalian cell (including human and non-human mammals).

In another preferred embodiment, the host cell is a prokaryotic cell, such as Escherichia coli.

In another preferred embodiment, the yeast cell is selected from yeast of one or more sources of the following group: Pichia pastoris, Kluyveromyces, or a combination thereof; preferably, the yeast cell includes: Kluyveromyces, more preferably Kluyveromyces marxianus, and/or Kluyveromyces lactis.

In another preferred embodiment, the host cell is selected from the following group: Escherichia coli, wheat germ cells, insect cells, SF9, Hela, HEK293, CHO, yeast cells, or a combination thereof.

The fourth aspect of the present invention provides an isolated nucleic acid molecule comprising or consisting of a sequence selected from the following:

(i) the sequence shown in SEQ ID NO: 3;

(ii) a sequence having one or more base substitutions, deletions or additions (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 base substitutions, deletions or additions) compared to the sequence shown in SEQ ID NO: 3;

(iii) a sequence having at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% sequence identity to the sequence set forth in SEQ ID NO: 3;

(iv) a sequence that hybridizes to the sequence described in any one of (i) to (iii) under stringent conditions; or

(v) a complementary sequence of the sequence described in any one of (i) to (iii);

Furthermore, the sequence described in any one of (ii) to (v) substantially retains the biological function of the sequence from which it is derived;

For example, the isolated nucleic acid molecule is RNA;

For example, the isolated nucleic acid molecule comprises a direct repeat sequence in a CRISPR/Cas system.

In another preferred embodiment, the nucleic acid molecule comprises one or more stem loops or optimized secondary structures;

For example, the sequence of any of (ii)-(v) retains the secondary structure of the sequence from which it is derived.

In another preferred embodiment, the nucleic acid molecule comprises a sequence selected from the following, or consists of a sequence selected from the following:

(a) The nucleotide sequence shown in SEQ ID NO: 3;

(b) a sequence that hybridizes under stringent conditions to the sequence described in (a); or

(c) The complementary sequence of the nucleotide sequence shown in SEQ ID NO: 3.

The fifth aspect of the present invention provides a guide RNA (gRNA), which includes a direct repeat (DR) sequence capable of binding to the gene editing protein described in the first aspect of the present invention and a spacer sequence capable of targeting the target sequence.

The sixth aspect of the present invention provides a composite comprising:

(i) a protein component selected from the group consisting of the gene editing protein described in the first aspect of the present invention, the fusion protein described in the second aspect of the present invention, or a combination thereof; and

(ii) a nucleic acid component selected from the group consisting of the guide RNA described in the fifth aspect of the present invention, a nucleic acid encoding the guide RNA described in the fifth aspect of the present invention, a precursor RNA of the guide RNA described in the fifth aspect of the present invention, a precursor RNA nucleic acid encoding the guide RNA described in the fifth aspect of the present invention, or a combination thereof;

Wherein, the protein component and the nucleic acid component are combined with each other to form a complex.

In another preferred embodiment, the direct repeat (DR) sequence in the guide RNA (gRNA) is connected to the 3’ end or 5’ end of the nucleic acid molecule.

In another preferred embodiment, the spacer sequence in the guide RNA (gRNA) comprises a complementary sequence to the target sequence.

The seventh aspect of the present invention provides a vector comprising the polynucleotide described in the third aspect of the present invention or the nucleic acid molecule described in the fourth aspect of the present invention.

In another preferred embodiment, the vector comprises:

(1) a first regulatory element, which is operably linked to a nucleotide sequence encoding the gene editing protein described in the first aspect of the present invention or a nucleotide sequence encoding the fusion protein described in the second aspect of the present invention; and

(2) a second regulatory element, the second regulatory element being operably linked to a nucleotide encoding a guide RNA Acid sequence, the guide RNA comprises:

(a) a spacer sequence capable of hybridizing to the target sequence, and

(b) a direct repeat (DR) sequence connected to the spacer sequence, capable of guiding the gene editing protein described in the first aspect of the present invention to bind to the guide RNA to form the complex described in the sixth aspect of the present invention that targets the target sequence.

In another preferred example, the first regulatory element and the second regulatory element are located on the same or different vectors.

In another preferred embodiment, the first regulatory element and/or the second regulatory element is a promoter, such as an inducible promoter.

In another preferred embodiment, the vector comprises one or more promoters, which are operably connected to the nucleic acid sequence, enhancer, transcription termination signal, polyadenylation sequence, replication origin, selective marker, nucleic acid restriction site, and/or homologous recombination site.

In another preferred embodiment, the vector includes a plasmid or a viral vector.

In another preferred embodiment, the viral vector is selected from the following group: adeno-associated virus (AAV), adenovirus, lentivirus, retrovirus, herpes virus, SV40, poxvirus, or a combination thereof.

In another preferred embodiment, the vector includes a cloning vector, a transformation vector, an expression vector, a shuttle vector, an integration vector, and a multifunctional vector.

The eighth aspect of the present invention provides a CRISPR-Cas composition, comprising:

(i) a first component selected from the group consisting of the gene editing protein described in the first aspect of the present invention, the fusion protein described in the second aspect of the present invention, a nucleotide sequence encoding the gene editing protein described in the first aspect of the present invention or the fusion protein described in the second aspect of the present invention, and any combination thereof; and

(ii) a second component, wherein the second component is a nucleotide sequence comprising one or more guide RNAs according to the fifth aspect of the present invention, or encoding the nucleotide sequence comprising one or more guide RNAs according to the fifth aspect of the present invention;

The guide RNA is capable of forming a complex with the protein, protein variant or fusion protein described in (i).

In another preferred embodiment, the guide RNA comprises a direct repeat sequence and a spacer sequence from the 5' to the 3' direction, and the spacer sequence is capable of hybridizing with the target sequence.

In another preferred embodiment, the direct repeat sequence is the nucleic acid molecule defined in the fourth aspect of the present invention.

In another preferred embodiment, the composition further comprises a pharmaceutically acceptable carrier.

In another preferred embodiment, the composition comprises a pharmaceutical composition.

In another preferred embodiment, the dosage form of the composition is selected from the following group: a lyophilized preparation, a liquid preparation, or a combination thereof.

In another preferred embodiment, the composition is in the form of a liquid preparation.

In another preferred embodiment, the composition is in the form of an injection.

In another preferred embodiment, the composition is a cell preparation.

A ninth aspect of the present invention provides a CRISPR-Cas system, comprising one or more vectors, wherein the one or more vectors comprise:

(i) a first nucleic acid, which is a nucleotide sequence encoding the gene editing protein described in the first aspect of the present invention or the fusion protein described in the second aspect of the present invention; optionally, the first nucleic acid is operably linked to a first regulatory element; and

(ii) a second nucleic acid encoding a nucleotide sequence comprising the guide RNA according to the fifth aspect of the present invention; Optionally the second nucleic acid is operably linked to a second regulatory element;

in:

The first nucleic acid and the second nucleic acid are present on the same or different vectors;

The guide RNA is capable of forming a complex with the protein or fusion protein described in (i).

In another preferred embodiment, the vector includes a plasmid or a viral vector.

In another preferred example, the guide RNA includes a spacer sequence capable of hybridizing with the target sequence; and a direct repeat (DR) sequence connected to the spacer sequence and capable of guiding the protein to bind to the guide RNA, thereby forming a CRISPR-Cas composition or complex targeting the target sequence.

In another preferred embodiment, the guide RNA includes unmodified and modified guide RNA.

In another preferred embodiment, the modified guide RNA includes chemical modification of the bases.

In another preferred embodiment, the chemical modification includes methylation modification, methoxy modification, fluorination modification or thio modification.

In another preferred embodiment, the directly repeated sequence is the nucleic acid molecule defined in claim 4.

In another preferred embodiment, the first regulatory element and/or the second regulatory element is a promoter, such as an inducible promoter.

In another preferred embodiment, at least one component in the composition is non-naturally occurring or modified.

In another preferred embodiment, the spacer sequence is connected to the 3’ end of the direct repeat (DR) sequence.

In another preferred example, the spacer sequence comprises a complementary sequence to the target sequence.

In another preferred embodiment, when the target sequence is DNA, the target sequence is located at the 3' end of the protospacer adjacent motif (PAM), and the PAM has a sequence represented by 5'-PAM being TTTN, and N is A, T, C or G.

In another preferred embodiment, the target sequence is a DNA from a prokaryotic cell or a eukaryotic cell, or a DNA sequence formed based on RNA reverse transcription; or, the target sequence is a non-naturally occurring DNA, or a DNA sequence formed based on RNA reverse transcription.

In another preferred example, the target sequence includes a cDNA sequence.

In another preferred embodiment, the target sequence includes single-stranded DNA and double-stranded DNA sequences.

In another preferred embodiment, the target sequence exists in cells.

In another preferred embodiment, the target sequence is present in the cell nucleus or cytoplasm (eg, organelle).

In another preferred embodiment, the cell is a eukaryotic cell.

In another preferred embodiment, the cell is a prokaryotic cell.

In another preferred embodiment, the target sequence exists outside the cell.

In another preferred embodiment, the gene editing protein in the first aspect of the present invention is connected to one or more NLS sequences, or the fusion protein contains one or more NLS sequences.

In another preferred embodiment, the NLS sequence is connected to the N-terminus or C-terminus of the gene editing protein described in the first aspect of the present invention.

In another preferred embodiment, the NLS sequence is fused to the N-terminus or C-terminus of the gene editing protein described in the first aspect of the present invention.

The tenth aspect of the present invention provides a kit comprising one or more components selected from the following: The gene editing protein described in the first aspect of the invention, the fusion protein described in the second aspect of the invention, the polynucleotide described in the third aspect of the invention, the complex described in the sixth aspect of the invention, the vector described in the seventh aspect of the invention, the CRISPR-Cas composition described in the eighth aspect of the invention, or the system described in the ninth aspect of the invention.

In another preferred embodiment, the kit further comprises a label or instructions.

In another preferred embodiment, the kit is used for one or more of gene or genome editing, disease treatment, target gene targeting, and cutting of target genes or non-target genes.

The eleventh aspect of the present invention provides a delivery composition comprising a delivery vector and one or more selected from the following: the gene editing protein described in the first aspect of the present invention, the fusion protein described in the second aspect of the present invention, the polynucleotide described in the third aspect of the present invention, the complex described in the sixth aspect of the present invention, the vector described in the seventh aspect of the present invention, the CRISPR-Cas composition described in the eighth aspect of the present invention, or the system described in the ninth aspect of the present invention.

In another preferred embodiment, the delivery vehicle is a particle.

In another preferred embodiment, the delivery vector is selected from lipid particles, sugar particles, metal particles, protein particles, liposomes, exosomes, microvesicles, gene guns or viral vectors (e.g., replication-defective retroviruses, lentiviruses, adenoviruses or adeno-associated viruses).

The twelfth aspect of the present invention provides a host cell, comprising the gene editing protein described in the first aspect of the present invention, the fusion protein described in the second aspect of the present invention, the polynucleotide described in the third aspect of the present invention, the nucleic acid molecule described in the fourth aspect of the present invention, the complex described in the sixth aspect of the present invention, the vector described in the seventh aspect of the present invention, the CRISPR-Cas composition described in the eighth aspect of the present invention, or the system described in the ninth aspect of the present invention, or the delivery composition described in the eleventh aspect of the present invention.

In another preferred embodiment, the host cell is a eukaryotic cell, such as a yeast cell, a plant cell or a mammalian cell (including human and non-human mammals).

In another preferred embodiment, the host cell is a prokaryotic cell, such as Escherichia coli.

In another preferred embodiment, the yeast cell is selected from yeast of one or more sources of the following group: Pichia pastoris, Kluyveromyces, or a combination thereof; preferably, the yeast cell includes: Kluyveromyces, more preferably Kluyveromyces marxianus, and/or Kluyveromyces lactis.

In another preferred embodiment, the host cell is selected from the following group: Escherichia coli, wheat germ cells, insect cells, SF9, Hela, HEK293, CHO, yeast cells, or a combination thereof.

The thirteenth aspect of the present invention provides an enzyme preparation, which includes the gene editing protein described in the first aspect of the present invention, the fusion protein described in the second aspect of the present invention, the complex described in the sixth aspect of the present invention, the CRISPR-Cas composition described in the eighth aspect of the present invention, or the system described in the ninth aspect of the present invention or the delivery composition described in the eleventh aspect of the present invention.

