CN116716277A

CN116716277A - Cas enzyme and application thereof

Info

Publication number: CN116716277A
Application number: CN202310823036.9A
Authority: CN
Inventors: 李珊珊; 赵庆芝; 刘锐恒
Original assignee: Shandong Shunfeng Biotechnology Co Ltd
Current assignee: Shandong Shunfeng Biotechnology Co Ltd
Priority date: 2022-07-07
Filing date: 2023-07-06
Publication date: 2023-09-08
Also published as: WO2024008145A1

Abstract

The invention belongs to the field of nucleic acid editing, in particular to the technical field of regularly clustered interval short palindromic repeat (CRISPR). Specifically, the invention provides a novel Cas enzyme, which belongs to a novel Cas protein and has wide application prospect.

Description

Cas enzyme and application thereof

Technical Field

The invention relates to the field of gene editing, in particular to the technical field of regular clustered interval short palindromic repeat (CRISPR). Specifically, the invention screens out a novel class of Cas enzymes, and develops a corresponding gene editing tool and application thereof based on the novel Cas enzymes.

Background

CRISPR/Cas technology is a widely used gene editing technology that uses RNA-guided specific binding of target sequences on the genome and cleavage of DNA to create double strand breaks, site-directed gene editing using biological non-homologous end joining or homologous recombination.

The CRISPR/Cas9 system is the most commonly used type II CRISPR system that recognizes the PAM motif of 3' -NGG and blunt-ends the target sequence. The CRISPR/Cas Type V system is a newly discovered class of CRISPR systems that have a 5' -TTN motif that performs cohesive end cleavage of a target sequence, e.g., cpf1, C2C1, casX, casY. However, the different CRISPR/Cas currently in existence each have different advantages and disadvantages. For example, cas9, C2C1 and CasX each require two RNAs for guide RNAs, whereas Cpf1 requires only one guide RNA and can be used for multiplex gene editing. CasX has a size of 980 amino acids, whereas common Cas9, C2C1, casY and Cpf1 are typically around 1300 amino acids in size. In addition, PAM sequences of Cas9, cpf1, casX, casY are all relatively complex and diverse, while C2C1 recognizes the stringent 5' -TTN, so its target site is easily predicted compared to other systems, thereby reducing potential off-target effects.

In summary, given that the currently available CRISPR/Cas systems are limited by several drawbacks, the development of a more robust new CRISPR/Cas system with versatile good performance is of great importance for the development of biotechnology.

Disclosure of Invention

The inventors of the present application have unexpectedly discovered a novel endonuclease (Cas enzyme) through a number of experiments and repeated fumbling. Based on this finding, the present inventors developed a new CRISPR/Cas system and a gene editing method and a nucleic acid detection method based on the same.

Cas effector proteins

In one aspect, the application provides a Cas protein, which is an effector protein in a CRISPR/Cas system, and in the application, is called Cas-sf2201, cas-sf4274, cas-sf2771 and Cas-sf2586, and the amino acid sequences of the proteins are respectively shown in SEQ ID nos. 1 to 4.

In one embodiment, the Cas protein amino acid sequence has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% sequence identity compared to any of SEQ ID nos. 1-4, and substantially retains the biological function of the sequence from which it is derived. Preferably, the Cas protein is derived from the same species as Cas-sf2201, cas-sf4274, cas-sf2771 or Cas-sf 2586.

In one embodiment, the Cas protein amino acid sequence has a sequence with one or more amino acid substitutions, deletions, or additions as compared to any of SEQ ID nos. 1-4; and substantially retains the biological function of the sequence from which it originates; the substitution, deletion or addition of one or more amino acids includes substitution, deletion or addition of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids. Preferably, the Cas protein is derived from the same species as Cas-sf2201, cas-sf4274, cas-sf2771 or Cas-sf 2586.

It will be apparent to those skilled in the art that the structure of a protein may be altered without adversely affecting its activity and functionality, for example, one or more conservative amino acid substitutions may be introduced into the amino acid sequence of the protein without adversely affecting the activity and/or three-dimensional structure of the protein molecule. Examples and embodiments of conservative amino acid substitutions are apparent to those skilled in the art. In particular, the 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 nonpolar amino acid residue, with a polar uncharged amino acid residue, with a basic amino acid residue, with an acidic amino acid residue. Such substituted amino acid residues may or may not be encoded by the genetic code. Conservative substitutions of one amino acid by another belonging to the same group are within the scope of the invention as long as the substitution does not result in inactivation of the biological activity of the protein. Thus, the proteins of the invention may comprise one or more conservative substitutions in the amino acid sequence, which are preferably made according to the following table. In addition, proteins that also contain one or more other non-conservative substitutions are also contemplated by the present invention, provided that the non-conservative substitutions do not significantly affect the desired function and biological activity of the proteins of the present invention.

Conservative amino acid substitutions may be made at one or more predicted nonessential amino acid residues. "nonessential" amino acid residues are amino acid residues that can be altered (deleted, substituted or substituted) without altering the biological activity, whereas "essential" amino acid residues are required for the biological activity. A "conservative amino acid substitution" is a substitution in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Amino acid substitutions may be made in non-conserved regions of the Cas proteins described above. In general, such substitutions are not made to conserved amino acid residues, or amino acid residues that are within a conserved motif, 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 the conserved regions.

It is well known in the art that one or more amino acid residues may be altered (substituted, deleted, truncated or inserted) from the N-and/or C-terminus of a protein while still retaining its functional activity. Thus, proteins that have one or more amino acid residues altered from the N and/or C terminus of the Cas protein while retaining their desired functional activity are also within the scope of the invention. These changes may include changes introduced by modern molecular methods such as PCR, including PCR amplification that alters or extends the protein coding sequence by including an amino acid coding sequence in the oligonucleotides used in the PCR amplification.

It will be appreciated that proteins may be altered in a variety of ways, including amino acid substitutions, deletions, truncations and insertions, and that methods for such manipulation are generally known in the art. For example, amino acid sequence variants of the above proteins can be prepared by mutation of DNA. Single or multiple amino acid substitutions, deletions and/or insertions may also be made by other forms of mutagenesis and/or by directed evolution, for example, using known mutagenesis, recombination and/or shuffling (shuffleling) methods, in combination with associated screening methods.

Those of skill in the art will appreciate that these minor amino acid changes in the Cas proteins of the invention may occur (e.g., naturally occurring mutations) or be generated (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 nature of the polypeptide may be altered, but the polypeptide may retain its activity. Smaller effects can be expected if mutations are present that are not close to the catalytic domain, active site or other functional domain.

The skilled artisan can identify the essential amino acids of the Cas protein of the invention according to methods known in the art, such as site-directed mutagenesis or protein evolution or analysis of bioinformatics. The catalytic, active or other functional domains of a 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, in combination with mutations in the amino acids at putative key sites.

In one embodiment, the Cas protein contains the amino acid sequence set forth in any one of SEQ ID nos. 1-4.

In one embodiment, the Cas protein is the amino acid sequence set forth in any one of SEQ ID nos. 1-4.

In one embodiment, the Cas protein is a derivatized protein having the same biological function as a protein having the sequence set forth in any one of SEQ ID nos. 1-4.

Such biological functions include, but are not limited to, activity of binding to a guide RNA, endonuclease activity, activity of binding to and cleaving at a specific site of a target sequence under the guidance of a guide RNA, including, but not limited to, cis cleavage activity and Trans cleavage activity.

The invention also provides a fusion protein comprising a Cas protein as described above and other modifying moieties.

In one embodiment, the modifying moiety is selected from the group consisting of an additional protein or polypeptide, a detectable label, or any combination thereof.

In one embodiment, the modifying moiety is selected from the group consisting of an epitope tag, a reporter gene sequence, a Nuclear Localization Signal (NLS) sequence, a targeting moiety, a transcriptional activation domain (e.g., VP 64), a transcriptional repression domain (e.g., KRAB domain or SID domain), a nuclease domain (e.g., fok 1), and a domain having an activity selected from the group consisting of: nucleotide deaminase, methylase activity, demethylase, transcriptional activation activity, transcriptional repression activity, transcriptional release factor activity, histone modification activity, nuclease activity, single-stranded RNA cleavage activity, double-stranded RNA cleavage activity, single-stranded DNA cleavage activity, double-stranded DNA cleavage activity and nucleic acid binding activity; and any combination thereof. The NLS sequences are well known to those skilled in the art, examples of which include, but are not limited to, the SV40 large T antigen, EGL-13, c-Myc, and TUS proteins.

In one embodiment, the NLS sequence is located at, near, or near the terminus (e.g., N-terminus, C-terminus, or both) of the Cas protein of the invention.

Such epitope tags (tags) are well known to those skilled in the art, including but not limited to His, V5, FLAG, HA, myc, VSV-G, trx, etc., and other suitable epitope tags (e.g., purification, detection, or labeling) may be selected by those skilled in the art.

Such reporter sequences are well known to those skilled in the art, examples of which include, but are not limited to GST, HRP, CAT, GFP, hcRed, dsRed, CFP, YFP, BFP, etc.

In one embodiment, the fusion proteins of the invention comprise a domain capable of binding to a DNA molecule or an intracellular molecule, such as Maltose Binding Protein (MBP), the DNA binding domain of Lex a (DBD), the DBD of GAL4, and the like.

In one embodiment, the fusion proteins of the invention comprise a detectable label, such as a fluorescent dye, e.g., FITC or DAPI.

In one embodiment, the Cas protein of the invention is coupled, conjugated or fused to the modifying moiety, optionally through a linker.

In one embodiment, the modification is directly linked to the N-terminus or C-terminus of the Cas protein of the invention.

In one embodiment, the modifying moiety is linked to the N-terminus or C-terminus of the Cas protein of the invention by a linker. Such linkers are well known in the art, examples of which include, but are not limited to, linkers comprising one or more (e.g., 1, 2, 3, 4, or 5) amino acids (e.g., glu or Ser) or amino acid derivatives (e.g., ahx, β -Ala, GABA, or Ava), or PEG, etc.

The Cas protein, protein derivative or fusion protein of the present invention is not limited by the manner of its production, and for example, it may be produced by genetic engineering methods (recombinant techniques) or by chemical synthesis methods.