In another preferred embodiment, the enzyme preparation includes an injection and/or a lyophilized preparation.

A fourteenth aspect of the present invention provides a medicine kit, comprising:

A first container, and the complex described in the sixth aspect of the present invention, the composition described in the eighth aspect of the present invention, or the system described in the ninth aspect of the present invention located in the first container, or a drug containing the complex described in the sixth aspect of the present invention, the composition described in the eighth aspect of the present invention, or the system described in the ninth aspect of the present invention.

In another preferred embodiment, the drug in the first container is a single preparation containing the complex described in the sixth aspect of the present invention, the composition described in the eighth aspect of the present invention, or the system described in the ninth aspect of the present invention.

In another preferred embodiment, the dosage form of the drug is selected from the following group: a lyophilized preparation, a liquid preparation, or a combination thereof.

In another preferred embodiment, the dosage form of the drug is an oral dosage form or an injection dosage form.

In another preferred embodiment, the medicine kit further contains instructions.

A fifteenth aspect of the present invention provides a medicine kit, comprising:

(a1) a first container, and the gene editing protein according to the first aspect of the present invention, or the fusion protein according to the second aspect of the present invention, or a gene encoding the same or an expression vector thereof, or a drug containing the gene editing protein according to the first aspect of the present invention, or the fusion protein according to the second aspect of the present invention, or a gene encoding the same or an expression vector thereof, located in the first container;

(b1) an optional second container, and the guide RNA or its expression vector according to the fifth aspect of the present invention, or a drug containing the guide RNA or its expression vector according to the fifth aspect of the present invention, located in the second container.

In another preferred embodiment, the first container and the second container are different containers.

In another preferred embodiment, the drug in the first container is a single preparation containing the gene editing protein described in the first aspect of the present invention, or the fusion protein described in the second aspect of the present invention, or its encoding gene or its expression vector.

In another preferred embodiment, the drug in the second container is a single preparation containing the guide RNA or its expression vector described in the fifth aspect of the present invention.

In another preferred embodiment, the dosage form of the drug is selected from the following group: a lyophilized preparation, a liquid preparation, or a combination thereof.

In another preferred embodiment, the dosage form of the drug is an oral dosage form or an injection dosage form.

In another preferred embodiment, the medicine kit further contains instructions.

The sixteenth aspect of the present invention provides a method for targeting and editing a target gene or cutting a target gene, comprising: the gene editing protein described in the first aspect of the present invention, or the fusion protein described in the second aspect of the present invention, or the complex described in the sixth aspect of the present invention, or the composition described in the eighth aspect of the present invention, or the system described in the ninth aspect of the present invention, or the delivery composition described in the eleventh aspect of the present invention, or the enzyme preparation described in the thirteenth aspect of the present invention, or the drug kit described in the fourteenth aspect of the present invention or the fifteenth aspect of the present invention is contacted with the target gene, or delivered to a cell containing the target gene, and the target sequence is present in the target gene.

In another preferred embodiment, the target gene exists in cells.

In another preferred embodiment, the cell is a prokaryotic cell.

In another preferred embodiment, the cell is a eukaryotic cell, such as a mammalian cell (eg, a human cell) or a plant cell.

In another preferred embodiment, the target gene exists in a nucleic acid molecule (eg, a plasmid) in vitro.

In another preferred example, the editing of the target gene or the cutting of the target gene includes the break of the target sequence, such as a double-strand break of DNA or a single-strand break of RNA, or the insertion of an exogenous nucleic acid into the break.

In another preferred embodiment, the target gene includes DNA.

In another preferred embodiment, the DNA includes single-stranded DNA and double-stranded DNA.

The seventeenth aspect of the present invention provides a method for inducing a change in a cell state, the method comprising contacting the gene editing protein described in the first aspect of the present invention, or the fusion protein described in the second aspect of the present invention, or the complex described in the sixth aspect of the present invention, or the composition described in the eighth aspect of the present invention, or the system described in the ninth aspect of the present invention, or the delivery composition described in the eleventh aspect of the present invention, or the enzyme preparation described in the thirteenth aspect of the present invention, or the drug kit described in the fourteenth aspect of the present invention or the fifteenth aspect of the present invention with a target gene in a cell.

The eighteenth aspect of the present invention provides a method for changing the expression of a gene product, comprising: modifying the gene editing protein described in the first aspect of the present invention, or the fusion protein described in the second aspect of the present invention, or the The complex described in the sixth aspect, the composition described in the eighth aspect of the present invention, the system described in the ninth aspect of the present invention, the delivery composition described in the eleventh aspect of the present invention, the enzyme preparation described in the thirteenth aspect of the present invention, or the drug kit described in the fourteenth aspect of the present invention or the fifteenth aspect of the present invention is contacted with a nucleic acid molecule encoding the gene product, or delivered to a cell containing the nucleic acid molecule, and the target sequence is present in the nucleic acid molecule.

In another preferred embodiment, the nucleic acid molecule is present in an in vitro nucleic acid molecule (eg, a plasmid).

In another preferred embodiment, the expression of the gene product is altered (eg, enhanced or decreased).

In another preferred embodiment, the gene product is a protein.

In another preferred embodiment, the protein, fusion protein, polynucleotide, isolated nucleic acid molecule, complex, vector or composition is contained in a delivery vehicle.

In another preferred embodiment, the delivery vector is selected from lipid particles, sugar particles, metal particles, protein particles, liposomes, exosomes, viral vectors (such as replication-defective retroviruses, lentiviruses, adenoviruses or adeno-associated viruses).

In another preferred embodiment, the method is used to modify cells, cell lines or organisms by changing one or more target sequences in a target gene or a nucleic acid molecule encoding a target gene product.

The nineteenth aspect of the present invention provides a cell or its progeny obtained by the method described in any one of the sixteenth to eighteenth aspects of the present invention, wherein the cell comprises a modification that is not present in its wild type.

The twentieth aspect of the present invention provides a cell product of the cell or its progeny described in the nineteenth aspect of the present invention.

The twenty-first aspect of the present invention provides an in vitro, ex vivo or in vivo cell or cell line or their progeny, wherein the cell or cell line or their progeny comprises: the gene editing protein described in the first aspect of the present invention, or the fusion protein described in the second aspect of the present invention, or the polynucleotide described in the third aspect of the present invention, or the complex described in the sixth aspect of the present invention, or the vector described in the seventh aspect of the present invention, or the composition described in the eighth aspect of the present invention, or the system described in the ninth aspect of the present invention, or the delivery composition described in the eleventh aspect of the present invention.

In another preferred embodiment, the cell is a prokaryotic cell.

In another preferred embodiment, the cell is a eukaryotic cell, such as a mammalian cell (eg, a human cell) or a plant cell.

In another preferred embodiment, the cells are stem cells or stem cell lines.

The twenty-second aspect of the present invention provides the use of the gene editing protein described in the first aspect of the present invention, or the fusion protein described in the second aspect of the present invention, or the polynucleotide described in the third aspect of the present invention, or the complex described in the sixth aspect of the present invention, or the vector described in the seventh aspect of the present invention, or the composition described in the eighth aspect of the present invention, or the system described in the ninth aspect of the present invention, or the kit described in the tenth aspect of the present invention, or the delivery composition described in the eleventh aspect of the present invention, or the enzyme preparation described in the thirteenth aspect of the present invention, or the medicine kit described in the fourteenth aspect of the present invention or the fifteenth aspect of the present invention, for preparing a drug or preparation, wherein the drug or preparation is used for nucleic acid editing (e.g., gene or genome editing).

In another preferred embodiment, the gene or genome editing includes modifying genes, knocking out genes, changing the expression of gene products, repairing mutations, and/or inserting polynucleotides.

The twenty-third aspect of the present invention provides the gene editing protein described in the first aspect of the present invention, or the fusion protein described in the second aspect of the present invention, or the polynucleotide described in the third aspect of the present invention, or the complex described in the sixth aspect of the present invention, or the vector described in the seventh aspect of the present invention, or the composition described in the eighth aspect of the present invention. or the system of the ninth aspect of the present invention, or the kit of the tenth aspect of the present invention, or the delivery composition of the eleventh aspect of the present invention, or the enzyme preparation of the thirteenth aspect of the present invention, or the kit of the fourteenth aspect of the present invention or the fifteenth aspect of the present invention, for preparing a drug or a preparation, wherein the drug or preparation is used for one or more selected from the group consisting of:

(i) ex vivo gene or genome editing;

(ii) Detection of single-stranded DNA in vitro;

(iii) editing a target sequence in a target locus to modify an organism or non-human organism;

(iv) treating a disorder caused by a defect in the target sequence in the target locus;

(v) treating a condition or disease in a subject in need thereof.

In another preferred embodiment, the disease or illness includes cancer, infectious disease, neurological disease, ophthalmic disease, hearing disease.

In another preferred embodiment, the disease or condition includes cystic fibrosis, atherosclerotic cardiovascular disease (ASCVD), progressive pseudohypertrophic muscular dystrophy (Duchenne muscular dystrophy, DMD), Becker muscular dystrophy, alpha-1-antitrypsin deficiency, Pompe disease (glycogen storage disease type II), myotonic dystrophy, Huntington's disease, fragile X syndrome, Friedreich's ataxia, amyotrophic lateral sclerosis, hereditary chronic kidney disease, sickle cell disease, primary hyperoxaluria (PH1), beta thalassemia, frontotemporal dementia, Leber's congenital amaurosis, hyperlipidemia, hypercholesterolemia (FH), hereditary angioedema (HAE), transthyretinopathy. ATTR, Hepatitis B, Retinal diseases, Macular degeneration, Wilms' tumor, Ewing's sarcoma, Neuroendocrine tumors, Glioblastoma, Neuroblastoma, Melanoma, Skin cancer, Breast cancer, Colon cancer, Rectal cancer, Prostate cancer, Liver cancer, Kidney cancer, Pancreatic cancer, Lung cancer, Biliary tract cancer, Cervical cancer, Endometrial cancer, Esophageal cancer, Gastric cancer, Head and neck cancer, Medullary thyroid cancer, Ovarian cancer, Glioma, Lymphoma, Leukemia, Myeloma, Acute lymphocytic leukemia, Acute myeloid leukemia, Chronic lymphocytic leukemia, Chronic myeloid leukemia, Hodgkin's lymphoma, Non-Hodgkin's lymphoma and Urinary bladder cancer.

In another preferred embodiment, the disorder or disease is caused by a pathogenic point mutation.

The twenty-fourth aspect of the present invention provides a method for detecting whether a target nucleic acid molecule is present in a sample, the method comprising contacting the sample with the gene editing protein described in the first aspect of the present invention, or the fusion protein described in the second aspect of the present invention, or the complex described in the sixth aspect of the present invention, or the composition described in the eighth aspect of the present invention, or the system described in the ninth aspect of the present invention, or the kit described in the tenth aspect of the present invention, or the delivery composition described in the eleventh aspect of the present invention, or the enzyme preparation described in the thirteenth aspect of the present invention and a non-target sequence, detecting a detectable signal generated by the cleavage of the non-target sequence, thereby detecting the target nucleic acid molecule, wherein the non-target sequence does not hybridize with the guide RNA.

In another preferred example, if the non-target sequence is cleaved by a protein in the complex or CRISPR-Cas composition or system or delivery composition, it indicates that the target nucleic acid molecule is present in the sample; and if the non-target sequence is not cleaved by a protein in the complex or CRISPR-Cas composition or system or delivery composition, it indicates that the target nucleic acid molecule is not present in the sample.

In another preferred embodiment, the target nucleic acid molecule is a target DNA.

In another preferred embodiment, the target DNA includes DNA formed based on RNA reverse transcription.

In another preferred embodiment, the target DNA includes cDNA.

In another preferred embodiment, the target DNA is selected from the following group: single-stranded DNA, double-stranded DNA, or a combination thereof.

It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the following (such as embodiments) The various technical features specifically described in the specification can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows an agarose gel electrophoresis diagram of the CasW1 insert fragment and the pET-28a(+) vector double-digested with EcoRI and NotI.

FIG2 shows the SDS-PAGE Coomassie Brilliant Blue staining of the newly purified CasW1 protein.

FIG3 shows an agarose gel electrophoresis diagram of dsDNA template prepared in vitro.

FIG4 shows a capillary electrophoresis diagram for identifying the in vitro cleavage effect of CasW1. Taking the in vitro cleavage effect of Cpf1 as a control, it can be seen that CasW1 can cleave a 450 bp dsDNA template into two dsDNA fragments, and the cleavage activity is close to 100%.

Figure 5 shows a capillary electrophoresis diagram for identifying the cleavage effect of CasW1 in vitro, with Cas12i.16 and S7R-Cas12i3 (M2869) in the prior art as controls.

FIG. 6 shows a plasmid map of pET-28a(+)-CasW1.

FIG. 7 shows a plasmid map of pET-28a(+)-LbCpf1.

Figure 8 shows the plasmid map of pET-28a(+)-HED Cas12i.16.

Figure 9 shows the plasmid map of pET-28a(+)-S7R Cas12i.3.

FIG10 shows a schematic diagram of the secondary structure of the DR sequence of CasW1.

DETAILED DESCRIPTION

After extensive and in-depth research, the inventor unexpectedly discovered a new gene editing protein for the first time. The gene editing protein of the present invention has very good gene editing activity, can effectively edit or cut the target gene, and can effectively treat the symptoms or diseases of subjects in need. On this basis, the inventor completed the present invention.

the term

The following examples are only used to describe the present invention, but not to limit the present invention. Unless otherwise specified, the experiments and methods described in the examples are basically carried out according to conventional methods well known in the art and described in various references.

In addition, if the specific conditions are not specified in the examples, they are carried out according to the conventional conditions or the conditions recommended by the manufacturer. If the manufacturer is not specified in the reagents or instruments used, they are all conventional products that can be obtained commercially. It is known to those skilled in the art that the embodiments describe the present invention by way of example and are not intended to limit the scope of the present invention. All public cases and other references mentioned herein are incorporated herein by reference in their entirety.

In order to more easily understand the present disclosure, some terms are first defined. As used in this application, unless otherwise expressly provided herein, each of the following terms should have the meaning given below. Other definitions are set forth throughout the application.

The term "about" can refer to a value or composition that is within an acceptable error range for a particular value or composition as determined by one of ordinary skill in the art, which will depend in part on how the value or composition is measured or determined. For example, as used herein, the expression "about 100" includes all values between 99 and 101 (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

As used herein, the term "comprising" or "including (comprising)" may be open, semi-closed and closed. In other words, the term also includes "consisting essentially of" or "consisting of".

Sequence identity (or homology) is determined by comparing two aligned sequences along a predetermined comparison window (which can be 50%, 60%, 70%, 80%, 90%, 95% or 100% of the length of the reference nucleotide sequence or protein) and determining the number of positions at which identical residues occur. Typically, this is expressed as a percentage. The measurement of sequence identity of nucleotide sequences is a method well known to those skilled in the art.

Gene editing proteins

In the present invention, the gene editing protein is the effector protein in the CRISPR/Cas system.

In the present invention, Cas protein, Cas enzyme, and Cas effector protein can be used interchangeably. Cas protein is taken in the broadest sense, including wild-type Cas protein, its derivatives or variants, analogs, and functional fragments thereof such as oligonucleotide binding fragments.

The term "wild type" has the meaning generally understood by those skilled in the art, which refers to the typical form of an organism, strain, gene, protein, or the characteristics that distinguish it from mutant or variant forms when it exists in nature, which can be isolated from a source in nature and has not been intentionally modified by man.

The terms "variant", "derivative" and "analog" refer to polypeptides that substantially retain the function or activity of the Cas protein of the present invention.

Typically, the derivatization of a protein does not adversely affect the desired activity of the protein (e.g., activity binding to a guide RNA, endonuclease activity, activity binding to and cutting a specific site of a target sequence under the guidance of a guide RNA), that is, the derivative of the protein has the same activity as the protein. A modified form of a "derivative" includes one or more amino acids of the protein that may be deleted, inserted, modified and/or substituted. The terms "non-naturally occurring" or "engineered" are used interchangeably and indicate artificial involvement.

In one aspect, the present invention provides a gene editing protein (Cas protein) comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the amino acid sequence of SEQ ID NO.1, and substantially retains the biological function of the sequence from which it is derived;

In one embodiment, the amino acid sequence of the Cas protein has one or more amino acid substitutions, deletions or additions compared to the amino acid sequence of SEQ ID NO.1, and substantially retains the biological function of the sequence from which it is derived;

In one embodiment, the Cas protein comprises the amino acid sequence shown in SEQ ID NO.1;

or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, deletions or additions) compared to the sequence shown in SEQ ID NO.1; or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity with the amino acid sequence shown in SEQ ID NO.1;

In one embodiment, the Cas protein has an amino acid sequence shown in SEQ ID NO.1.

It is clear to those skilled in the art that the structure of a protein can be changed without adversely affecting its activity and functionality, for example, by introducing one or more conservative amino acid substitutions into the amino acid sequence of the protein without adversely affecting the activity and/or three-dimensional structure of the protein molecule.

Those skilled in the art will be aware of examples and embodiments of conservative amino acid substitutions. Specifically, an amino acid residue may be substituted with another amino acid residue belonging to the same group as the site to be substituted, i.e., with a non- A polar amino acid residue replaces another non-polar amino acid residue, a polar uncharged amino acid residue replaces another polar uncharged amino acid residue, a basic amino acid residue replaces another basic amino acid residue, and an acidic amino acid residue replaces another acidic amino acid residue. Such substituted amino acid residues may or may not be encoded by the genetic code. As long as the substitution does not lead to the inactivation of the biological activity of the protein, conservative substitutions in which one amino acid is replaced by other amino acids belonging to the same group fall within the scope of the present invention. Therefore, the protein of the present invention may contain one or more conservative substitutions in the amino acid sequence, and these conservative substitutions are preferably produced by substitution according to Table A. In addition, the present invention also encompasses proteins that also contain one or more other non-conservative substitutions, as long as the non-conservative substitutions do not significantly affect the desired functions and biological activities of the protein of the present invention.

Conservative amino acid substitutions can be performed at one or more predicted non-essential amino acid residues. "Non-essential" amino acid residues are amino acid residues that can be changed (deleted, substituted or replaced) without changing biological activity, while "essential" amino acid residues are required for biological activity. "Conservative amino acid substitutions" are substitutions in which amino acid residues are replaced by amino acid residues with similar side chains. Amino acid substitutions can be performed in non-conserved regions of Cas enzymes. In general, such substitutions are not performed on conserved amino acid residues, or on amino acid residues located within conserved motifs, where such residues are required for protein activity. However, it will be appreciated by those skilled in the art that functional variants may have fewer conservative or non-conservative changes in conserved regions.

Table A

It is known to those skilled in the art that one or more amino acid residues can be changed (replaced, deleted, truncated or inserted) from the N and/or C terminus of a protein while still retaining its functional activity. Therefore, proteins that have changed one or more amino acid residues from the N and/or C terminus of the Cas protein of the present invention while retaining its desired functional activity are also within the scope of the present invention. These changes may include changes introduced by modern molecular methods such as PCR, which includes PCR amplification of a protein coding sequence by means of an amino acid coding sequence included in the oligonucleotides used in the PCR amplification to change or extend the PCR amplification.

It will be appreciated that proteins can be altered in a variety of ways, including amino acid substitutions, deletions, truncations, and insertions, and methods for such manipulations are generally known to those skilled in the art.

For example, amino acid sequence variants of Cas proteins can be prepared by mutation of DNA. It can also be accomplished by other forms of mutagenesis and/or by directed evolution, for example, using known mutagenesis, recombination and/or shuffling methods, combined with relevant screening methods, to perform one or more amino acid substitutions; or one to more amino acid deletions and/or one to more amino acid insertions.

Those skilled in the art will appreciate that these minor amino acid changes in the Cas proteins of the present invention can occur (e.g., naturally occurring mutations) or be produced (e.g., using r-DNA technology) without loss of protein function or activity. If these mutations occur in the catalytic domain, active site, or other functional domain of the protein, the properties of the polypeptide may be changed, but the polypeptide may retain its activity. If the mutations present are not close to the catalytic domain, active site, or other functional domain, it can be expected that the impact will be small.

Those skilled in the art can identify the essential amino acids of the Cas protein according to methods known in the art, such as site-directed mutagenesis or protein evolution or analysis of bioinformatics systems. The catalytic domain, active site or other functional domain of the protein can also be determined by physical analysis of the structure, such as by the following techniques: such as nuclear magnetic resonance, crystallography, electron diffraction or photoaffinity labeling, combined with mutations of amino acids at putative key sites.

Orthologue (ortholog)

As used herein, the term "orthologue" has the meaning commonly understood by those skilled in the art. As a further guide, an "orthologue" of a protein as described herein refers to a protein belonging to a different species that performs the same or similar function as its orthologue.

The nucleic acid cleavage disclosed herein includes: DNA or RNA breakage in the target nucleic acid produced by the Cas protein (Cis cleavage), DNA or RNA breakage in the side branch nucleic acid substrate (single-stranded nucleic acid substrate) caused by the Cas protein side cutting activity (i.e., non-specific or non-targeted, Trans cleavage). In some embodiments, the cleavage is a double-stranded DNA break. In some embodiments, the cleavage is a single-stranded DNA break or a single-stranded RNA break.

Trans cutting refers to that in certain environments, the activated Cas12 family protein remains active after binding to the target sequence, and continues to non-specifically cut non-target oligonucleotides. The side cutting activity can detect the presence of specific target oligonucleotides using the Cas system. For example, the Cas12i system is engineered to non-specifically cut ssDNA or transcripts. Side cutting activity is used for a highly sensitive and specific nucleic acid detection platform called SHERLOCK, which can be used for many clinical diagnoses (Gootenberg, JS et al., Nucleic acid detection with CRISPR-Cas13a/C2c2. Science 356, 438-442 (2017)).

Fusion Protein

In one aspect, the present invention provides a fusion protein, comprising the Cas protein described in any one of the foregoing and one or more functional domains.

In one embodiment, the functional domain includes one or more of a localization signal, a reporter protein, a Cas protein targeting portion, a DNA binding domain, an epitope tag, a transcription activation domain, a transcription repression domain, a nuclease, a deamination domain, a methylase, a demethylase, a transcription release factor, an HDAC, a cleavage active polypeptide, and a ligase;

In one embodiment, "methylase" is exemplified by HhaIDNA m5c-methyltransferase (M.HhaI), DNA methyltransferase 1 (DNMT1), DNA methyltransferase 3a (DNMT3a), DNA methyltransferase 3b (DNMT3b), METI, DRM3, ZMET2, CMT1, CMT2, etc.

"Demethylase" refers to an enzyme that removes methyl (CH3-) groups from nucleic acids, proteins (e.g., histones), and other molecules. Demethylases are important in epigenetic modification mechanisms. Demethylase proteins change the transcriptional regulation of the genome by controlling the methylation levels that occur on DNA and histones, and in turn regulate the chromatin state at specific loci in the organism, such as TET1 (ten-eleven translocation 1), ten-eleven translocation (TET) dioxygenase 1 (TET1CD), DME, DML1, DML2, ROS1, etc.

In another preferred embodiment, the transcriptional release factor, for example, is eukaryotic release factor 1 (ERF1) activity or eukaryotic release factor 3 (ERF3).

In one embodiment, the functional domain is selected from an adenosine deaminase catalytic domain or a cytidine deaminase catalytic domain.

In one embodiment, the localization signal comprises a nuclear localization signal and/or a nuclear export signal;

Preferably, the nuclear export signal comprises human protein tyrosine kinase 2;

Preferably, the reporter protein comprises one or more of glutathione-S-transferase, horseradish peroxidase, chloramphenicol acetyltransferase, β-galactosidase, β-glucuronidase or autofluorescent protein;

Preferably, the autofluorescent protein includes one or more of green fluorescent protein, HcRed, DsRed, cyan fluorescent protein, yellow fluorescent protein or blue fluorescent protein;

Preferably, the DNA binding domain comprises one or more of methylation binding protein, LexADBD or Gal4DBD;

Preferably, the epitope tag comprises one or more of a histidine tag, a V5 tag, a FLAG tag, an influenza virus hemagglutinin tag, a Myc tag, a VSV-G tag or a thioredoxin tag;

Preferably, the transcriptional activation domain comprises VP64 and/or VPR;

Preferably, the transcriptional repression domain comprises KRAB and/or SID;

Preferably, the nuclease comprises FokI;

Preferably, the deamination domain comprises one or more of ADAR1, ADAR2, APOBEC, AID or TAD;

Preferably, the cleavage active polypeptide includes a polypeptide having single-stranded RNA cleavage activity, a polypeptide having double-stranded RNA cleavage activity, a polypeptide having single-stranded DNA cleavage activity or a polypeptide having double-stranded DNA cleavage activity;

Preferably, the ligase comprises DNA ligase and/or RNA ligase.