Nucleic acid of Cas protein

In another aspect, the invention provides an isolated polynucleotide comprising:

(a) Polynucleotide sequences encoding Cas proteins or fusion proteins of the invention;

(b) A polynucleotide having a sequence as shown in any one of SEQ ID No. 5-12;

(c) A sequence having a substitution, deletion, or addition of one or more bases (e.g., a substitution, deletion, or addition of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 bases) as compared to the sequence set forth in any one of SEQ ID Nos. 5-12;

(d) The nucleotide sequence has homology of 80% (preferably 90%, more preferably 95%, most preferably 98%) with any one of SEQ ID No.5-12, and encodes a polypeptide of any one of SEQ ID No. 1-4; or alternatively, the process may be performed,

(e) A polynucleotide complementary to the polynucleotide of any one of (a) - (d).

In one embodiment, the nucleotide sequence set forth in any one of (a) - (e) is codon optimized for expression in a prokaryotic cell. In one embodiment, the nucleotide sequence set forth in any one of (a) - (e) is codon optimized for expression in a eukaryotic cell.

In one embodiment, the polynucleotide is preferably single-stranded or double-stranded.

Direct Repeat (Direct Repeat) sequence

In another aspect, the invention provides an engineered homodromous repeat sequence that forms a complex with the Cas protein described above.

The orthostatic repeat sequence is linked to a guide sequence capable of hybridizing to a target sequence to form a guide RNA (guide RNA or gRNA).

Hybridization of the target sequence to the gRNA, representing at least 70%,75%,80%,85%,90%,91%,92%,93%,94%,95%,96%,97%,98%,99%, or 100% identity of the target sequence to the nucleic acid sequence of the gRNA, such that hybridization can form a complex; or at least 12, 15, 16, 17, 18, 19, 20, 21, 22, or more bases of the nucleic acid sequence representing the target sequence and the gRNA may be complementarily paired to form a complex.

In some embodiments, the orthostatic sequence has at least 90% sequence identity to the sequence set forth in SEQ ID Nos. 13-17. In some embodiments, the orthostatic repeat sequence has a substitution, deletion, or addition of one or more bases (e.g., substitution, deletion, or addition of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 bases) as compared to the sequence set forth in SEQ ID nos. 13-17.

In some embodiments, the orthostatic repeat sequence is as shown in any one of SEQ ID Nos. 13-17.

Guide RNA (gRNA)

In another aspect, the invention provides a gRNA comprising a first segment and a second segment; the first segment is also known as a "framework region", "protein binding segment", "protein binding sequence", or "Direct Repeat (Direct Repeat) sequence"; the second segment is also referred to as a "targeting sequence of a targeting nucleic acid" or a "targeting segment of a targeting nucleic acid", or a "targeting sequence of a targeting nucleic acid".

The first segment of the gRNA is capable of interacting with the Cas protein of the invention, thereby forming a complex of Cas protein and gRNA.

In one embodiment, the first segment is a homeotropic repeat as described above.

The targeting sequence of the targeting nucleic acid or targeting segment of the targeting nucleic acid of the invention comprises a nucleotide sequence complementary to a sequence in the target nucleic acid. In other words, the targeting sequence of the targeting nucleic acid or targeting segment of the targeting nucleic acid of the invention interacts with the target nucleic acid in a sequence-specific manner via hybridization (i.e., base pairing). Thus, the targeting sequence of the targeting nucleic acid or targeting segment of the targeting nucleic acid may be altered, or may be modified to hybridize to any desired sequence within the target nucleic acid. The nucleic acid is selected from DNA or RNA.

The targeting sequence of the targeting nucleic acid or the percentage of complementarity between the targeting segment of the targeting nucleic acid and the target sequence of the target nucleic acid can be at least 60% (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100%).

The "framework region", "protein binding segment", "protein binding sequence", or "cognate repeat" of the gRNA of the invention can interact with a CRISPR protein (or Cas protein). The gRNA of the invention directs its interacting Cas protein to a specific nucleotide sequence within the target nucleic acid through the action of the targeting sequence of the targeting nucleic acid.

Preferably, the guide RNA comprises a first segment and a second segment in the 5 'to 3' direction.

In the context of the present invention, the second segment is also understood as a guide sequence which hybridizes to the target sequence.

The gRNA of the invention is capable of forming a complex with the Cas protein.

The gRNA of the Cas-sf4274 protein of the invention comprises a guide sequence that hybridizes to a target nucleic acid, wherein the target nucleic acid comprises a sequence located 3' of a Protospacer Adjacent Motif (PAM); the PAM sequence was 5'-TTN-3', where n=a/T/C/G.

The gRNA of the Cas-sf2201 protein of the invention comprises a guide sequence that hybridizes to a target nucleic acid, wherein the target nucleic acid comprises a sequence located 3' of a Protospacer Adjacent Motif (PAM); the PAM sequence was 5'-TTN-3', where n=a/T/C/G.

The gRNA of the Cas-sf2771 protein of the invention comprises a guide sequence that hybridizes to a target nucleic acid, wherein the target nucleic acid comprises a sequence located 3' of a Protospacer Adjacent Motif (PAM); the PAM sequence was 5'-TTN-3', where n=a/T/C/G.

The gRNA of the Cas-sf2586 protein of the invention comprises a guide sequence that hybridizes to a target nucleic acid, wherein the target nucleic acid comprises a sequence located 3' of a Protospacer Adjacent Motif (PAM); the PAM sequence is 5'-ATT-3' or 5'-ATC-3'.

Carrier body

The invention also provides a vector comprising a Cas protein, an isolated nucleic acid molecule, or a polynucleotide as described above; preferably, it further comprises a regulatory element operably linked thereto.

In one embodiment, the regulatory element is selected from one or more of the following group: enhancers, transposons, promoters, terminators, leader sequences, polyadenylation sequences, and marker genes.

In one embodiment, the vector comprises a cloning vector, an expression vector, a shuttle vector, an integration vector.

In some embodiments, the vectors included in the system are viral vectors (e.g., retroviral vectors, lentiviral vectors, adenoviral vectors, adeno-associated vectors, and herpes simplex vectors), but may also be of the plasmid, viral, cosmid, phage, etc. type, which are well known to those skilled in the art.

CRISPR system

The present invention provides an engineered non-naturally occurring vector system, or CRISPR-Cas system, comprising a Cas protein or a nucleic acid sequence encoding the Cas protein and a nucleic acid encoding one or more guide RNAs.

In one embodiment, the nucleic acid sequence encoding the Cas protein and the nucleic acid encoding one or more guide RNAs are synthetic.

In one embodiment, the nucleic acid sequence encoding the Cas protein and the nucleic acid encoding one or more guide RNAs do not co-occur naturally.

The one or more guide RNAs target one or more target sequences in the cell. The one or more target sequences hybridize to a genomic locus of a DNA molecule encoding one or more gene products and the Cas protein is directed to the genomic locus of the DNA molecule of the one or more gene products, and the Cas protein, upon reaching the target sequence position, modifies, edits or cleaves the target sequence, whereby expression of the one or more gene products is altered or modified.

The cells of the invention include one or more of animals, plants or microorganisms.

In some embodiments, the Cas protein is codon optimized for expression in a cell.

In some embodiments, the Cas protein directs cleavage of one or both strands at the target sequence position.

In some embodiments, the Cas protein cleaves the complementary strand and/or the non-complementary strand of the target nucleic acid under the mediation of the gRNA.

Preferably, the Cas protein cleaves both the complementary strand and the non-complementary strand of the target nucleic acid.

Preferably, the Cas protein preferentially cleaves the non-complementary strand of the target nucleic acid.

In the present invention, the gRNA directs Cas protein recognition and binding on the complementary strand, and the non-complementary strand is a nucleic acid strand paired with the complementary strand. The PAM sequence is located on a non-complementary strand that contains a PAM complementary sequence paired with the PAM sequence described above.

In one embodiment, the cleavage site of the complementary strand of the Cas-sf4274 to the target sequence is between 22nt and 23nt at the 5 'end of the PAM complementary sequence, and the cleavage site of the non-complementary strand of the Cas-sf4274 to the target sequence is between 23nt and 24nt or between 28nt and 29nt or between 30nt and 31nt at the 3' end of the PAM sequence, and the gRNA directs Cas-sf4274 protein to recognize and bind to the complementary strand on which the non-complementary strand is a DNA strand paired with the complementary strand.

In one embodiment, the cleavage site of the complementary strand of Cas-sf2201 to the target sequence is between 22nt and 23nt at the 5 'end of the PAM complementary sequence, the cleavage site of the non-complementary strand of Cas-sf2201 to the target sequence is between 25nt and 26nt or between 28nt and 29nt at the 3' end of the PAM sequence, and the gRNA directs Cas-sf2201 protein to recognize and bind to the complementary strand, which is the DNA strand paired with the complementary strand.

In one embodiment, the cleavage site of the complementary strand of the target sequence by Cas-sf2771 is between 22nt and 23nt of the 5 'end of the PAM complementary sequence, the cleavage site of the non-complementary strand of the target sequence by Cas-sf2771 is between 18nt and 19nt of the 3' end of the PAM sequence, and the gRNA directs Cas-sf2771 protein to recognize and bind to the complementary strand, which is the DNA strand paired with the complementary strand.

In one embodiment, the cleavage site of Cas-sf2586 for the non-complementary strand of the target sequence, which is the DNA strand paired with the complementary strand, is between 24nt and 25nt at the 3' end of the PAM sequence, and the gRNA directs Cas-sf2586 protein to recognize and bind to the complementary strand.

The invention also provides an engineered non-naturally occurring carrier system that can include one or more carriers comprising:

a) A first regulatory element operably linked to the gRNA,

b) A second regulatory element operably linked to the Cas protein;

wherein components (a) and (b) are on the same or different supports of the system.

The first and second regulatory elements include promoters (e.g., constitutive promoters or inducible promoters), enhancers (e.g., 35S promoter or 35S enhanced promoter), internal Ribosome Entry Sites (IRES), and other expression control elements (e.g., transcriptional termination signals, such as polyadenylation signals and poly U sequences).

In some embodiments, the vectors in the system are viral vectors (e.g., retroviral vectors, lentiviral vectors, adenoviral vectors, adeno-associated vectors, and herpes simplex vectors), but may also be of the plasmid, viral, cosmid, phage, etc. type, which are well known to those skilled in the art.

In some embodiments, the systems provided herein are in a delivery system. In some embodiments, the delivery system is a nanoparticle, liposome, exosome, microvesicle, or gene gun.

In one embodiment, the target sequence is a DNA or RNA sequence from a prokaryotic or eukaryotic cell. In one embodiment, the target sequence is a non-naturally occurring DNA or RNA sequence.

In one embodiment, the target sequence is present in a cell. In one embodiment, the target sequence is present in the nucleus or in the cytoplasm (e.g., organelle). In one embodiment, the cell is a eukaryotic cell. In other embodiments, the cell is a prokaryotic cell.

In one embodiment, the Cas protein has one or more NLS sequences attached. In one embodiment, the fusion protein comprises one or more NLS sequences. In one embodiment, the NLS sequence is linked to the N-terminus or C-terminus of the protein. In one embodiment, the NLS sequence is fused to the N-terminus or C-terminus of the protein.

In another aspect, the invention relates to an engineered CRISPR system comprising the Cas protein described above and one or more guide RNAs, wherein the guide RNAs comprise a homodromous repeat sequence and a spacer sequence capable of hybridizing to a target nucleic acid, the Cas protein is capable of binding to the guide RNAs and targeting a target nucleic acid sequence complementary to the spacer sequence.

In one embodiment, the Cas enzyme is a Cas-sf4274 protein, the target nucleic acid is DNA (preferably, double-stranded DNA), the target nucleic acid is located at the 3' end of a protospacer adjacent to a motif (PAM), and the PAM has a sequence shown as 5' -TTN-3', where n=a/T/C/G.

In one embodiment, the Cas enzyme is a Cas-sf2201 protein, the target nucleic acid is DNA (preferably, double-stranded DNA), the target nucleic acid is located at the 3' end of a protospacer adjacent to a motif (PAM), and the PAM has a sequence shown as 5' -TTN-3', where n=a/T/C/G.

In one embodiment, the Cas enzyme is a Cas-sf2771 protein, the target nucleic acid is DNA (preferably, double-stranded DNA), the target nucleic acid is located at the 3' end of a protospacer adjacent to a motif (PAM), and the PAM has a sequence shown as 5' -TTN-3', where n=a/T/C/G.

In one embodiment, the Cas enzyme is Cas-sf2586 protein, the target nucleic acid is DNA (preferably, double-stranded DNA), the target nucleic acid is located at the 3' end of a protospacer adjacent to a motif (PAM), and the PAM has a sequence shown as 5' -ATT-3' or 5' -ATC-3 '.

Protein-nucleic acid complexes/compositions

In another aspect, the invention provides a complex or composition comprising:

(i) A protein component selected from the group consisting of: the Cas protein, the derivatized protein, or the fusion protein described above, and any combination thereof; and

(ii) A nucleic acid component comprising (a) a guide sequence capable of hybridizing to a target sequence; and (b) a homeotropic repeat capable of binding to the Cas protein of the invention.

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

In one embodiment, the nucleic acid component is a guide RNA in a CRISPR-Cas system.

In one embodiment, the complex or composition is non-naturally occurring or modified. In one embodiment, at least one component of the complex or composition is non-naturally occurring or modified. In one embodiment, the first component is non-naturally occurring or modified; and/or, the second component is non-naturally occurring or modified.

Activated CRISPR complexes

In another aspect, the present invention also provides an activated CRISPR complex comprising: (1) a protein component selected from the group consisting of: the Cas protein, the derivatized protein, or the fusion protein of the invention, and any combination thereof; (2) A gRNA comprising (a) a guide sequence capable of hybridizing to a target sequence; and (b) a homeotropic repeat capable of binding to the Cas protein of the invention; and (3) a target sequence that binds to the gRNA. Preferably, the binding is binding to the target nucleic acid through a targeting sequence of the targeting nucleic acid on the gRNA.

The term "activated CRISPR complex", "activated complex" or "ternary complex" as used herein refers to a complex in a CRISPR system where Cas protein, gRNA bind to or are modified with a target nucleic acid.

The Cas proteins and grnas of the invention can form binary complexes that are activated upon binding to a nucleic acid substrate that is complementary to a spacer sequence in the gRNA (or, referred to as, a guide sequence that hybridizes to a target nucleic acid) to form an activated CRISPR complex. In some embodiments, the spacer sequence of the gRNA is perfectly matched to the target substrate. In other embodiments, the spacer sequence of the gRNA matches a portion (continuous or discontinuous) of the target substrate.

In preferred embodiments, the activated CRISPR complex may exhibit a sidebranch nuclease cleavage activity, which refers to the nonspecific cleavage activity or the nicking activity of the activated CRISPR complex on single stranded nucleic acids, also known in the art as trans cleavage activity.

Delivery and delivery compositions

Cas proteins, grnas, fusion proteins, nucleic acid molecules, vectors, systems, complexes, and compositions of the invention may be delivered by any method known in the art. Such methods include, but are not limited to, electroporation, lipofection, nuclear transfection, microinjection, sonoporation, gene gun, calcium phosphate mediated transfection, cationic transfection, lipofection, dendritic transfection, heat shock transfection, nuclear transfection, magnetic transfection, lipofection, puncture transfection, optical transfection, reagent enhanced nucleic acid uptake, and delivery via liposomes, immunoliposomes, virosomes, artificial virosomes, and the like.

Accordingly, in another aspect, the present invention provides a delivery composition comprising a delivery vehicle, and one or more selected from any of the following: cas proteins, fusion proteins, nucleic acid molecules, vectors, systems, complexes, and compositions of the invention.

In one embodiment, the delivery vehicle is a particle.

In one embodiment, the delivery vehicle is selected from the group consisting of a lipid particle, a sugar particle, a metal particle, a protein particle, a liposome, an exosome, a microbubble, a gene gun, or a viral vector (e.g., replication defective retrovirus, lentivirus, adenovirus, or adeno-associated virus).

Host cells

The invention also relates to an in vitro, ex vivo or in vivo cell or cell line or their progeny comprising: cas proteins, fusion proteins, nucleic acid molecules, protein-nucleic acid complexes, activated CRISPR complexes, vectors, delivery compositions of the invention are described herein.

In certain embodiments, the cell is a prokaryotic cell.

In certain embodiments, the cell is a eukaryotic cell. In certain embodiments, the cell is a mammalian cell. In certain embodiments, the cell is a human cell. In certain embodiments, the cell is a non-human mammalian cell, e.g., a non-human primate, bovine, ovine, porcine, canine, simian, rabbit, rodent (e.g., rat or mouse) cell. In certain embodiments, the cells are non-mammalian eukaryotic cells, such as cells of poultry birds (e.g., chickens), fish, or crustaceans (e.g., clams, shrimps). In certain embodiments, the cell is a plant cell, e.g., a cell of a monocot or dicot or a cell of a cultivated plant or a food crop such as tapioca, corn, sorghum, soybean, wheat, oat, or rice, e.g., an algae, tree, or production plant, fruit, or vegetable (e.g., a tree such as citrus, nut, eggplant, cotton, tobacco, tomato, grape, coffee, cocoa, etc.).

In certain embodiments, the cell is a stem cell or stem cell line.

In certain instances, a host cell of the invention comprises a modification of a gene or genome that is not present in its wild type.

Gene editing method and application

The Cas protein, nucleic acid, the above composition, the above CIRSPR/Cas system, the above vector system, the above delivery composition or the above activated CRISPR complex or the above host cell of the present invention may be used for any one or any of several of the following uses: targeting and/or editing a target nucleic acid; cleaving double-stranded DNA, single-stranded DNA, or single-stranded RNA; nonspecific cleavage and/or degradation of collateral nucleic acids; nonspecifically cleaving the single-stranded nucleic acid; detecting nucleic acid; detecting nucleic acid in a target sample; editing the double-stranded nucleic acid specifically; base editing double-stranded nucleic acid; base editing single stranded nucleic acids. In other embodiments, it may also be used to prepare reagents or kits for any one or any of several of the uses described above.

The invention also provides the use of the Cas protein, nucleic acid, composition, CIRSPR/Cas system, vector system, delivery composition or activated CRISPR complex described above in gene editing, gene targeting or gene cleavage; alternatively, use in the preparation of a reagent or kit for gene editing, gene targeting or gene cleavage.

In one embodiment, the gene editing, gene targeting or gene cleaving is performed intracellularly and/or extracellularly.

The invention also provides a method of editing, targeting, or cleaving a target nucleic acid comprising contacting the target nucleic acid with the Cas protein, nucleic acid, composition, CIRSPR/Cas system, vector system, delivery composition, or activated CRISPR complex described above. In one embodiment, the method is editing, targeting, or cleaving a target nucleic acid inside or outside a cell.

The gene editing or editing target nucleic acids include modifying genes, knocking out genes, altering expression of gene products, repairing mutations, and/or inserting polynucleotides, gene mutations.

The editing may be performed in prokaryotic and/or eukaryotic cells.

In another aspect, the invention also provides the use of the Cas protein, the nucleic acid, the composition, the CIRSPR/Cas system, the vector system, the delivery composition, or the activated CRISPR complex described above in nucleic acid detection, or in the preparation of a reagent or kit for nucleic acid detection.

In another aspect, the invention also provides a method of cleaving a single-stranded nucleic acid, the method comprising contacting a nucleic acid population with the Cas protein and the gRNA described above, wherein the nucleic acid population comprises a target nucleic acid and a plurality of non-target single-stranded nucleic acids, the Cas protein cleaving the plurality of non-target single-stranded nucleic acids.

The gRNA is capable of binding to the Cas protein.

The gRNA is capable of targeting the target nucleic acid.

The contacting may be inside a cell in vitro, ex vivo or in vivo.

Preferably, the cleavage of single-stranded nucleic acid is a nonspecific cleavage.

In another aspect, the invention also provides the use of the Cas protein, the nucleic acid, the composition, the CIRSPR/Cas system, the vector system, the delivery composition or the activated CRISPR complex described above for non-specific cleavage of single stranded nucleic acids, or for the preparation of a reagent or kit for non-specific cleavage of single stranded nucleic acids.