In one embodiment, the functional domain is the full length or a functional fragment of TadA8e.

Polynucleotide

In one aspect, the present invention provides a polynucleotide, which is a polynucleotide sequence encoding the gene editing protein (Cas protein), or a polynucleotide sequence encoding the aforementioned fusion protein.

In one embodiment, the polynucleotide (DNA molecule) includes nucleotides that have more than 70%, preferably more than 90%, more preferably more than 95%, further preferably 99%, and further preferably 100% identity with the nucleotide sequence described in SEQ ID NO.2.

In one embodiment, the polynucleotide is a DNA molecule that is codon-optimized according to the codon preference of the host cell.

The optimization described in the present disclosure may require mutations in the nucleotide sequence of the coded protein (e.g., the Cas protein of the present disclosure) to simulate the codon preference of the expected host organism or cell when encoding the same protein at the same time. Therefore, the codon can be changed, but the encoded protein remains unchanged. For example, if the expected target cell is a human cell, a nucleotide sequence of the coded protein optimized by human codons can be used. As another non-limiting embodiment, if the expected host cell is an animal cell (e.g., a mouse cell, an insect cell), a nucleotide sequence of the coded protein optimized by the animal codon can be generated. As another non-limiting embodiment, if the expected host cell is a plant cell, a nucleotide sequence of the coded protein optimized by plant codons can be generated.

A selection list of codons is readily available, for example, the "Codon Usage Database" available at www.kazusa.or.jp/codon. In some cases, the nucleic acid of the present disclosure comprises a nucleotide sequence encoding CasY7 or a variant thereof or a fusion protein thereof, the nucleotide sequence being codon-optimized for expression in eukaryotic cells. In some cases, the nucleic acid of the present disclosure comprises a nucleotide sequence encoding CasW1 or a variant thereof or a fusion protein thereof, the nucleotide sequence being codon-optimized for expression in animal cells. In some cases, the nucleic acid of the present disclosure comprises a nucleotide sequence encoding CasW1 or a variant thereof or a fusion protein thereof, the nucleotide sequence being codon-optimized for expression in fungal cells. In some cases, the nucleic acid of the present disclosure comprises a nucleotide sequence encoding CasW1 or a variant thereof or a fusion protein thereof, the nucleotide sequence being codon-optimized for expression in plant cells.

In one embodiment, the host cell comprises a prokaryotic cell or a eukaryotic cell.

CRISPR system

The terms “Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR-associated (Cas) (CRISPR-Cas) system” or “CRISPR system” are used interchangeably and have the meaning commonly understood by those skilled in the art, which generally includes transcription products or other elements related to the expression of CRISPR-associated (“Cas”) genes, or transcription products or other elements capable of directing the activity of the Cas genes.

CRISPR-Cas Composition

In one aspect, the present invention also provides a CRISPR-Cas composition, comprising:

(1) Protein component: the aforementioned gene editing protein (Cas protein), or the aforementioned fusion protein; or a nucleic acid molecule encoding the gene editing protein (Cas protein) or the aforementioned fusion protein;

(2) RNA component: guide RNA, or one or more nucleic acids encoding the guide RNA, or a precursor RNA of the guide RNA, or a nucleic acid encoding a precursor RNA of the guide RNA;

The protein component and the nucleic acid component are combined with each other to form a complex.

In one embodiment, the composition is an activated CRISPR complex, and the activated CRISPR complex further comprises: a target sequence of a target nucleic acid bound to the guide RNA.

In one embodiment, the CRISPR-Cas composition comprises one or more vectors, wherein the one or more vectors comprise:

(1) a first regulatory element, which is operably linked to a nucleotide sequence encoding the gene editing protein (Cas protein) or a nucleotide sequence encoding the fusion protein; and

(2) a second regulatory element, wherein the second regulatory element is operably linked to a nucleotide sequence encoding the guide RNA, wherein the guide RNA comprises:

(a) a spacer sequence capable of hybridizing with a target sequence of a target nucleic acid, and

(b) a direct repeat (DR) sequence connected to the spacer sequence and capable of guiding the gene editing protein (Cas protein) to bind to the guide RNA to form a CRISPR-Cas complex targeting the target sequence;

Wherein the first regulatory element and the second regulatory element are located on the same or different vectors of the CRISPR-Cas vector system.

In one embodiment, the first regulatory element or the second regulatory element comprises a promoter, and the promoter comprises one or more of an inducible promoter, a constitutive promoter, or a tissue-specific promoter;

In one embodiment, the promoter comprises one or more of T7, SP6, T3, CMV, EF1a, SV40, PGK1, human β-actin, CAG, U6, H1, T7, T7lac, araBAD, trp, lac or Ptac;

In one embodiment, the first regulatory element and the second regulatory element are located on the same or different vectors.

In one embodiment, the vector comprises a retroviral vector, a lentiviral vector, an adenoviral vector, an adeno-associated viral vector, a herpes simplex vector, or a phagemid vector;

In one embodiment, the vector comprises a plasmid vector.

In one embodiment, the target nucleic acid comprises DNA derived from a eukaryotic organism or DNA derived from a prokaryotic organism;

In one embodiment, the eukaryotic organism includes an animal or a plant;

In one embodiment, the target nucleic acid comprises non-human mammal DNA, human DNA, insect DNA, bird DNA, reptile DNA, amphibian DNA, rodent DNA, fish DNA, worm DNA, nematode DNA, or yeast DNA;

In one embodiment, the non-human mammalian DNA comprises non-human primate DNA.

CRISPR/Cas complex

The term "CRISPR/Cas complex" refers to a complex formed by the binding of guide RNA, gRNA (guide RNA) or mature crRNA (or guide RNA) and gene editing protein (Cas protein), which contains a co-directional repeat sequence that hybridizes to the guide sequence of the target sequence and binds to the gene editing protein (Cas protein), and the complex can recognize and cut the target nucleotide that can hybridize with the guide RNA or mature crRNA.

Guide RNA (gRNA)

The terms "guide RNA (gRNA)", "mature crRNA", "crRNA", "guide sequence", and "guide RNA" are used interchangeably and have the meanings commonly understood by those skilled in the art. In general, a guide RNA may include a direct repeat (DR) sequence and a spacer. The sequence may consist essentially of or consist of a direct repeat (DR) sequence and a spacer sequence.

In some cases, the spacer sequence is any polynucleotide sequence that has sufficient complementarity with the target sequence to hybridize with the target sequence and guide the CRISPR-Cas complex to specifically bind to the target sequence. In one embodiment, when optimally aligned, the degree of complementarity between the spacer sequence and its corresponding target sequence is at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99%. The guide sequence comprises a sequence (e.g., a direct repeat (DR) sequence) that has sufficient complementarity with the target nucleic acid sequence to hybridize with the target nucleic acid sequence and guide the sequence-specific binding of the complex to the target nucleic acid sequence.

It is known in the art that on the basis of sufficient complementarity to function, complete complementarity is not required, and therefore, if necessary, the adjustment of the cleavage efficiency can be achieved by introducing mismatches (e.g., one or more mismatches between the spacer sequence and the target nucleic acid, such as 1 or 2 nucleotide mismatches (including the position of the mismatch along the spacer sequence/target sequence)). For example, if a cleavage rate of less than 100% of the target is desired (e.g., in a cell population), 1 or 2 mismatches between the spacer sequence and the target sequence can be introduced into the spacer sequence.

In one aspect, the present invention provides a guide RNA, which includes a direct repeat (DR) sequence capable of binding to the Cas protein and a spacer sequence capable of targeting a target sequence.

In one embodiment, the direct repeat sequence, the direct repeat sequence (Direct Repeat, DR) comprises the sequence shown in SEQ ID NO.3.

In one embodiment, the 3' end of the direct repeat sequence comprises a stem-loop structure, and further comprises a stem of the stem-loop structure formed by hybridization of a first stem nucleotide chain and a second stem nucleotide chain, and the ring nucleotide chain forms a loop of the stem-loop structure;

In one embodiment, the direct repeat sequence comprises a nucleotide sequence having at least 80% identity with the nucleotide sequence described in SEQ ID NO.3;

In one embodiment, the direct repeat sequence comprises a nucleotide sequence having at least 85% or more, more preferably more than 90%, and further preferably more than 95% identity with the nucleotide sequence described in SEQ ID NO.3;

In one embodiment, the homeotropic repeat sequence includes the nucleotide sequence described in SEQ ID NO.3.

In one embodiment, more than 80% of the spacer sequence is complementary to the target nucleic acid;

In one embodiment, more than 90%, more preferably more than 95%, further preferably more than 99%, and further preferably 100% of the spacer sequence is complementary to the target nucleic acid;

In one embodiment, the length of the spacer sequence is 18-41 nt, for example 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 nt, more preferably 18 to 27 nucleotides, more preferably 18 to 24 nucleotides, and most preferably 18 to 22 nucleotides.

In one embodiment, the spacer sequence is 20 nt in length.

Target nucleic acid

In the present invention, target nucleic acid is used interchangeably with target sequence or target nucleic acid sequence or target nucleic acid molecule, and refers to a specific nucleic acid that comprises a nucleic acid sequence that is fully or partially complementary to the spacer sequence in the guide RNA. "Target sequence" refers to a polynucleotide targeted by a spacer sequence in a guide RNA, such as a sequence having complementarity with the spacer sequence, wherein hybridization between the target sequence and the spacer sequence will promote the formation of a CRISPR-Cas complex (including Cas protein and guide RNA). Complete complementarity is not required, as long as there is enough complementarity to cause hybridization and promote the formation of a CRISPR-Cas complex. In some embodiments, the target nucleic acid comprises a non-coding region (e.g., a promoter or terminator). In some embodiments, the target nucleic acid is single-stranded, or double-stranded.

The target sequence can comprise any polynucleotide, such as DNA. In some cases, the target sequence is located in a cell or outside the cell. In some cases, the target sequence is located in the nucleus, cytoplasm, or organelle (e.g., mitochondria or chloroplast) of the cell.

The target nucleic acid can be a sequence encoding a gene product (e.g., a protein) or a non-coding sequence (e.g., a regulatory polynucleotide or junk DNA). In some cases, the target sequence should be associated with a protospacer adjacent motif (PAM).

Donor template

In the present invention, donor template nucleic acid or donor template are used interchangeably and refer to a nucleic acid molecule that can be used by one or more cellular proteins to change the structure of the target nucleic acid after the gene editing protein (Cas protein) described in this article changes the target nucleic acid.

In some embodiments, the donor template nucleic acid is a double-stranded nucleic acid or a single-stranded nucleic acid. In some embodiments, the donor template nucleic acid is linear or circular (e.g., plasmid). In some instances, the donor template nucleic acid is an exogenous nucleic acid molecule. In some instances, the donor template nucleic acid is an endogenous nucleic acid molecule (e.g., chromosome). In some embodiments, the donor template can be used to realize genetic recombination, and the recombination is homologous recombination.

Cutting

Cutting refers to DNA breaks in target nucleic acids produced by gene editing proteins (Cas proteins) described herein. In some embodiments, cutting is double-stranded DNA breaks. In some embodiments, cutting is single-stranded DNA breaks.

In the present invention, the meanings of cleaving a target nucleic acid or modifying a target nucleic acid may overlap. Modifying a target nucleic acid includes not only modification of a single nucleotide, but also insertion or deletion of a nucleic acid fragment.

Reporter nucleic acid

Reporter nucleic acid refers to a molecule that can be cut or otherwise deactivated by an activated CRISPR system protein as described herein. Reporter nucleic acid comprises a nucleic acid element that can be cut by a CRISPR protein (e.g., using a single-stranded non-targeted nucleic acid molecule, with different reporter groups or labeling molecules at both ends). The cutting of the nucleic acid element produces a detectable signal. Before cutting, or when the reporter nucleic acid is in an "active" state, the reporter nucleic acid prevents the generation or detection of a positive detectable signal. It will be understood that in certain example embodiments, a minimum background signal can be generated in the presence of an active reporter nucleic acid. A positive detectable signal can be any signal that can be detected using optics, fluorescence, chemiluminescence, electrochemistry, or other detection methods known in the art. For example, in certain embodiments, when a reporter nucleic acid is present, a first signal (i.e., a negative detectable signal) can be detected, which is then converted to a second signal (e.g., a positive detectable signal) after the target molecule is detected and cut or deactivated by an activated CRISPR protein. The reporter nucleic acid can be a single-stranded DNA molecule, a single-stranded RNA molecule, or a single-stranded DNA-RNA hybrid.