In another aspect, the invention also provides a kit for gene editing, gene targeting or gene cleavage comprising the Cas protein, gRNA, nucleic acid, the composition, the CIRSPR/Cas system, the vector system, the delivery composition, the activated CRISPR complex or the host cell.

In another aspect, the invention also provides a kit for detecting a target nucleic acid in a sample, the kit comprising: (a) a Cas protein, or a nucleic acid encoding the Cas protein; (b) A guide RNA, or a nucleic acid encoding the guide RNA, or a precursor RNA comprising the guide RNA, or a nucleic acid encoding the precursor RNA; and (c) a single stranded nucleic acid detector that is single stranded and does not hybridize to the guide RNA.

It is known in the art that precursor RNAs can be cleaved or processed into the mature guide RNAs described above.

In another aspect, the invention provides the use of the Cas protein, the nucleic acid, the composition, the CIRSPR/Cas system, the vector system, the delivery composition, the activated CRISPR complex, or the host cell described above in the preparation of a formulation or kit for:

(i) Gene or genome editing;

(ii) Target nucleic acid detection and/or diagnosis;

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

(iv) Treatment of disease;

(iv) Targeting the target gene.

Preferably, the gene or genome editing is performed in or out of a cell.

Preferably, the target nucleic acid detection and/or diagnosis is performed in vitro.

Preferably, the treatment of the disease is treatment of a condition caused by a defect in the target sequence in the target locus.

In another aspect, the invention provides a method of detecting a target nucleic acid in a sample, the method comprising contacting a sample with the Cas protein, a gRNA (guide RNA) comprising a region that binds to the Cas protein and a guide sequence that hybridizes to the target nucleic acid, and a single stranded nucleic acid detector; detecting a detectable signal generated by the Cas protein cleaving single-stranded nucleic acid detector, thereby detecting a target nucleic acid; the single stranded nucleic acid detector does not hybridize to the gRNA.

Method for specifically modifying target nucleic acid

In another aspect, the invention also provides a method of specifically modifying a target nucleic acid, the method comprising: contacting a target nucleic acid with the Cas protein, nucleic acid, composition, CIRSPR/Cas system, vector system, delivery composition, or activated CRISPR complex.

The specific modification may occur in vivo or in vitro.

The specific modification may occur either intracellularly or extracellularly.

In some cases, the cell is selected from a prokaryotic cell or a eukaryotic cell, e.g., an animal cell, a plant cell, or a microbial cell.

In one embodiment, the modification refers to cleavage of the target sequence, e.g., single/double strand cleavage of DNA, or single strand cleavage of RNA.

In some cases, the method further comprises contacting the target nucleic acid with a donor polynucleotide, wherein the donor polynucleotide, a portion of the donor polynucleotide, a copy of the donor polynucleotide, or a portion of a copy of the donor polynucleotide is integrated into the target nucleic acid.

In one embodiment, the modification further comprises inserting an editing template (e.g., an exogenous nucleic acid) into the break.

In one embodiment, the method further comprises: contacting an editing template with the target nucleic acid, or delivering into a cell comprising the target nucleic acid. In this embodiment, the method repairs the disrupted target gene by homologous recombination with an exogenous template polynucleotide; in some embodiments, the repair results in a mutation, including an insertion, deletion, or substitution of one or more nucleotides of the target gene, in other embodiments, the mutation results in one or more amino acid changes in a protein expressed from a gene comprising the target sequence.

Detection (non-specific cleavage)

In another aspect, the invention provides a method of detecting a target nucleic acid in a sample, the method comprising contacting the sample with the Cas protein, the nucleic acid, the composition, the CIRSPR/Cas system, the vector system, the delivery composition, or the activated CRISPR complex and a single stranded nucleic acid detector; detecting a detectable signal generated by the Cas protein cleaving single stranded nucleic acid detector, thereby detecting a target nucleic acid.

In the present invention, the target nucleic acid comprises ribonucleotides or deoxyribonucleotides; including single-stranded nucleic acids, double-stranded nucleic acids, e.g., single-stranded DNA, double-stranded DNA, single-stranded RNA, double-stranded RNA.

In one embodiment, the target nucleic acid is derived from a sample of a virus, bacterium, microorganism, soil, water source, human, animal, plant, or the like. Preferably, the target nucleic acid is a product of enrichment or amplification by methods such as PCR, NASBA, RPA, SDA, LAMP, HAD, NEAR, MDA, RCA, LCR, RAM.

In one embodiment, the target nucleic acid is a viral nucleic acid, a bacterial nucleic acid, a specific nucleic acid associated with a disease, such as a specific mutation site or SNP site, or a nucleic acid that differs from a control; preferably, the virus is a plant virus or an animal virus, for example, papilloma virus, hepadnavirus, herpes virus, adenovirus, poxvirus, parvovirus, coronavirus; preferably, the virus is a coronavirus, preferably SARS, SARS-CoV2 (COVID-19), HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1, mers-CoV.

In the present invention, the gRNA has a degree of match of at least 50%, preferably at least 60%, preferably at least 70%, preferably at least 80%, preferably at least 90% with the target sequence on the target nucleic acid.

In one embodiment, when the target sequence contains one or more characteristic sites (e.g., specific mutation sites or SNPs), the characteristic sites are perfectly matched to the gRNA.

In one embodiment, the detection method may comprise one or more grnas with different targeting sequences to different target sequences.

In the present invention, the single-stranded nucleic acid detector includes, but is not limited to, single-stranded DNA, single-stranded RNA, DNA-RNA hybrids, nucleic acid analogs, base modifications, single-stranded nucleic acid detectors containing abasic spacers, and the like; "nucleic acid analogs" include, but are not limited to: locked nucleic acids, bridged nucleic acids, morpholino nucleic acids, ethylene glycol nucleic acids, hexitol nucleic acids, threose nucleic acids, arabinose nucleic acids, 2' oxymethyl RNAs, 2' methoxyacetyl RNAs, 2' fluoro RNAs, 2' amino RNAs, 4' thio RNAs, and combinations thereof, including optional ribonucleotide or deoxyribonucleotide residues.

In the present invention, the detectable signal is realized by: visual-based detection, sensor-based detection, color detection, fluorescent signal-based detection, gold nanoparticle-based detection, fluorescence polarization, colloidal phase change/dispersion, electrochemical detection, and semiconductor-based detection.

In the present invention, it is preferable that both ends of the single-stranded nucleic acid detector are provided with a fluorescent group and a quenching group, respectively, and that the single-stranded nucleic acid detector may exhibit a detectable fluorescent signal when cleaved. The fluorescent group is selected from one or more of FAM, FITC, VIC, JOE, TET, CY, CY5, ROX, texas Red or LC RED 460; the quenching group is selected from one or more of BHQ1, BHQ2, BHQ3, dabcy1 or Tamra.

In other embodiments, the 5 'end and the 3' end of the single-stranded nucleic acid detector are respectively provided with different labeling molecules, and colloidal gold test results before the single-stranded nucleic acid detector is cleaved by the Cas protein and after the single-stranded nucleic acid detector is cleaved by the Cas protein are detected by a colloidal gold detection mode; the single-stranded nucleic acid detector will exhibit different color development results on the detection line and the quality control line of colloidal gold before being cleaved by Cas protein and after being cleaved by Cas protein.

In some embodiments, the method of detecting a target nucleic acid may further comprise comparing the level of the detectable signal to a reference signal level, and determining the amount of the target nucleic acid in the sample based on the level of the detectable signal.

In some embodiments, the method of detecting a target nucleic acid may further comprise using RNA reporter nucleic acid and DNA reporter nucleic acid (e.g., fluorescent colors) on different channels, and sampling based on combining (e.g., using a minimum or product) the levels of the detectable signals by measuring the signal levels of the RNA and DNA reporter molecules, and determining the levels of the detectable signals by measuring the amounts of target nucleic acid in the RNA and DNA reporter molecules.

In one embodiment, the target gene is present in a cell.

In one embodiment, the cell is a prokaryotic cell.

In one embodiment, the cell is a eukaryotic cell.

In one embodiment, the cell is an animal cell.

In one embodiment, the cell is a human cell.

In one embodiment, the cell is a plant cell, such as a cell of a cultivated plant (e.g., cassava, maize, sorghum, wheat, or rice), algae, tree, or vegetable.

In one embodiment, the target gene is present in an in vitro nucleic acid molecule (e.g., a plasmid).

In one embodiment, the target gene is present in a plasmid.

Definition of terms

In the present invention, unless otherwise indicated, scientific and technical terms used herein have the meanings commonly understood by one of ordinary skill in the art. Further, the procedures of molecular genetics, nucleic acid chemistry, molecular biology, biochemistry, cell culture, microbiology, cell biology, genomics and recombinant DNA, etc., as used herein, are all conventional procedures widely used in the corresponding field. Meanwhile, in order to better understand the present invention, definitions and explanations of related terms are provided below.

In the present invention, amino acid residues may be represented by single letters or by three letters, for example: alanine (Ala, A), valine (Val, V), glycine (Gly, G), leucine (Leu, L), glutamine (Gln, Q), phenylalanine (Phe, F), tryptophan (Trp, W), tyrosine (Tyr, Y), aspartic acid (Asp, D), asparagine (Asn, N), glutamic acid (Glu, E), lysine (Lys, K), methionine (Met, M), serine (Ser, S), threonine (Thr, T), cysteine (Cys, C), proline (Pro, P), isoleucine (Ile, I), histidine (His, H), arginine (Arg, R).

Cas proteins

In the present invention, cas protein, cas enzyme, cas effector protein may be used interchangeably; the present inventors have first discovered and identified a Cas effector protein having an amino acid sequence selected from the group consisting of:

(i) Any one of SEQ ID No. 1-4;

(ii) 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) as compared to the sequence set forth in any of SEQ ID nos. 1-4; or (b)

(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 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the sequence set forth in any one of SEQ ID nos. 1-4.

Nucleic acid cleavage or cleavage nucleic acids herein include DNA or RNA cleavage (Cis cleavage), cleavage of DNA or RNA in a side branch nucleic acid substrate (single-stranded nucleic acid substrate) in a target nucleic acid produced by a Cas enzyme described herein (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 cleavage or a single-stranded RNA cleavage.

CRISPR system

As used herein, the term "regularly clustered, spaced short palindromic repeats (CRISPR) -CRISPR-associated (Cas) (CRISPR-Cas) system" or "CRISPR system" is used interchangeably and has the meaning commonly understood by those skilled in the art, which generally comprises transcripts or other elements related to the expression of a CRISPR-associated ("Cas") gene, or transcripts or other elements capable of directing the activity of the Cas gene.

CRISPR/Cas complexes

As used herein, the term "CRISPR/Cas complex" refers to a complex formed by directing RNA (guide RNA) or mature crRNA to bind to a Cas protein, comprising a direct repeat sequence that hybridizes to a target sequence and binds to a Cas protein, which complex is capable of recognizing and cleaving a polynucleotide that hybridizes to the guide RNA or mature crRNA.

Guide RNA (guide RNA, gRNA)

As used herein, the terms "guide RNA", "mature crRNA", "guide sequence" are used interchangeably and have the meaning commonly understood by those skilled in the art. In general, the guide RNA can comprise, consist essentially of, or consist of a direct repeat (direct repeat) and a guide sequence.

In certain instances, the guide sequence is any polynucleotide sequence that has sufficient complementarity to a target sequence to hybridize to the target sequence and direct specific binding of the CRISPR/Cas complex to the target sequence. In one embodiment, the degree of complementarity between a guide 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% when optimally aligned. It is within the ability of one of ordinary skill in the art to determine the optimal alignment. For example, there are published and commercially available alignment algorithms and programs such as, but not limited to, the Smith-Waterman algorithm (Smith-Waterman), bowtie, geneious, biopython, and SeqMan in ClustalW, matlab.

Target sequence

"target sequence" refers to a polynucleotide targeted by a guide sequence in a gRNA, e.g., a sequence that has complementarity to the guide sequence, wherein hybridization between the target sequence and the guide sequence will promote the formation of a CRISPR/Cas complex (including Cas proteins and grnas). Complete complementarity is not necessary so long as sufficient complementarity exists to cause hybridization and promote the formation of a CRISPR/Cas complex.

The target sequence may comprise any polynucleotide, such as DNA or RNA. In some cases, the target sequence is located either inside or outside the cell. In some cases, the target sequence is located in the nucleus or cytoplasm of the cell. In some cases, the target sequence may be located within an organelle of a eukaryotic cell, such as a mitochondria or chloroplast. Sequences or templates that can be used for recombination into a target locus comprising the target sequence are referred to as "editing templates" or "editing polynucleotides" or "editing sequences". In one embodiment, the editing template is an exogenous nucleic acid. In one embodiment, the recombination is homologous recombination.

In the present invention, a "target sequence" or "target polynucleotide" or "target nucleic acid" may be any polynucleotide that is endogenous or exogenous to a cell (e.g., a eukaryotic cell). For example, the target polynucleotide may be a polynucleotide that is present in the nucleus of a eukaryotic cell. The target polynucleotide may be a sequence encoding a gene product (e.g., a protein) or a non-coding sequence (e.g., a regulatory polynucleotide or unwanted DNA). In some cases, the target sequence should be related to the Protospacer Adjacent Motif (PAM).

Single-stranded nucleic acid detector

The single-stranded nucleic acid detector according to the present invention means a detector comprising a sequence of 2 to 200 nucleotides, preferably 2 to 150 nucleotides, preferably 3 to 100 nucleotides, preferably 3 to 30 nucleotides, preferably 4 to 20 nucleotides, more preferably 5 to 15 nucleotides. Preferably a single-stranded DNA molecule, a single-stranded RNA molecule or a single-stranded DNA-RNA hybrid.

The single-stranded nucleic acid detector comprises different reporter groups or marker molecules at both ends, which do not exhibit a reporter signal when in an initial state (i.e., not cleaved), and which exhibit a detectable signal when cleaved, i.e., a detectable distinction between cleaved and pre-cleaved.

In one embodiment, the reporter or marker molecule comprises a fluorophore and a quencher, wherein the fluorophore is selected from one or more of FAM, FITC, VIC, JOE, TET, CY, CY5, ROX, texas Red or LC RED 460; the quenching group is selected from one or more of BHQ1, BHQ2, BHQ3, dabcy1 or Tamra.

In one embodiment, the single stranded nucleic acid detector has a first molecule (e.g., FAM or FITC) attached to the 5 'end and a second molecule (e.g., biotin) attached to the 3' end. The reaction system containing the single-stranded nucleic acid detector is matched with a flow strip to detect target nucleic acid (preferably, a colloidal gold detection mode). The flow strip is designed with two capture lines, with an antibody binding to a first molecule (i.e., a first molecular antibody) at the sample contact end (colloidal gold), an antibody binding to the first molecular antibody at the first line (control line), and an antibody binding to a second molecule (i.e., a second molecular antibody, such as avidin) at the second line (test line). When the reaction flows along the strip, the first molecular antibody binds to the first molecule carrying the cleaved or uncleaved oligonucleotide to the capture line, the cleaved reporter will bind to the antibody of the first molecular antibody at the first capture line, and the uncleaved reporter will bind to the second molecular antibody at the second capture line. Binding of the reporter group at each line will result in a strong readout/signal (e.g., color). As more reporter is cut, more signal will accumulate at the first capture line and less signal will appear at the second line. In certain aspects, the invention relates to the use of a flow strip as described herein for detecting nucleic acids. In certain aspects, the invention relates to a method of detecting nucleic acids using a flow strip as defined herein, e.g. a (lateral) flow test or a (lateral) flow immunochromatographic assay. In certain aspects, the molecules in the single stranded nucleic acid detector may be interchanged or the positions of the molecules may be changed, so long as the reporting principle is the same or similar to that of the present invention, and the modified manner is also included in the present invention.

The detection method provided by the invention can be used for quantitative detection of target nucleic acid to be detected. The quantitative detection index can be quantified according to the signal intensity of the reporter group, such as the luminous intensity of the fluorescent group, the width of the color-developing strip, and the like.

Wild type

As used herein, the term "wild-type" has the meaning commonly understood by those skilled in the art, which refers to a typical form of an organism, strain, gene, or a characteristic that, when it exists in nature, differs from a mutant or variant form, which may be isolated from a source in nature and not intentionally modified by man.

Derivatization

As used herein, the term "derivatization" refers to a chemical modification of an amino acid, polypeptide, or protein in which one or more substituents have been covalently attached to the amino acid, polypeptide, or protein. Substituents may also be referred to as side chains.

A derivatized protein is a derivative of the protein, in general, derivatization of the protein does not adversely affect the desired activity of the protein (e.g., binding to a guide RNA, endonuclease activity, binding to a specific site of a target sequence under the guidance of a guide RNA and cleavage activity), that is, the derivative of the protein has the same activity as the protein.

Derivatizing proteins

Also referred to as "protein derivatives" refers to modified forms of a protein, for example, wherein one or more amino acids of the protein may be deleted, inserted, modified and/or substituted.

Non-naturally occurring

As used herein, the terms "non-naturally occurring" or "engineered" are used interchangeably and refer to human involvement. When these terms are used to describe a nucleic acid molecule or polypeptide, it means that the nucleic acid molecule or polypeptide is at least substantially free from at least one other component to which it is associated in nature or as found in nature.

Orthologs (orthologs)

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

Identity of

As used herein, the term "identity" is used to refer to the match of sequences between two polypeptides or between two nucleic acids. When a position in both sequences being compared is occupied by the same base or amino acid monomer subunit (e.g., a position in each of two DNA molecules is occupied by adenine, or a position in each of two polypeptides is occupied by lysine), then the molecules are identical at that position. The "percent identity" between two sequences is a function of the number of matched positions shared by the two sequences divided by the number of positions to be compared x 100. For example, if 6 out of 10 positions of two sequences match, then the two sequences have 60% identity. For example, the DNA sequences CTGACT and CAGGTT share 50% identity (3 out of 6 positions in total are matched). Typically, the comparison is made when two sequences are aligned to produce maximum identity. Such alignment may be conveniently performed using, for example, a computer program such as the Align program (DNAstar, inc.) Needleman et al (1970) j.mol.biol.48: 443-453. The percent identity between two amino acid sequences can also be determined using the algorithms of E.Meyers and W.Miller (Comput. Appl biosci.,4:11-17 (1988)) which have been integrated into the ALIGN program (version 2.0), using the PAM120 weight residue table (weight residue table), the gap length penalty of 12 and the gap penalty of 4. Furthermore, percent identity between two amino acid sequences can be determined using the Needleman and Wunsch (J MoI biol.48:444-453 (1970)) algorithm that has been incorporated into the GAP program of the GCG software package (available on www.gcg.com), using the Blossum 62 matrix or PAM250 matrix, and GAP weights (GAP weights) of 16, 14, 12, 10, 8, 6, or 4, and length weights of 1, 2, 3, 4, 5, or 6.

Carrier body

The term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid molecule linked thereto. Vectors include, but are not limited to, single-stranded, double-stranded, or partially double-stranded nucleic acid molecules; a nucleic acid molecule comprising one or more free ends, free ends (e.g., circular); a nucleic acid molecule comprising DNA, RNA, or both; and other diverse polynucleotides known in the art. The vector may be introduced into a host cell by transformation, transduction or transfection such that the genetic material elements carried thereby are expressed in the host cell. A vector may be introduced into a host cell to thereby produce a transcript, protein, or peptide, including from a protein, fusion protein, isolated nucleic acid molecule, or the like (e.g., a CRISPR transcript, such as a nucleic acid transcript, protein, or enzyme) as described herein. A vector may contain a variety of elements that control expression, including, but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. In addition, the vector may also contain a replication origin.

One type of vector is a "plasmid", which refers to a circular double stranded DNA loop into which additional DNA fragments may be inserted, for example, by standard molecular cloning techniques.

Another type of vector is a viral vector in which a virus-derived DNA or RNA sequence is present in a vector used to package a virus (e.g., retrovirus, replication-defective retrovirus, adenovirus, replication-defective adenovirus, and adeno-associated virus). Viral vectors also comprise polynucleotides carried by a virus for transfection into a host cell. Certain vectors (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors) are capable of autonomous replication in a host cell into which they are introduced.

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

Host cells

As used herein, the term "host cell" refers to a cell that can be used to introduce a vector, including, but not limited to, prokaryotic cells such as e.g. escherichia coli or bacillus subtilis, eukaryotic cells such as microbial cells, fungal cells, animal cells and plant cells.