The detection method of the present invention can be used for quantitative detection of target nucleic acid to be detected. The measurement index can be quantified according to the signal strength of the reporter group, such as the luminescence intensity of the fluorescent group, or the width of the color band.

Functional domain

The functional domain herein takes its broadest meaning, including proteins such as enzymes or factors themselves or having specific functional fragments/domains. A gene editing protein (e.g., a dCas protein) is connected/concluded with one or more functional domains, and the functional domains are selected from one or more of a localization signal, a reporter protein, a Cas protein targeting portion, a DNA binding domain, an epitope tag, a transcription activation domain, a transcription inhibition domain, a nuclease, a deamination domain, a methylase, a demethylase, a transcription release factor, an HDAC, a cleavage active polypeptide, and a ligase. When more than one functional domain is included, the functional domains may be the same or different.

Deamination domain

In the present invention, the deamination domain includes a deaminase (e.g., an adenosine deaminase or a cytidine deaminase) catalytic domain. As used herein, "adenosine deaminase" or "adenosine deaminase protein" refers to a protein, a polypeptide, or one or more functional domains of a protein or polypeptide that is capable of catalyzing a hydrolytic deamination reaction that converts adenine (or the adenine portion of a molecule) into hypoxanthine (or the hypoxanthine portion of a molecule).

In some embodiments, the adenine-containing molecule is adenosine (A), and the hypoxanthine-containing molecule is inosine (I). The adenine-containing molecule can be deoxyribonucleic acid (DNA) or ribonucleic acid (RNA).

Adenosine deaminases include, but are not limited to, members of the enzyme family known as adenosine deaminases acting on RNA (ADAR), members of the enzyme family known as adenosine deaminases acting on tRNA (ADAT), and other family members containing adenosine deaminase domains (ADAD). According to the present disclosure, adenosine deaminases are capable of targeting adenine in RNA/DNA and RNA duplexes. In a specific embodiment, adenosine deaminases have been modified to increase their ability to edit DNA in RNA/DNA heteroduplexes of RNA duplexes.

In some embodiments, the deaminase is a cytidine deaminase. The term "cytidine deaminase" or "cytidine deaminase protein" refers to a protein, a polypeptide, or one or more functional domains of a protein or polypeptide that is capable of catalyzing a hydrolytic deamination reaction that converts cytosine (or the cytosine portion of a molecule) into uracil (or the uracil portion of a molecule). In some embodiments, the cytosine-containing molecule is cytidine (C), and the uracil-containing molecule is uridine (U). The cytosine-containing molecule can be deoxyribonucleic acid (DNA) or ribonucleic acid (RNA).

Cytidine deaminases include, but are not limited to, members of the enzyme family known as apolipoprotein B mRNA editing complex (APOBEC) family deaminases, activation-induced deaminases (AID), or cytidine deaminase 1 (CDA1). In a specific embodiment, an APOBEC family deaminase is included.

In some embodiments, the cytidine deaminase comprises a wild-type amino acid sequence of a cytidine deaminase. In some embodiments, the cytidine deaminase comprises one or more mutations in the cytidine deaminase sequence such that the editing efficiency and/or substrate editing preference of the cytidine deaminase is changed according to specific needs.

Identity

"Identity" is used to refer to the matching of sequences between two polypeptides or between two nucleic acids. "Identity" means the percentage of the number of identical residues between the polypeptide or nucleic acid sequences to the total number of residues, and the calculation of the total number of residues is determined based on the type of mutation. Mutation types include insertions (extensions) at either or both ends of the sequence, deletions (truncations) at either or both ends of the sequence, substitutions/alternations of one or more amino acids/nucleotides, insertions within the sequence, and deletions within the sequence.

Taking a polypeptide sequence as an example, if the mutation type is one or more of the following: substitution/replacement of one or more amino acids/nucleotides, insertion within the sequence, and deletion within the sequence, the total number of residues is calculated as the larger of the molecules being compared. If the mutation type also includes an insertion (extension) at either or both ends of the sequence or a deletion (truncation) at either or both ends of the sequence, the number of amino acids inserted or deleted at either or both ends (for example, the number of insertions or deletions at both ends is less than 20) is not included in the total number of residues. When calculating the percent identity, the sequences being compared are aligned in a manner that produces the maximum match between the sequences, and gaps in the alignment (if any) are resolved by a specific algorithm. The same applies to the calculation of nucleotide identity.

Carrier

A vector is a nucleic acid molecule that is capable of transporting another nucleic acid molecule to which it has been linked.

Vectors include, but are not limited to, single-stranded, double-stranded, or partially double-stranded nucleic acid molecules; nucleic acid molecules including one or more free ends, or no free ends (e.g., circular); nucleic acid molecules including DNA, RNA, or both; and other various polynucleotides known in the art. Vectors can be introduced into host cells by transformation, transduction, or transfection so that the genetic material elements they carry are expressed in the host cells. A vector can be introduced into a host cell to produce transcripts, proteins, or peptides, including proteins, fusion proteins, isolated nucleic acid molecules, etc. as described herein (e.g., CRISPR transcripts, such as nucleic acid transcripts, proteins, or enzymes). A vector can contain a variety of elements that control expression, including, but not limited to, promoter sequences, transcription start sequences, enhancer sequences, selection elements, and reporter genes. The vector may also contain a replication initiation site.

Vectors include plasmids and viral vectors, wherein the plasmid refers to a circular double-stranded DNA loop into which other DNA fragments can be inserted, for example, by standard molecular cloning techniques. Viral vectors, wherein virally derived DNA or RNA sequences are present in vectors for packaging viruses, and viruses include, for example, retroviruses, replication-defective retroviruses, adenoviruses, replication-defective adenoviruses, and adeno-associated viruses. Viral vectors also include polynucleotides carried by viruses for transfection into a host cell. Some vectors (for example, bacterial vectors and episomal mammalian vectors with bacterial replication origins) can replicate autonomously in the host cells into which they are introduced.

Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of the host cell after introduction into the host cell, and are replicated together with the host genome. Moreover, some vectors are capable of directing the expression of genes to which they are operably linked. Such vectors are referred to as "expression vectors."

In some embodiments, the vector (e.g., a viral vector or a non-viral vector, such as a lentiviral vector or a plasmid) can be delivered to the target tissue by, for example, intramuscular injection, intravenous administration, transdermal administration, intranasal administration, oral administration, or mucosal administration. The above delivery can be carried out via a single dose or multiple doses. It will be appreciated by those skilled in the art that the actual dose to be delivered herein can vary to a large extent according to a variety of factors, including but not limited to vector selection, target cells, organisms, tissues, the general condition of the subject to be treated, the degree of transformation/modification sought, the route of administration, the mode of administration, and the type of transformation/modification sought.

Regulatory elements

Herein, "regulatory elements" include promoters, enhancers, internal ribosome entry sites (IRES) and other expression control elements (e.g., transcription termination signals, such as polyadenylation signals, poly-U sequences), and their detailed descriptions can be found in Goeddel, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif (1990). In some cases, regulatory elements include those that direct constitutive expression of a nucleotide sequence in many types of host cells and those that direct the nucleotide sequence to be expressed only in certain host cells (e.g., tissue-specific regulatory sequences). Tissue-specific promoters may primarily direct expression in desired tissues of interest, such as muscle, neurons, bone, skin, blood, specific organs (e.g., liver, pancreas), or special cell types (e.g., lymphocytes). In other cases, regulatory elements may also direct expression in a timing-dependent manner (e.g., in a cell cycle-dependent or developmental stage-dependent manner), which may or may not be tissue- or cell-type-specific.

The term "promoter" refers to a non-coding nucleotide sequence that is located upstream of a gene and can initiate downstream gene expression. A constitutive promoter is a nucleotide sequence that, when operably linked to a polynucleotide that encodes or limits a gene product, will result in the production of a gene product in the cell under most or all physiological conditions of the cell. An inducible promoter refers to a promoter that selectively expresses a coding sequence or functional RNA in response to the presence of endogenous or exogenous stimuli, such as by responding to chemical compounds (chemical inducers), or to environmental, hormone, chemical, and/or developmental signals. Inducible or regulated promoters include promoters that are induced or regulated, for example, by light, heat, stress, flooding or drought, salt stress, osmotic stress, plant hormones, wounds, or chemicals (such as ethanol, abscisic acid (ABA), jasmonates, salicylic acid, or safeners).

Host cells

As used herein, "host cell" refers to a eukaryotic cell (e.g., an animal cell, a plant cell, a fungal cell, etc.), a prokaryotic cell (e.g., some microbial cells, Escherichia coli, Bacillus subtilis, etc.), or a cell from a multicellular organism cultured as a unicellular entity (e.g., a cell line), which serves as a recipient of nucleic acid (e.g., an expression vector) and includes the descendants of the original cell that has been genetically modified by the nucleic acid.

It is understood that the progeny of a single cell may not necessarily have completely the same morphology or genome, etc. as the original parent cell due to natural, accidental, or deliberate mutation. A "recombinant host cell" (also called a "genetically modified host cell") is a host cell into which a heterologous nucleic acid, such as an expression vector, has been introduced.

Those skilled in the art will appreciate that the design of the expression vector may depend on factors such as the choice of the host cell to be transformed, the level of expression desired, and the like.

In another aspect, the present invention also provides a host cell or its progeny, wherein the host cell comprises the aforementioned gene editing protein (Cas protein), or the aforementioned fusion protein, or the aforementioned polynucleotide, or the aforementioned vector system, or the aforementioned CRISPR-Cas system, or the aforementioned composition.

In one embodiment, the host cell comprises a non-human mammal, human, insect, bird, reptile, amphibian, rodent, fish, worm, nematode, or yeast cell.

In one aspect, the present invention also provides a multicellular organism, comprising the aforementioned cell or its progeny.

In one embodiment, the multicellular organism is an animal model or a plant model for a relevant disease.

NLS

NLS refers to "nuclear localization sequence" or "nuclear localization signal", which refers to an amino acid sequence that causes a protein to enter the cell nucleus. Nuclear localization sequences are known in the art (e.g., International PCT Application No. PCT/EP2000/011690 filed by Plank et al. on November 23, 2000 and published as WO/2001/038547), which is incorporated herein by reference for its disclosure of exemplary nuclear localization sequences. In other embodiments, the NLS is an optimized NLS, e.g., as described in Koblan et al., Nature Biotech. 2018 doi: 10.1038/nbt.4172.

Operatively connected

"Operably linked" means that the target nucleotide sequence is linked to the regulatory elements in a manner that allows the nucleotide sequence to be expressed (e.g., in an in vitro transcription/translation system or in a host cell when the vector is introduced into the host cell). Advantageous vectors include lentiviruses and adeno-associated viruses, and the type of these vectors can also be selected to target specific types of cells.

Complementarity

"Complementarity" refers to the ability of one nucleic acid sequence to form one or more hydrogen bonds with another nucleic acid sequence by means of traditional Watson-Crick or other non-traditional types. The percentage of complementarity represents the percentage of residues in one nucleic acid molecule that can form hydrogen bonds (e.g., Watson-Crick base pairing) with another nucleic acid sequence (e.g., 5, 6, 7, 8, 9, 10 out of 10 are complementary, then the percentage of complementarity is 50%, 60%, 70%, 80%, 90% and 100%). "Complete complementarity" means that all consecutive residues of one nucleic acid sequence form hydrogen bonds with the same number of consecutive residues in another nucleic acid sequence. "Substantially complementary" refers to a degree of complementarity of at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% over a region of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50 or more nucleotides, or to two nucleic acids that hybridize under stringent conditions.

The term "stringent conditions" in relation to hybridization refers to conditions under which a nucleic acid having complementarity to a target sequence predominantly hybridizes to the target sequence and substantially does not hybridize to non-target sequences. Stringent conditions are generally sequence-dependent and depend on many factors. In general, the longer the sequence, the higher the temperature at which the sequence specifically hybridizes to its target sequence.