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

Regulatory element

As used herein, the term "regulatory element" is intended to include promoters, enhancers, internal Ribosome Entry Sites (IRES), and other expression control elements (e.g., transcription termination signals, such as polyadenylation signals and poly U sequences), the detailed description of which may be found in goldel (Goeddel), gene expression techniques: methods of enzymology (GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY) 185, academic Press (Academic Press), san Diego (San Diego), calif. (1990). In some cases, regulatory elements include those sequences that direct constitutive expression of a nucleotide sequence in many types of host cells as well as those sequences that direct expression of the nucleotide sequence in only certain host cells (e.g., tissue-specific regulatory sequences). Tissue-specific promoters may primarily direct expression in a desired tissue of interest, such as muscle, neurons, bone, skin, blood, specific organs (e.g., liver, pancreas), or specific cell types (e.g., lymphocytes). In some cases, regulatory elements may also direct expression in a time-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. In certain instances, the term "regulatory element" encompasses enhancer elements, such as WPRE; a CMV enhancer; the R-U5' fragment in the LTR of HTLV-I (mol. Cell. Biol., volume 8 (1), pages 466-472, 1988), the SV40 enhancer, and the intron sequence between exons 2 and 3 of rabbit beta-globin (Proc. Natl. Acad. Sci. USA., volume 78 (3), pages 1527-31, 1981).

Promoters

As used herein, the term "promoter" has a meaning well known to those skilled in the art and refers to a non-coding nucleotide sequence located upstream of a gene that is capable of initiating expression of a downstream gene. Constitutive (constitutive) promoters are nucleotide sequences of: when operably linked to a polynucleotide encoding or defining a gene product, it results in the production of the gene product in the cell under most or all physiological conditions of the cell. An inducible promoter is a nucleotide sequence which, when operably linked to a polynucleotide encoding or defining a gene product, results in the production of the gene product in a cell, essentially only when an inducer corresponding to the promoter is present in the cell. Tissue specific promoters are nucleotide sequences that: when operably linked to a polynucleotide encoding or defining a gene product, it results in the production of the gene product in the cell substantially only if the cell is a cell of the tissue type to which the promoter corresponds.

NLS

A "nuclear localization signal" or "nuclear localization sequence" (NLS) is an amino acid sequence that "tags" a protein for introduction into the nucleus by nuclear transport, i.e., a protein with NLS is transported to the nucleus. Typically, NLS contains positively charged Lys or Arg residues exposed at the protein surface. Exemplary nuclear localization sequences include, but are not limited to, NLS from: SV40 large T antigen, EGL-13, c-Myc, and TUS proteins. In some embodiments, the NLS comprises PKKKRKV sequence. In some embodiments, the NLS comprises the AVKRPAATKKAGQAKKKKLD sequence. In some embodiments, the NLS comprises the PAAKRVKLD sequence. In some embodiments, the NLS comprises the MSRRRKANPTKLSENAKKLAKEVEN sequence. In some embodiments, the NLS comprises the KLKIKRPVK sequence. Other nuclear localization sequences include, but are not limited to, the acidic M9 domain of hnRNP A1, the sequences KIPIK and PY-NLS in yeast transcription repressor Mat. Alpha.2.

Operatively connected to

As used herein, the term "operably linked" is intended to mean that the nucleotide sequence of interest is linked to the one or more regulatory elements in a manner that allows for expression of the nucleotide sequence (e.g., in an in vitro transcription/translation system or in a host cell when the vector is introduced into the host cell).

Complementarity and method of detecting complementary

As used herein, the term "complementarity" refers to the ability of a nucleic acid to form one or more hydrogen bonds with another nucleic acid sequence by means of a conventional watson-crick or other non-conventional type. Percent complementarity means the percentage of residues in a nucleic acid molecule that can form hydrogen bonds (e.g., watson-Crick base pairing) with a second nucleic acid sequence (e.g., 5, 6, 7, 8, 9, 10 of 10 are 50%, 60%, 70%, 80%, 90%, and 100% complementary). "fully complementary" means that all consecutive residues of one nucleic acid sequence form hydrogen bonds with the same number of consecutive residues in one second nucleic acid sequence. "substantially complementary" as used herein 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.

Stringent conditions

As used herein, "stringent conditions" for hybridization refers to conditions under which a nucleic acid having complementarity to a target sequence hybridizes predominantly to the target sequence and does not substantially hybridize to non-target sequences. Stringent conditions are typically sequence-dependent and will vary depending on a number of factors. In general, the longer the sequence, the higher the temperature at which the sequence specifically hybridizes to its target sequence.

Hybridization

The term "hybridization" or "complementary" or "substantially complementary" means that a nucleic acid (e.g., RNA, DNA) comprises a nucleotide sequence that enables it to bind non-covalently, i.e., form base pairs and/or G/U base pairs with another nucleic acid in a sequence-specific, antiparallel manner (i.e., the nucleic acid specifically binds to the complementary nucleic acid), "anneal" or "hybridize".

Hybridization requires that the two nucleic acids contain complementary sequences, although there may be mismatches between bases. Suitable conditions for hybridization between two nucleic acids depend on the length of the nucleic acids and the degree of complementarity, variables well known in the art. Typically, the hybridizable nucleic acid is 8 nucleotides or more in length (e.g., 10 nucleotides or more, 12 nucleotides or more, 15 nucleotides or more, 20 nucleotides or more, 22 nucleotides or more, 25 nucleotides or more, or 30 nucleotides or more).

It will be appreciated that the sequence of a polynucleotide need not be 100% complementary to the sequence of its target nucleic acid to specifically hybridize. Polynucleotides may comprise 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, 99% or more, 99.5% or more, or 100% sequence complementarity to a target region in a target nucleic acid sequence to which it hybridizes.

Hybridization of the target sequence to the gRNA represents that at least 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of the target sequence and the nucleic acid sequence of the gRNA can hybridize to form a complex; or at least 12, 15, 16, 17, 18, 19, 20, 21, 22 or more bases of the nucleic acid sequence representing the target sequence and the gRNA may be complementarily paired and hybridized to form a complex.

Expression of

As used herein, the term "expression" refers to a process whereby a polynucleotide is transcribed from a DNA template (e.g., into mRNA or other RNA transcript) and/or a process whereby the transcribed mRNA is subsequently translated into a peptide, polypeptide, or protein. Transcripts and encoded polypeptides may be collectively referred to as "gene products". If the polynucleotide is derived from genomic DNA, expression may include splicing of mRNA in eukaryotic cells.

Joint

As used herein, the term "linker" refers to a linear polypeptide formed from multiple amino acid residues joined by peptide bonds. The linker of the invention may be an amino acid sequence that is synthesized artificially, or a naturally occurring polypeptide sequence, such as a polypeptide having the function of a hinge region. Such linker polypeptides are well known in the art (see, e.g., holliger, P. Et al (1993) Proc. Natl. Acad. Sci. USA90:6444-6448; poljak, R.J. Et al (1994) Structure 2:1121-1123).

Treatment of

As used herein, the term "treating" refers to treating or curing a disorder, delaying the onset of symptoms of a disorder, and/or delaying the progression of a disorder.

A subject

As used herein, the term "subject" includes, but is not limited to, various animals, plants, and microorganisms.

Animals

Such as mammals, e.g., bovine, equine, ovine, porcine, canine, feline, lagomorph (e.g., mice or rats), non-human primate (e.g., macaque or cynomolgus) or human. In certain embodiments, the subject (e.g., human) has a disorder (e.g., a disorder resulting from a disease-related gene defect).

Plants and methods of making the same

The term "plant" is understood to mean any differentiated multicellular organism capable of photosynthesis, including crop plants at any stage of maturity or development, in particular monocotyledonous or dicotyledonous plants, vegetable crops, including artichoke, broccoli, sesame seed, leek, asparagus, lettuce (e.g., head lettuce, leaf lettuce), cabbage (bok choy), yellow arrowroot, melons (e.g., melon, watermelon, columbian melon (crenhaw), white melon, cantaloupe), rape crops (e.g., cabbage, broccoli, chinese cabbage, kohlrabi, chinese cabbage), artichoke, carrot, cabbage (napa), okra, onion, celery, parsley, chick pea, parsnip, chicory, pepper, potato, cucurbit (e.g., zucchini, cucumber, zucchini, melon, pumpkin), radish, dried onion, turnip cabbage, purple eggplant (also known as eggplant), salon, chicory, shallot, chicory, garlic, spinach, green onion, melon, green leafy vegetables (greens), beet (sugar beet and fodder beet), sweet potato, lettuce, horseradish, tomato, turnip, spice; fruit and/or vining crops, such as apples, apricots, cherries, nectarines, peaches, pears, plums, prunes, cherries, quince, almonds, chestnuts, hazelnuts, pecans, pistachios, walnuts, oranges, blueberries, boysenberries (boysenberries), redberries, currants, rowfruits, raspberries, strawberries, blackberries, grapes, avocados, bananas, kiwi fruits, persimmons, pomegranates, pineapple, tropical fruits, pome fruits, melons, mangoes, papaya, and litchis; field crops, such as clover, alfalfa, evening primrose, white mango, corn/maize (forage maize, sweet maize, popcorn), hops, jojoba, peanuts, rice, safflower, small grain cereal crops (barley, oat, rye, wheat, etc.), sorghum, tobacco, kapok, leguminous plants (beans, lentils, peas, soybeans), oleaginous plants (rape, mustard, poppy, olives, sunflower, coconut, castor oil plants, cocoa beans, groundnut), arabidopsis, fibrous plants (cotton, flax, hemp, jute), camphorridae (cinnamon, camphordons), or a plant such as coffee, sugar cane, tea, and natural rubber plants; and/or flower bed plants, such as flowering plants, cactus, fleshy plants and/or ornamental plants, and trees, such as forests (broadleaf and evergreen trees, e.g., conifers), fruit trees, ornamental trees, and nut-bearing trees, and shrubs and other seedlings.

Advantageous effects of the application

The application discovers a novel Cas enzyme, and Blast results show that the Cas enzyme has lower consistency with the reported Cas enzyme, belongs to a novel Cas protein, and has wide application prospect.