"Hybridization" refers to a reaction in which one or more polynucleotides react to form a complex that is stabilized via hydrogen bonding of the bases between the nucleotide residues. The complex may comprise two strands forming a duplex, three or more strands forming a multi-stranded complex, a single self-hybridizing strand, or any combination of these. A hybridization reaction may constitute a step in a broader process such as the initiation of PCR, or cleavage of a polynucleotide by an enzyme. Sequences that are capable of hybridizing to a given sequence are referred to as the "complement" of the given sequence.

Hybridization of the target sequence with the gRNA means that at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of the nucleic acid sequences of the target sequence and the gRNA can hybridize to form a complex; or represents that at least 12, 15, 16, 17, 18, 19, 20 or more bases of the nucleic acid sequences of the target sequence and the gRNA can complementarily pair and hybridize to form a complex.

Express

Nucleic acid expression includes one or more of generation of an RNA template from a DNA sequence (e.g., transcription), processing of the RNA transcript (e.g., by splicing, editing, 5' cap formation, and/or 3' end processing), translation of the RNA into a polypeptide or protein, or post-translational modification of the polypeptide or protein.

deliver

"Delivery" refers to providing an entity (such as a drug) to a destination, for example, the components of the CRISPR-Cas system/composition of the present invention can be delivered in various forms, such as a combination of DNA/RNA or RNA/RNA or protein RNA. For example, a gene editing protein (Cas protein) can be delivered as a polynucleotide encoding DNA or a polynucleotide encoding RNA or as a protein.

In one aspect, the present invention also provides a delivery system, which comprises the gene editing protein (Cas protein) or the fusion protein, or the polynucleotide, or the CRISPR-Cas composition.

In one embodiment, the delivery system further comprises a delivery vehicle, and the delivery vehicle comprises nanoparticles, liposomes, exosomes, microbubbles, a gene gun or an electroporation device.

In addition, when the delivery object is a plant cell, a method such as cell penetrating peptide (CPP) delivery is also adopted. For example, in a specific embodiment, a gene editing protein (Cas protein) and/or at least one guide RNA is coupled to one or more CPPs, so as to effectively transport the CPP coupled with a gene editing protein (Cas protein) and/or a guide RNA into a plant cell (e.g., in a protoplast). CPP has a short peptide of less than 35 amino acids, which is derived from a protein or derived from a chimeric sequence, and can transport biomolecules across the cell membrane in a non-receptor-dependent manner. CPP can be a cationic peptide, a peptide with a hydrophobic sequence, an amphipathic peptide, a peptide rich in proline and an antimicrobial sequence, and a chimeric or dichotomous peptide. CPP can penetrate the biomembrane, and thus trigger different biomolecules to move across the cell membrane into the cytoplasm, and can improve their intracellular pathways, and thus promote the interaction between biomolecules and targets.

Exemplarily, CPPs include Tat (a nuclear transcription activating protein required for viral replication by HIV type 1), penetratin, Kaposi fibroblast growth factor (FGF) signal peptide sequence, integrin β3 signal peptide sequence, poly-arginine peptide Arg sequence, guanine-rich molecular transporter, sweet arrow peptide, etc.

Connectors

Herein, "linker" refers to a chemical group or molecule that connects two molecules or parts, such as two domains of a fusion protein, such as a gene editing protein (Cas protein) and a deaminase. In some connection modes, the linker is located between or flanking two groups, molecules or other parts, and connects the two by covalent bonds.

In some embodiments, the joint is a linear polypeptide formed by amino acids or multiple amino acid residues connected by peptide bonds. In some embodiments, the joint is an organic molecule, a group, a polymer or a chemical part. The length and type of the joint can be designed as needed. In some embodiments, the joint can select an artificially synthesized amino acid sequence or a naturally occurring peptide sequence.

Detection

In one aspect, the present invention also provides a method for targeting and editing a target nucleic acid, the method comprising contacting the target nucleic acid with any one of the aforementioned CRISPR-Cas systems or compositions.

In one aspect, the present invention also provides a method for non-specifically degrading single-stranded DNA after recognizing a target nucleic acid, the method comprising contacting the target nucleic acid with the aforementioned CRISPR-Cas composition.

In one aspect, the present invention also provides a method for targeting a non-spacer complementary strand of a double-stranded target nucleic acid and causing a nick therein after recognizing a spacer complementary strand of the double-stranded target nucleic acid, the method comprising contacting the double-stranded target nucleic acid with the aforementioned CRISPR-Cas system or composition.

In one aspect, the present invention also provides a method for targeting and cleaving a double-stranded target nucleic acid, the method comprising contacting the double-stranded target nucleic acid with the aforementioned CRISPR-Cas system or composition.

In one embodiment, the non-spacer sequence complementary strand of the double-stranded target nucleic acid is nicked before the spacer complementary strand of the double-stranded DNA is nicked.

In one aspect, the present invention also provides a method for specifically editing a double-stranded nucleic acid, the method comprising contacting the following under sufficient conditions and for a sufficient amount of time,

(1) the aforementioned gene editing protein (Cas protein), or fusion protein, another enzyme with sequence-specific nicking activity, and the guide RNA, wherein the guide RNA guides the gene editing protein (Cas protein) or the fusion protein to produce a nick on the relative strand relative to the activity of the other sequence-specific nicking enzyme; and (2) the double-stranded nucleic acid; the method results in the formation of a double-strand break.

In one aspect, the invention also provides a method of editing a double-stranded nucleic acid, the method comprising contacting the following under sufficient conditions and for a sufficient amount of time:

(1) the aforementioned gene editing protein (Cas protein), or a fusion protein, and a fusion protein of a protein domain having DNA modification activity, and the RNA guide targeting the double-stranded nucleic acid; and (2) the double-stranded nucleic acid;

The gene editing protein (Cas protein) of the fusion protein is modified to produce a nick in the non-target strand of the double-stranded nucleic acid.

In one embodiment, the two strands of the double-stranded nucleic acid are cleaved at different sites, resulting in staggered cleavage.

In one embodiment, both strands of the double-stranded nucleic acid are cleaved at the same site, resulting in a blunt double-strand break.

In one aspect, the present invention also provides a method for targeting and cleaving a single-stranded target nucleic acid, the method comprising contacting the target nucleic acid with any of the aforementioned CRISPR-Cas compositions.

In one aspect, the present invention also provides a method for inducing a change in a cell state, the method comprising contacting the aforementioned CRISPR-Cas composition with the target nucleic acid in a cell.

In one embodiment, the cell state comprises apoptosis or dormancy;

In one embodiment, the cell comprises a eukaryotic cell or a prokaryotic cell;

In one embodiment, the cell comprises a mammalian cell or a plant pathogenic cell;

In one embodiment, the cell comprises a cancer cell;

In one embodiment, the cell comprises an infectious cell or a cell infected by an infectious agent;

In one embodiment, the cells include virus-infected cells, prion-infected cells;

In one embodiment, the cell comprises a fungal cell, a protozoan, or a parasite cell.

In one aspect, the present invention also provides a method for detecting a target nucleic acid in a sample, the method comprising contacting the sample with the aforementioned gene editing protein (Cas protein), guide RNA and non-target sequence; detecting a detectable signal generated by the gene editing protein (Cas protein) cutting the non-target sequence, thereby detecting the target nucleic acid; the non-target sequence does not hybridize with the guide RNA.

Reagent test kit

In one aspect, the present invention provides a kit, comprising the aforementioned gene editing protein (Cas protein), the aforementioned fusion protein, the aforementioned polynucleotide, the aforementioned CRISPR-Cas composition, and the use of the aforementioned host cell in preparing a kit, wherein the components of the kit are in the same or different containers.

In one aspect, the present invention also provides a container, comprising the aforementioned kit.

In one embodiment, the container comprises a sterile container;

In one embodiment, the container comprises a syringe.

In some embodiments, the kit also includes instructions for using the kit, such as instructions in more than one language. The kit may also include one or more reagents for use in the process of utilizing one or more of the above components. The reagents may be provided in any suitable container. For example, the kit may provide one or more reaction or storage buffers. The above reagents may be provided in a form (e.g., in a concentrated or lyophilized form) requiring the addition of one or more other components before use; the buffer may be any buffer, including but not limited to sodium carbonate buffer, sodium bicarbonate buffer, borate buffer, Tris buffer, MOPS buffer, HEPES buffer, and combinations thereof. The buffer may have a suitable pH value, for example, may be alkaline. In some embodiments, the pH of the buffer is between about 7-10.

treat

"Treatment" refers to treating or curing a subject's condition, delaying the onset of symptoms of a condition, and/or delaying the severity of a condition. The term "subject" includes, but is not limited to, various animals, plants, and microorganisms. Animals include mammals, such as bovines, equines, ovines, swine, canines, felines, lagomorphs, rodents (e.g., mice or rats), non-human primates (e.g., macaques or cynomolgus monkeys), or humans. In certain embodiments, the subject (e.g., a human) suffers from a condition (e.g., a condition caused by a disease-related genetic defect). "Plant" is any differentiated multicellular organism capable of photosynthesis, including crop plants at any maturity or developmental stage.

In one aspect, the present invention also provides the use of the aforementioned gene editing protein (Cas protein), the aforementioned fusion protein, the aforementioned polynucleotide, the aforementioned CRISPR-Cas composition, and the aforementioned host cell in the preparation of a drug for treating a condition or disease in a subject in need thereof.

In one embodiment, the use comprises administering the CRISPR-Cas composition to the subject or to an ex vivo cell of the subject;

In one embodiment, the spacer sequence is complementary to at least 15 nucleotides of the target nucleic acid associated with the condition or disease, and the Cas protein or the fusion protein cleaves the target nucleic acid;

In one embodiment, the condition or disease comprises cancer or an infectious disease;

In one embodiment, the cancer comprises one or more of Wilms tumor, Ewing sarcoma, neuroendocrine tumor, glioblastoma, neuroblastoma, melanoma, skin cancer, breast cancer, colon cancer, rectal cancer, prostate cancer, liver cancer, kidney cancer, pancreatic cancer, lung cancer, biliary tract cancer, cervical cancer, endometrial cancer, esophageal cancer, gastric cancer, head and neck cancer, medullary thyroid cancer, ovarian cancer, glioma, lymphoma, leukemia, myeloma, acute lymphocytic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia, Hodgkin's lymphoma, non-Hodgkin's lymphoma, or urinary bladder cancer;

In one embodiment, the condition or disease comprises one or more of cystic fibrosis, atherosclerotic cardiovascular disease (ASCVD), pseudohypertrophic muscular dystrophy, Becker muscular dystrophy, alpha-1-antitrypsin deficiency, Pompe disease, myotonic dystrophy, Huntington disease, fragile X syndrome, Friedreich's ataxia, amyotrophic lateral sclerosis, frontotemporal dementia, hereditary chronic kidney disease, hyperlipidemia, Leber's congenital amaurosis, sickle cell disease, primary hyperoxaluria (PH1), hypercholesterolemia (FH), hereditary angioedema (HAE), retinal disease, macular degeneration, transthyretin amyloidosis, or beta thalassemia;

In one embodiment, the infectious agent of the infectious disease includes one or more of human immunodeficiency virus, herpes simplex virus-1, hepatitis B or herpes simplex virus-2.

The main advantages of the present invention include:

(a) The present invention discovers a new gene editing protein (Cas protein) for the first time. The gene editing protein (Cas protein) of the present invention has very good gene editing activity, can effectively edit or cut the target gene, and can effectively treat the symptoms or diseases of subjects in need (for example, one or more of cystic fibrosis, pseudohypertrophic muscular dystrophy, Becker muscular dystrophy, alpha-1-antitrypsin deficiency, Pompe disease, myotonic dystrophy, Huntington's disease, fragile X syndrome, Friedreich's ataxia, amyotrophic lateral sclerosis, frontotemporal dementia, hereditary chronic kidney disease, hyperlipidemia, Leber's congenital amaurosis, sickle cell disease, hypercholesterolemia, transthyretin amyloidosis or beta-thalassemia).

(b) The present invention has discovered a new Cas protein, which has a lower homology with the reported Cas enzymes and exhibits excellent DNA nuclease activity compared to the Cas enzymes in the prior art, and has broad application prospects.

The present invention is further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not used to limit the scope of the present invention. The experimental methods in the following examples where specific conditions are not specified are usually carried out under conventional conditions, such as the conditions described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and weight parts.

Unless otherwise specified, the reagents and materials in the examples of the present invention are all commercially available products.