Embodiments of the present application will be described in detail below with reference to the accompanying drawings and examples, but it will be understood by those skilled in the art that the following drawings and examples are only for illustrating the present application and are not to be construed as limiting the scope of the present application. Various objects and advantageous aspects of the present application will become apparent to those skilled in the art from the following detailed description of the preferred embodiments and the accompanying drawings.

Drawings

FIG. 1 results of detection of single stranded nucleic acids by Cas-sf 4274.

FIG. 2 results of detection of single stranded nucleic acids by Cas-sf 2201.

FIG. 3 results of detection of single stranded nucleic acids by Cas-sf 2771.

The PAM structure of cas-sf 4274.

Fig. 5 PAM structure of cas-sf 2201.

FIG. 6 PAM structure of Cas-sf 2771.

FIG. 7 cleavage results of double-stranded nucleic acid by Cas-sf 4274.

FIG. 8 cleavage results of double-stranded nucleic acid by Cas-sf 2201.

FIG. 9 cleavage results of double-stranded nucleic acid by Cas-sf 2771.

FIG. 10 cleavage results of double-stranded nucleic acid by Cas-sf 2586.

FIG. 11. Position of Cas-sf4274 cleavage of target nucleic acid.

FIG. 12. Position of Cas-sf2201 cleavage of target nucleic acid.

FIG. 13. Position of cleavage of target nucleic acid by Cas-sf 2771.

FIG. 14. Position of Cas-sf2586 cleavage target nucleic acid.

FIG. 15-Cas-sf 4274 complementary strand (TS) and non-complementary strand (NTS) cleavage efficiency.

FIG. 16-Cas-sf 2201 complementary strand (TS) and non-complementary strand (NTS) cleavage efficiency.

FIG. 17 Cas-sf2771 complementary strand (TS) and non-complementary strand (NTS) cleavage efficiency.

FIG. 18-Cas-sf 2586 complementary strand (TS) and non-complementary strand (NTS) cleavage efficiency.

FIG. 19 shows the results of target gene sequencing after editing eukaryotic cells with Cas-sf 2201.

Sequence information

Detailed Description

The following examples are only intended to illustrate the invention and are not intended to limit it. The experiments and methods described in the examples were performed substantially in accordance with conventional methods well known in the art and described in various references unless specifically indicated. For example, for the conventional techniques of immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics and recombinant DNA used in the present invention, reference may be made to Sambrook (Sambrook), friech (Fritsch) and manitis (Maniatis), molecular cloning: laboratory Manual (MOLECULAR CLONING: A LABORATORY MANUAL), edit 2 (1989); the handbook of contemporary molecular biology (CURRENT PROTOCOLS IN MOLECULAR BIOLOGY) (edited by f.m. ausubel (f.m. ausubel) et al, (1987)); series (academic publishing company) of methods in enzymology (METHODS IN ENZYMOLOGY): PCR 2: practical methods (PCR 2:A PRACTICAL APPROACH) (m.j. Maxfresen (m.j. Macpherson), b.d. black ms (b.d. hames) and g.r. taylor (1995)), harlow and Lane (Lane) edits (1988), antibodies: laboratory Manual (ANTIBODIES, ALABORATORY Manual), animal cell CULTURE (ANIMAL CELL CULTURE) (R.I. French Lei Xieni (R.I. Freshney) eds. (1987)).

In addition, the specific conditions are not specified in the examples, and the process is carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention. Those skilled in the art will appreciate that the examples describe the invention by way of example and are not intended to limit the scope of the invention as claimed. All publications and other references mentioned herein are incorporated by reference in their entirety.

EXAMPLE 1 Cas protein acquisition

The inventors analyzed the macrogenome of the uncultured, and identified 4 new Cas enzymes by redundancy removal, protein cluster analysis. Blast results show that Cas proteins have lower sequence identity to the reported Cas proteins, which are designated Cas-sf2201, cas-sf4274, cas-sf2771 and Cas-sf2586 in the present invention; the amino acid sequence, coding nucleic acid sequence and codon optimized nucleic acid of the proteins are shown in tables 1-3 below, and the homologous repeat sequences of the gRNAs corresponding to the different proteins are shown in Table 4.

TABLE 1 amino acid sequence of Cas protein

TABLE 2 nucleic acid sequence of Cas proteins

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TABLE 3 nucleic acid sequence after codon optimization of Cas protein

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TABLE 4 homologous repeat of gRNA corresponding to Cas protein

Example 2 application of Cas-sf4274 protein in nucleic acid detection

This example demonstrates the trans-cleavage activity of Cas-sf4274 by in vitro assays. The Cas-sf4274 protein is guided in this example to recognize and bind to the target nucleic acid using a gRNA that can be paired with the target nucleic acid; subsequently, the Cas-sf4274 protein triggers trans cleavage activity on any single stranded nucleic acid, thereby cleaving the single stranded nucleic acid detector in the system; fluorescent groups and quenching groups are respectively arranged at two ends of the single-stranded nucleic acid detector, and if the single-stranded nucleic acid detector is cut, fluorescence is excited; in other embodiments, both ends of the single-stranded nucleic acid detector may be provided with a label that can be detected by colloidal gold.

In this example, the target nucleic acid was selected to be single-stranded DNA, and N-B-i3g1-ssDNA0, which has the sequence: CGACATTCCGAAGAACGCTGAAGCGCTGGGGGCAAATTGTGCAATTTGCGGC;

the gRNA sequence is:

GUUGCAAUGCCUAAUCAAAUUGUGUCGAUAUGGACACCCCCCAGCGCUUCAGCGUUC(the underlined region is the region targeted to the target nucleic acid);

the single-stranded nucleic acid reporter molecule sequence is FAM-TTATT-BHQ1;

the following reaction system is adopted: cas-sf4274 final concentration is 100nM, gRNA final concentration is 200nM, target nucleic acid final concentration is 200nM, single-stranded nucleic acid reporter final concentration is 500nM. FAM fluorescence/20 s was read by incubation at 37 ℃. The control group had no target nucleic acid added.

As shown in FIG. 1, cas-sf4274 is able to cleave a single stranded nucleic acid reporter molecule for detection in a system that rapidly reports fluorescence in the presence of target nucleic acid as compared to a control without target nucleic acid; in the absence of target nucleic acid, there is no change in fluorescent signal. The above experiments reflect that Cas-sf4274, in combination with a single stranded nucleic acid reporter, can be used for detection of target nucleic acids. In FIG. 1, 1 is an experimental group to which a target nucleic acid was added, and 2 is a control group to which no target nucleic acid was added.

EXAMPLE 3 application of Cas protein in nucleic acid detection

The application of Cas-sf2201, cas-sf2771 in nucleic acid detection was verified using the same method as example 2.

The results of Cas-sf2201 are shown in fig. 2, with Cas-sf2201 being able to cleave a single-stranded nucleic acid reporter molecule for detection in a system, rapidly reporting fluorescence, in the presence of a target nucleic acid, compared to a control without target nucleic acid; in the absence of target nucleic acid, there is no change in fluorescent signal. The results of Cas-sf2771 are shown in fig. 3, where Cas-sf2771 is able to cleave a single-stranded nucleic acid reporter molecule for detection in a system in the presence of a target nucleic acid, reporting fluorescence rapidly, compared to a control without target nucleic acid; in the absence of target nucleic acid, there is no change in fluorescent signal. The above experiments reflect that Cas-sf2201, cas-sf2771, in combination with a single stranded nucleic acid reporter, can be used for detection of target nucleic acids. In FIGS. 2 and 3, 1 is an experimental group to which a target nucleic acid was added, and 2 is a control group to which no target nucleic acid was added.

EXAMPLE 4 PAM identification of Cas-sf4274 protein

Construction of Cas-sf4274 protein expression plasmid: the nucleic acid sequence is subjected to genetic synthesis after being subjected to humanized codon optimization, and is connected with an escherichia coli expression vector PeT28 (a) +vector. The addition of the JM23119 promoter to vector PeT28 (a) + -Cas-sf4274 initiates transcription of Cas-sf4274 gRNA. Forming a carrier: peT28 (a) + -Cas-sf4274-JM23119-gRNA, gRNA sequence: GUUGCAAUGCCUAAUCAAAUUGUGUCGAUAUGGACACUCCCCUACGUGCUGCUGAAGUUGCUnderlined is the target sequence; construction of PAM library: synthetic sequence CGTGTTTCGTAAAGTCTGGAAACGCGGAAGCCCCCAGCGCTTCAGCGTTCNNNNNNTCCCCTACGTGCTGCTGAAGTTGCCCGCAA, N is a random deoxynucleotide, underlined is the target sequence. The vector was filled in with Klenow enzyme and ligated into the pacyc184 vector. And (3) converting escherichia coli, and extracting plasmids to form a PAM library.

PAM library subtraction experiment: preparation of competence: BL21 (DE 3) -PeT28 (a) + -Cas-sf4274-JM23119-gRNA. PAM library plasmid transformation competence: BL21 (DE 3) -PeT28 (a) + -Cas-sf4274-JM23119-gRNA is coated on LB plates containing kanamycin and chloramphenicol, bacterial cells are collected after overnight culture at 37 ℃, the bacterial liquid concentration is adjusted to OD 600.6-0.8, IPTG 0.2mM is added, and induction is carried out at 37 ℃ for 4 hours. FastPure EndoFree Plasmid Maxi Kit (vazyme) plasmid extraction was performed to obtain post-subtraction PAM libraries. Primer: PAM-F: GGTCTTCGGTTTCCGTGTT; PAM-R: TGGCGTTGACTCTCAGTCAT. The control samples were obtained by PCR using 30 ng/. Mu.L of plasmid (PAM library) as a template primer, and the experimental samples were obtained by PCR using 30 ng/. Mu.L of plasmid (PAM library after subtraction) as a template. Data analysis was performed on control samples and experimental samples by second generation sequencing to obtain the PAM sequence for Cas-sf4274, and weblog was used to map, and PAM structure was found to be 5'-TTN-3', where n=a/T/C/G, as shown in fig. 4.

EXAMPLE 5 PAM identification of Cas-sf2201, cas-sf2771 protein

PAM of Cas-sf2201, cas-sf2771 proteins was identified using the same method as example 4.