Example 1 Identification of new Cas proteins

First, the inventors mined the metagenomic data using a computer program and analyzed the metagenomics of uncultured plants. Through redundancy removal and protein clustering analysis, a new Cas protease was identified and named CasW1. Its amino acid sequence is shown in SEQ ID No. 1 and its nucleotide coding sequence is shown in SEQ ID No. 2. After sequence alignment, CasW1 was identified as a member of the Cas12 family.

By analyzing, predicting and screening the metagenome, proteins and related elements related to the novel CRISPR-Cas system were obtained. The CRISPR-Cas effector protein of the present invention was compared with existing effector proteins, and it was found that it had a low similarity with known Cas proteins.

Through analysis, it was found that the PAM corresponding to the Cas protein obtained in the present invention is 5’-TTTN, where N represents A/T/G/C. The CRISPR locus of the sample containing CasW1 was annotated by PILER-CR, and the corresponding DNA encoding the direct repeat (DR) sequence was obtained as shown in SEQ ID No. 3, and its secondary structure schematic diagram is shown in Figure 10.

Example 2 In vitro enzyme cleavage experiments verified the cleavage activity of CasW1.

To verify whether CasW1 is a double-stranded DNA nuclease activity, the inventors verified its cutting activity.

1. Construct the expression vector of CasW1.

The above-mentioned nucleotide sequence fragment encoding CasW1 was synthesized and cloned into the prokaryotic protein expression vector pET-28a(+) (Biolabio, #QN1060) by restriction endonuclease digestion and T4 DNA ligase ligation to obtain a ligation product, as shown in Figure 6.

The inventors performed PCR on the obtained CasW1 fragment, and used EcoRI/NotI double digestion, and at the same time performed EcoRI/NotI double digestion on the prokaryotic protein expression vector pET-28a(+). Afterwards, the CasW1 fragment double digestion product and the prokaryotic protein expression vector pET-28a(+) double digestion product were subjected to agarose gel electrophoresis, and the results are shown in Figure 1, showing that the digestion product CasW1 nucleotide fragment of the correct size and the pET-28a(+) vector fragment with 11bp in the middle of the EcoRI/NotI digestion site were obtained.

The inventors then transformed the ligation product into E. coli competent cells DH5a, and then inoculated the competent cells DH5a on a LB solid plate with kanamycin resistance. After inverted culture in a 37°C incubator overnight, a single colony was picked for Sanger sequencing. The sequence results showed that the plasmid clone with the correct sequence was subjected to plasmid extraction, and the pET-28a(+)-CasW1 expression vector was obtained.

2. In vitro purification of CasW1 protein.

The experimental steps are as follows:

(1) Transformation: Take out a tube of competent Escherichia coli BL21 (DE3) (Shanghai Weidi Biotechnology Co., Ltd., EC1002) from the -80℃ refrigerator and place it on ice to dissolve for 5 minutes, then pipette 1ng pET-28a(+)-CasW1 expression vector plasmid into the competent cells, flick the bottom of the tube with your finger to mix, and let it stand on ice for 25 minutes. Heat shock in a 45℃ water bath for 45 seconds, quickly put it back on ice and let it stand for 2 minutes, add 700ul of antibiotic-free LB medium to the centrifuge tube, and resuscitate at 37℃, 220rpm for 60 minutes. After the recovery is complete, centrifuge at 5000rpm for 1 minute to collect the bacteria, discard 600μl of the supernatant, then gently mix the remaining liquid and competent cells and spread them on the LB plate, and use glass beads to coat the plate.

(2) Induction: At 9 am the next day, pick a single clone from the transformed LB plate and culture it in 3 ml LB liquid culture medium containing kanamycin (10 mg/ml), then place it on a shaker and culture it at 37°C and 220 rpm for 8 hours. At 5 pm, transfer the bacteria in 3 ml LB to 300 ml 2x YT medium containing kanamycin, add 150 ul IPTG (final concentration 0.5 mM) to the bacterial solution, and induce it overnight at 16°C for 13-14 hours.

(3) Harvesting bacteria: The bacterial solution obtained after induction was centrifuged at 4500 rpm and 4°C for 10 min, and the supernatant was discarded. The bacteria were resuspended in a 50 ml centrifuge tube with 15 ml of imidazole (concentration of 10 mM) (imidazole was added for competitive elution of CasW1 protein), and then 150 μl of PMSF protease inhibitor was added (to inhibit protein degradation and improve yield).

(4) Ultrasound: Fix a 50 ml centrifuge tube vertically in a beaker filled with ice water, and adjust the position so that the ultrasonic probe is below the bacterial liquid surface. Ultrasound mode: work for 3 seconds, rest for 12 seconds, 200w, 60 times. After the end of ultrasound, add 150 μl of PMSF protease inhibitor, and then centrifuge at 11000 rpm and 4°C for 25 minutes.

(5) Beads pretreatment: Pipette 300 μl Ni-NTA Agarose beads (QIAGEN, #30230) into a 15 ml centrifuge tube, add 10 ml PBS, rotate at room temperature for 5 min, centrifuge at 1000 g for 2 min, and 4 °C, remove the supernatant with a rubber pipette, and repeat the washing with PBS. Then add 10 ml imidazole (concentration of 10 mM), rotate at room temperature for 5 min, centrifuge at 1000 g for 2 min, and 4 °C, carefully remove the supernatant with a rubber pipette, and place the 15 ml centrifuge tube containing the washed beads on ice for later use.

(6) Transfer all the supernatant of the bacterial solution obtained after centrifugation in step (4) into the centrifuge tube containing Ni-NTA Agarose beads obtained in step (5), and incubate with rotation at 4°C for 1 hour.

(7) Wash: Ni-NTA Agarose beads were eluted twice with 10 ml imidazole (40 mM concentration). After adding imidazole each time, rotate at 4°C for 5 min, and then centrifuge at 1000 × g for 2 min at 4°C. Resuspend Ni-NTA Agarose beads with 500 μl imidazole (250 mM concentration), then transfer to a pre-cooled affinity chromatography column (MedChemExpress, #HY-K0221), equilibrate for 5 min, and collect the protein fraction eluted with 250 mM imidazole in a 1.5 ml centrifuge tube. Repeat the above elution steps three times.

(8) After measuring the protein concentration of each tube with NanoDrop OD280, the protein solution was collected together, and the protein was replaced into PBS buffer through a 30 kd ultrafiltration tube. Glycerol with a final concentration of 10% was added, and the aliquots were quickly frozen in liquid nitrogen and stored in a -80 °C refrigerator. SDS-PAGE polyacrylamide gel electrophoresis was used to identify the protein size and purity. As shown in Figure 2, the size of the CasW1 protein was about 130 kd, indicating that a well-purified CasW1 protein had been obtained.

3. In vitro cleavage verification

3.1 Preparation of in vitro cleaved dsDNA template.

Using the HepG2 cell (ATCC, catalog number HB-8065) genome as a template, forward and reverse primers were prepared based on the hHPRT1 gene (Genebank, NG_012329.2) dsDNA template. The upstream and downstream primers are as follows:

hHPRT1-dsDNA-F:gtagtgtcaactcattgctg(SEQ ID NO.5);

hHPRT1-dsDNA-R: gtcaagggcatatcctacaa (SEQ ID NO. 6).

Taq enzyme was used for PCR amplification, and the reaction system was as follows:

1 μl of genomic DNA (as template) (total amount 100 ng), 10 μl of 2×Taq PCR mix, 0.5 μl of upstream and downstream primers respectively, and ddH ₂ O was added to make up the total volume to 20 μl.

The PCR reaction program was as follows: 95°C for 5 min; 94°C for 30 s, 55°C for 30 s, 72°C for 20 s, 35 cycles; 72°C for 10 min; and insulation at 12°C.

The PCR reaction solution was then subjected to agarose gel electrophoresis, and the electrophoresis results are shown in Figure 3. Then, an agarose gel DNA recovery kit (TIANGEN, DP219-02) was used for gel recovery, and finally, enzyme-free water was used for elution to obtain an in vitro cleaved dsDNA template.

3.2 In vitro enzyme digestion reaction.

To detect the cutting activity of CasW1, the inventors designed two groups of comparative experiments to compare their cutting activities. The first group was a comparison of the cutting activities of CasW1 and LbCpf1, and the second group was a comparison of the cutting activities of CasW1 with HED Cas 12i.16 and S7R-Cas12i.3.

Among them, the amino acid sequence of LbCpf1 protein is shown in SEQ ID NO.12, and the nucleotide coding sequence is shown in SEQ ID NO.11; the amino acid sequence of HED Cas12i.16 protein is shown in SEQ ID NO.10, and the nucleotide sequence is shown in SEQ ID NO.9; the amino acid sequence of S7R-Cas12i.3 is shown in SEQ ID NO.8, and the nucleotide sequence is shown in SEQ ID NO.7.

The method of step 1 in Example 2 was used to construct LbCpf1, HED Cas12i.16, and S7R-Cas 12i.3 expression vectors, respectively. The maps of the recombinant expression vectors are shown in Figures 7, 8, and 9.

3.2.1 Comparison of cleavage activity of CasW1 and LbCpf1

The specific steps are as follows:

(1) Prepare CasW1-crRNA and LbCpf1-crRNA respectively.

A targeting sequence (spacer) was designed based on the hHPRT1 gene and named hHPRT1-spacer: GGTTAAAGATGGTTAAATGAT (SEQ ID NO.4).

According to the DR sequences of CasW1 and LbCpf1, the crRNA sequences of the above Cas proteins were designed and named CasW1-hHPRT1-crRNA and LbCpf1-hHPRT1-crRNA, respectively, as follows:

CasW1-hHPRT1-crRNA:

GTCTAAATGACCTATAAATTTCTACTATGTGTAGAT GGTTAAAGATGGTTAAATGAT (SEQ ID NO. 13), wherein the underlined sequence is the DR sequence of CasW1;

LbCpf1-hHPRT1-crRNA:

TAATTTCTACTAAGTGTAGAT GGTTAAAGATGGTTAAATGAT (SEQ ID NO. 14), wherein the underlined sequence is the DR sequence of LbCpf1;

CasW1-hHPRT1-crRNA and LbCpf1-hHPRT1-crRNA sequence fragments were chemically synthesized (by Nanjing GenScript Biotechnology Co., Ltd.) respectively. Then, a mixed solution of CasW1 and CasW1-hHPRT1-crRNA was prepared, and the components of the mixed solution were as follows:

20 μl of enzyme-free H ₂ O, 3 μl of NEBuffer r2.1 (10×, NEB, #B6002S), 3 μl of crRNA (CasW1-hHPRT1-crRNA or LbCpf1-hHPRT1-crRNA) (concentration: 30 nM), 1 μl of Cas protein (CasW1 or LbCpf1) (concentration: 30 nM), and the total volume of the reaction system was 27 μl.

The mixed solution of LbCpf1 and LbCpf1-hHPRT1-crRNA was prepared in the same manner.

Then, the mixed solution of CasW1 and CasW1-hHPRT1-crRNA, and the mixed solution of LbCpf1 and LbCpf1-hHPRT1-crRNA were placed in a PCR instrument respectively and reacted at 25°C for 10 minutes.

(2) Add 3uL 60nM dsDNA solution (final concentration is 6nM) to the mixed solution of CasW1 and CasW1-hHPRT1-crRNA and the mixed solution of LbCpf1 and LbCpf1-hHPRT1-crRNA respectively. The total reaction system is 30μl. Mix thoroughly, centrifuge briefly, and incubate in a PCR instrument at 37°C for 10 minutes.

(3) Add 1uL Proteinase K to the reaction system in the previous step, mix thoroughly, centrifuge, and incubate at room temperature for 10 minutes to digest the protein components in the reaction system;

(4) Use magnetic beads to purify the DNA fragments after enzyme digestion reaction.

Take out the DNA sorting magnetic bead solution (Novozyme, catalog number N411-02) from the 4°C refrigerator 20 minutes in advance and equilibrate to room temperature. Mix the magnetic beads by turning them upside down, draw 3 times the volume (about 150 μl) of the magnetic bead solution and add it to the dsDNA cleavage product obtained in step (3), and use a pipette to gently blow 10 times to mix, incubate at room temperature for 10 minutes to allow the dsDNA cleavage product to bind to the magnetic beads. Place the PCR tube containing the dsDNA cleavage product sample solution on the magnetic rack, and carefully remove the supernatant after the solution is clarified. Keep the aforementioned PCR tube on the magnetic rack at all times, add 200 μl of freshly prepared 80% ethanol to rinse the magnetic beads, incubate at room temperature for 30 seconds, and carefully remove the supernatant. Repeat the rinse once. Open the lid and dry the magnetic beads at room temperature for 5 minutes, remove the PCR tube from the magnetic rack, add 15 μl of enzyme-free water, vortex or use a pipette to mix thoroughly, and let it stand at room temperature for 2 minutes. Then place the PCR tube on the magnetic rack and let it stand for 5 minutes. After the solution is clarified, carefully pipette the supernatant into a new nuclease-free PCR tube.

(5) The dsDNA cleavage products were then analyzed using a portable bioanalyzer (Houze Biotechnology, Qsep1), and the enzyme cleavage effect was detected by referring to the S1 high-resolution cartridge (Houze Biotechnology, C105102) detection scheme. The analysis results are shown in Figure 4. The Smear analysis option provided by Qsep1 showed that the cleavage activity of CasW1 was 99.5%, and the cleavage activity of LbCpf1 was 95%. The cleavage activity of CasW1 was higher than that of LbCpf1.

3.2.1 Comparison of cleavage activity of CasW1, HED Cas12i.16, and S7R-Cas12i3

The specific steps are as follows:

(1) Prepare crRNAs of CasW1, HED Cas12i.16 and S7R-Cas12i3 respectively.

The hHPRT1-spacer sequence fragment was synthesized, and the crRNA sequences of the above Cas proteins were designed according to the DR sequences of CasW1, HED Cas12i.16 and S7R-Cas12i3, and named as CasW1-hHPRT1-crRNA, HED Cas12i.16-hHPRT1-crRNA and S7R-Cas12i3-hHPRT1-crRNA, respectively. The specific sequences are as follows:

CasW1-hHPRT1-crRNA:

GTCTAAATGACCTATAAATTTCTACTATGTGTAGAT GGTTAAAGATGGTTAAATGAT (SEQ ID NO. 13), wherein the underlined sequence is the DR sequence of CasW1;

HED Cas12i.16-hHPRT1-crRNA：

CTAGCAATGACTCAGAAATGTGTCCCCAGTTGACAC GGTTAAAGATGGTTAAATGAT (SEQ ID NO. 15), wherein the underlined sequence is the DR sequence of HED Cas12i.16;

S7R-Cas12i3-hHPRT1-crRNA:

AGAGAATGTGTGCATAGTCACAC GGTTAAAGATGGTTAAATGAT (SEQ ID NO. 16), wherein the underlined sequence is the DR sequence of S7R-Cas12i3;

CasW1-hHPRT1-crRNA, HED Cas12i.16-hHPRT1-crRNA and S7R-Cas12i3-hHPRT1-crRNA sequence fragments were chemically synthesized (by Nanjing GenScript Biotech Co., Ltd.), respectively.

(2) Referring to the cutting activity comparison experiment in step 3.2.1, in vitro enzyme cleavage experiments were performed with CasW1, HED Cas12i.16 and S7R-Cas12i3 respectively, and finally the DNA fragments after the enzyme cleavage reaction were purified using magnetic beads.

(3) The dsDNA cleavage products were then analyzed using a portable bioanalyzer (Houze Bio, Qsep1), and the enzyme cleavage effect was detected by referring to the S1 high-resolution card clamp (Houze Bio, C105102) detection scheme. The analysis results are shown in Figure 5. The Smear analysis option provided by Qsep1 showed that the cleavage activity of CasW1 was 100%, the cleavage activity of HED Cas12i.16 was 14%, and the cleavage activity of S7R-Cas12i3 was 58%. The cleavage activity of CasW1 was much higher than that of HED Cas12i.16 and S7R-Cas12i3.

Sequence information:

All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

Claims (22)

A gene editing protein, characterized in that the protein is selected from the following group: (a) a polypeptide having an amino acid sequence as shown in SEQ ID NO: 1; (b) a polypeptide having ≥80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5% homology (or identity) with the amino acid sequence of SEQ ID NO:1, and the polypeptide has the biological function of SEQ ID NO:1; (c) A derivative polypeptide formed by replacing, deleting or adding one or more (preferably 1-20, more preferably 1-10, and more preferably 1-5) amino acid residues of any amino acid sequence shown in SEQ ID NO:1, and retaining the biological function of SEQ ID NO:1. A fusion protein, characterized in that it comprises the gene editing protein according to claim 1; and one or more functional domains. An isolated polynucleotide, characterized in that the polynucleotide encodes the gene editing protein according to claim 1 or the fusion protein according to claim 2. An isolated nucleic acid molecule, characterized in that it comprises a sequence selected from the following, or consists of a sequence selected from the following: (i) the sequence shown in SEQ ID NO: 3; (ii) a sequence having one or more base substitutions, deletions or additions (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 base substitutions, deletions or additions) compared to the sequence shown in SEQ ID NO: 3; (iii) a sequence having at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% sequence identity to the sequence set forth in SEQ ID NO: 3; (iv) a sequence that hybridizes to the sequence described in any one of (i) to (iii) under stringent conditions; or (v) a complementary sequence of the sequence described in any one of (i) to (iii); Furthermore, the sequence described in any one of (ii) to (v) substantially retains the biological function of the sequence from which it is derived; For example, the isolated nucleic acid molecule is RNA; For example, the isolated nucleic acid molecule comprises a direct repeat sequence in a CRISPR/Cas system. A guide RNA (gRNA), characterized in that the guide RNA includes a direct repeat (DR) sequence capable of binding to the gene editing protein described in claim 1 and a spacer sequence capable of targeting a target sequence. A composite, characterized in that it comprises: (i) a protein component selected from the group consisting of the gene editing protein of claim 1, the fusion protein of claim 2, or a combination thereof; and (ii) a nucleic acid component selected from the group consisting of the guide RNA of claim 5, a nucleic acid encoding the guide RNA of claim 5, a precursor RNA of the guide RNA of claim 5, a precursor RNA nucleic acid encoding the guide RNA of claim 5, or a combination thereof; Wherein, the protein component and the nucleic acid component are combined with each other to form a complex. A vector, characterized in that it comprises the polynucleotide according to claim 3 or the nucleic acid molecule according to claim 4. A CRISPR-Cas composition, comprising: (i) a first component selected from the group consisting of the gene editing protein of claim 1, the fusion protein of claim 2, a nucleotide sequence encoding the gene editing protein of claim 1 or the fusion protein of claim 2, and any combination thereof; and (ii) a second component, which comprises one or more guide RNAs according to claim 5, or a nucleotide sequence encoding the one or more guide RNAs according to claim 5; The guide RNA is capable of forming a complex with the protein, protein variant or fusion protein described in (i). A CRISPR-Cas system, characterized in that it comprises one or more vectors, wherein the one or more vectors comprise: (i) a first nucleic acid, which is a nucleotide sequence encoding the gene editing protein of claim 1 or the fusion protein of claim 2; optionally, the first nucleic acid is operably linked to a first regulatory element; and (ii) a second nucleic acid encoding a nucleotide sequence comprising the guide RNA of claim 5; optionally, the second nucleic acid is operably linked to a second regulatory element; in: The first nucleic acid and the second nucleic acid are present on the same or different vectors; The guide RNA is capable of forming a complex with the protein or fusion protein described in (i). A kit, characterized in that it comprises one or more components selected from the following: the gene editing protein of claim 1, the fusion protein of claim 2, the polynucleotide of claim 3, the complex of claim 6, the vector of claim 7, the CRISPR-Cas composition of claim 8, or the system of claim 9. A delivery composition, characterized in that it comprises a delivery vector and one or more selected from the following: the gene editing protein of claim 1, the fusion protein of claim 2, the polynucleotide of claim 3, the complex of claim 6, the vector of claim 7, the CRISPR-Cas composition of claim 8, or the system of claim 9. A host cell, characterized in that it comprises the gene editing protein of claim 1, the fusion protein of claim 2, the polynucleotide of claim 3, the nucleic acid molecule of claim 4, the complex of claim 6, the vector of claim 7, the CRISPR-Cas composition of claim 8, the system of claim 9, or the delivery composition of claim 11. An enzyme preparation, characterized in that the enzyme preparation comprises the gene editing protein of claim 1, the fusion protein of claim 2, the complex of claim 6, the CRISPR-Cas composition of claim 8, the system of claim 9, or the delivery composition of claim 11. A medicine box, characterized in that it comprises: A first container, and the complex according to claim 6, the composition according to claim 8, or the system according to claim 9, or a drug containing the complex according to claim 6, the composition according to claim 8, or the system according to claim 9 located in the first container. A medicine box, characterized in that it comprises: (a1) a first container, and the gene editing protein according to claim 1, or the fusion protein according to claim 2, or the gene encoding it or its expression vector, or a drug containing the gene editing protein according to claim 1, or the fusion protein according to claim 2, or the gene encoding it or its expression vector, located in the first container; (b1) an optional second container, and the guide according to claim 5 located in the second container RNA or its expression vector, or a drug containing the guide RNA or its expression vector according to claim 5. A method for targeting and editing a target gene or cutting a target gene, characterized in that the gene editing protein according to claim 1, or the fusion protein according to claim 2, or the complex according to claim 6, or the composition according to claim 8, or the system according to claim 9, or the delivery composition according to claim 11, or the enzyme preparation according to claim 13, or the drug kit according to claim 14 or 15 is contacted with the target gene, or delivered to a cell containing the target gene, and the target sequence is present in the target gene. A method for inducing a change in a cell state, characterized in that the method comprises contacting the gene editing protein of claim 1, or the fusion protein of claim 2, or the complex of claim 6, or the composition of claim 8, or the system of claim 9, or the delivery composition of claim 11, or the enzyme preparation of claim 13, or the drug kit of claim 14 or 15 with a target gene in a cell. A method for changing the expression of a gene product, comprising: contacting the gene editing protein of claim 1, or the fusion protein of claim 2, or the complex of claim 6, or the composition of claim 8, or the system of claim 9, or the delivery composition of claim 11, or the enzyme preparation of claim 13, or the drug kit of claim 14 or 15 with a nucleic acid molecule encoding the gene product, or delivering it to a cell containing the nucleic acid molecule, wherein the target sequence is present in the nucleic acid molecule. An in vitro, ex vivo or in vivo cell or cell line or their progeny, characterized in that the cell or cell line or their progeny comprises: the gene editing protein of claim 1, or the fusion protein of claim 2, or the polynucleotide of claim 3, or the complex of claim 6, or the vector of claim 7, or the composition of claim 8, or the system of claim 9, or the delivery composition of claim 11. Use of the gene editing protein of claim 1, or the fusion protein of claim 2, or the polynucleotide of claim 3, or the nucleic acid molecule of claim 4, or the complex of claim 6, or the vector of claim 7, or the composition of claim 8, or the system of claim 9, or the kit of claim 10, the delivery composition of claim 11, or the enzyme preparation of claim 13, or the kit of claim 14 or 15, characterized in that it is used for the preparation of a drug or a preparation for nucleic acid editing (e.g., gene or genome editing). Use of the gene editing protein of claim 1, or the fusion protein of claim 2, or the polynucleotide of claim 3, or the complex of claim 6, or the vector of claim 7, or the composition of claim 8, or the system of claim 9, or the kit of claim 10, or the delivery composition of claim 11, or the enzyme preparation of claim 13, or the kit of claim 14 or 15, characterized in that it is used for preparing a drug or a preparation, wherein the drug or preparation is used for one or more selected from the following group: (i) ex vivo gene or genome editing; (ii) Detection of single-stranded DNA in vitro; (iii) editing a target sequence in a target locus to modify an organism or non-human organism; (iv) treating a disorder caused by a defect in the target sequence in the target locus; (v) treating a condition or disease in a subject in need thereof. A method for detecting whether a target nucleic acid molecule is present in a sample, characterized in that the method comprises: mixing the sample with the gene editing protein according to claim 1, or the fusion protein according to claim 2, Or the complex of claim 6, the composition of claim 8, the system of claim 9, the kit of claim 10, the delivery composition of claim 11, or the enzyme preparation of claim 13, contacting the non-target sequence with the non-target sequence, detecting the detectable signal generated by the cleavage of the non-target sequence, thereby detecting the target nucleic acid molecule, wherein the non-target sequence does not hybridize with the guide RNA.