The PAM structure of Cas-sf2201 is 5'-TTH-3', where h=a/T/C, as shown in fig. 5. The PAM structure of Cas-sf2771 is 5'-TTN-3', where n=a/T/C/G, as shown in fig. 6.

EXAMPLE 6 application of Cas-sf4274 protein in double-stranded nucleic acid editing

This example detects the cis-cleavage activity of double-stranded DNA of Cas-sf4274 in vitro. In this example, the Cas-sf4274 protein is recognized and bound to the target nucleic acid by using the gRNA that can be paired with the target nucleic acid, thereby cleaving the target nucleic acid in the system, and performing agarose electrophoresis detection on the cleaved target nucleic acid.

In this example, the target nucleic acid was selected to be double-stranded DNA (plasmid), 5space 1-PAM, which has the sequence: CATTAGATCTGTGTGGCCAANNNTCCCCTACGTGCTGCTGAAGTTGCLigating into Vector T-Vector-pEASY-Blunt Simple Cloning Vector; the italic part is PAM sequence, n=a/T/C/G, the underlined region is the targeting region.

gRNA:Cas-sf4274-5spacer1:

GUUGCAAUGCCUAAUCAAAUUGUGUCGAUAUGGACACUCCCCUACGUGCUGCUGAAG(underlined region is the targeting region)

The following reaction system is adopted: 20. Mu.L of system, cas-sf4274 final concentration 100nM, gRNA final concentration 200nM, double-stranded target nucleic acid final concentration 5 ng/. Mu.L. Incubation at 37℃for 1h and 85℃for 20min. The cleavage products were subjected to agarose electrophoresis to detect Cas-sf4274 cleavage capacity. The experimental group added Cas-sf4274 protein, gRNA and target nucleic acid, and the control group (CK) did not add gRNA.

The results are shown in fig. 7, where Cas-sf4274 is able to cleave double stranded nucleic acids in the system, showing a distinct cleavage band in the experimental group with PAM of 5'-TTN-3' (where n=a/T/C/G) compared to the control without gRNA. This suggests that Cas-sf4274 may be used for cleavage and editing of double stranded target nucleic acids with PAM of 5'-TTN-3', where n=a/T/C/G.

Example 7 application of Cas-sf2201, cas-sf2771, cas-sf2586 proteins in double-stranded nucleic acid editing

The cis cleavage activity of double-stranded DNA of Cas-sf2201, cas-sf2771, cas-sf2586 was detected in vitro using the same method as in example 4.

The results of cis cleavage activity of double stranded DNA of Cas-sf2201 protein are shown in fig. 8, with PAM of 5'-TTN-3' (where n=a/T/C/G) in the experimental group able to cleave double stranded nucleic acid in the system, showing a distinct cleavage band compared to the control without gRNA.

The results of cis cleavage activity of double stranded DNA of Cas-sf2771 protein are shown in fig. 9, where Cas-sf2771 is able to cleave double stranded nucleic acids in the system in the experimental group with PAM of 5'-TTN-3' (where n=a/T/C/G) compared to the control without gRNA, showing a distinct cleavage band.

The results of cis cleavage activity of double stranded DNA of Cas-sf2586 protein are shown in fig. 10, and Cas-sf2586 in the experimental group with PAM of 5'-ATT-3', 5'-ATC-3' is able to cleave double stranded nucleic acid in the system, showing a distinct cleavage band, compared to the control without gRNA.

This suggests that Cas-sf2201, cas-sf2771 can be used for cleavage and editing of double-stranded target nucleic acid with PAM of 5'-TTN-3' (where n=a/T/C/G), cas-sf2586 can be used for cleavage and editing of double-stranded target nucleic acid with PAM of 5'-ATT-3', 5 '-ATC-3'.

Example 8 Cas-sf4274 proteinCutting characteristics-cutting position of (a)

The cleavage site of Cas-sf4274 protein on the complementary strand TS and non-complementary strand NTS of the double-stranded target nucleic acid was determined by in vitro detection in this example. The Cas-sf4274 protein is guided by gRNA to recognize and bind to double stranded target nucleic acid in this example; cas proteins trigger cis cleavage activity on double-stranded target nucleic acids, thereby cleaving double-stranded target nucleic acids in the system. And (3) adding the adaptor with the T after the supplement A to the double-stranded target nucleic acid after cutting, and carrying out sanger sequencing after PCR enrichment on the connected product.

In this example, the target nucleic acid was selected to be double-stranded DNA (plasmid), the sequence: CATTAGATCTGTGTGGCCAATTCTCCC CTACGTGCTGCTGAAGTTGC was ligated into Vector T-Vector-pEASY-Blunt Simple Cloning Vector, with the PAM sequence in italics and the targeting region in underlined.

gRNA：Cas-sf4274-5spacer1:

GUUGCAAUGCCUAAUCAAAUUGUGUCGAUAUGGACACUCCCCUACGUGCUGCUGAAG(underlined regions are targeting regions).

The following reaction system is adopted: 50. Mu.L of system, cas-sf4274 100nM, gRNA 250nM, double-stranded target nucleic acid 10 ng/. Mu.L (plasmid). Cas protein, gRNA, incubation for 10min at 25 ℃; adding double-stranded target nucleic acid, incubating for 1h at 37 ℃ and incubating for 5min at 85 ℃; 50uL 2X Taq DNA Polymerase Mix (Nuozan) (1:1) is added into the system, and the reaction is carried out for 30min at 72 ℃; the reaction liquid is subjected to liquid recovery; the recovery liquid was added to 2. Mu.L of annealed primer 2. Mu.M (TK-117: CGGCATTCCTGAACCGTCTCTTTCCGATCT, TK-111: GATCGGAAGAGCGGTTCAGCAGGAATGCCG), T4 (NEB) ligase at 22℃for 1h. 10. Mu.L of ligation product was taken, primer S1-PAM-after: ACTCAGCGGCATTCCTGCTGAACCGC, PQ0275-F: CCGTATTACCGCCTTTGAG,2X Taq DNA Polymerase Mix (Northenzan) were subjected to PCR reactions. The PCR products were subjected to sanger sequencing. As a result, as shown in FIG. 11, the Cas-sf4274 protein cleaves the target nucleic acid with cleavage sites in the middle of 23-24, 28-29, 30-31nt of NTS and 22-23nt of TS. That is, the cleavage site of the complementary strand of the target sequence by Cas-sf4274 is between 22nt and 23nt at the 5 'end of the PAM complementary sequence, the cleavage site of the non-complementary strand of the target sequence by Cas-sf4274 is between 23nt and 24nt or between 28nt and 29nt or between 30nt and 31nt at the 3' end of the PAM sequence, and the gRNA directs Cas-sf4274 protein to recognize and bind to the complementary strand on which the non-complementary strand is a DNA strand paired with the complementary strand.

Example 9 cleavage Properties of Cas-sf2201, cas-sf2771, cas-sf2586 protein-cleavage position

The positions of the complementary strand and the non-complementary strand of the double-stranded target nucleic acid were cleaved by the proteins Cas-sf2201, cas-sf2771, cas-sf2586 in vitro using the same method as in example 8.

Results for Cas-sf2201 protein as shown in figure 12, cas-sf2201 protein cleaves target nucleic acid at a position intermediate 25-26, 28-29nt of NTS and 22-23nt of TS. That is, the cleavage site of the complementary strand of Cas-sf2201 to the target sequence is between 22nt and 23nt at the 5 'end of the PAM complementary sequence, the cleavage site of the non-complementary strand of Cas-sf2201 to the target sequence is between 25nt and 26nt or between 28nt and 29nt at the 3' end of the PAM sequence, and the gRNA directs Cas-sf2201 protein to recognize and bind to the complementary strand, which is the DNA strand paired with the complementary strand.

The results for the Cas-sf2771 protein are shown in fig. 13, with the Cas-sf2771 protein cleavage site in between 18-19nt of NTS and 22-23nt of TS when cleaving the target nucleic acid. That is, the cleavage site of the complementary strand of the target sequence by Cas-sf2771 is between 22nt and 23nt at the 5 'end of the PAM complementary sequence, the cleavage site of the non-complementary strand of the target sequence by Cas-sf2771 is between 18nt and 19nt at the 3' end of the PAM sequence, and the gRNA directs Cas-sf2771 protein to recognize and bind to the complementary strand, which is the DNA strand paired with the complementary strand.

The results for the Cas-sf2586 protein are shown in figure 14, with the Cas-sf2586 protein cleavage site in the middle of 24-25nt of NTS when cleaving the target nucleic acid. That is, the cleavage site of Cas-sf2586 for the non-complementary strand of the target sequence, which is the DNA strand paired with the complementary strand, is between 24nt and 25nt at the 3' end of the PAM sequence, and the gRNA directs Cas-sf2586 protein to recognize and bind to the complementary strand.

EXAMPLE 10 cleavage Property of Cas-sf4274 protein-NTS/TS cleavage efficiency

This example detects the cleavage efficiency of Cas-sf4274 on target nucleotide double-stranded DNA complementary strand (TS), non-complementary strand (NTS). 5'6-FAM labeled non-complementary strand (NTS), 5' ROX labeled complementary strand (TS), and gRNA directs the recognition and binding of Cas-sf4274 protein to the target nucleic acid, thereby cleaving the target nucleic acid in the system, and performing capillary electrophoresis detection (ABI 3730xl genetic analyzer) of the cleaved target nucleic acid. The DNA fragments migrate from the cathode to the anode in the gel, are arranged according to the length of the fragments, when migrating to the scanning window of the laser scanner at the anode end, the fluorescent dye is excited to emit light with a certain wavelength, the light is recorded according to the fluorescence intensity, the electrophoresis track of each DNA fragment with the fluorescent dye is recorded according to the actual time of each DNA fragment passing through the laser scanning window, and each fragment is represented by a fluorescence absorption peak. The higher the peak, the more the fragment amount; the time at which the peak occurs has a direct relationship with the fragment size, the smaller the fragment the earlier the peak occurs. FAM fluorescence, cas-sf4274 uncleaved NTS fragment size was 380nt, cas-sf4274 cleaved NTS fragment approximately 126nt. ROX fluorescence, cas-sf4274 uncleaved TS fragment size was 380nt, cas-sf4274 cleaved TS fragment was about 254nt. Fragment cutting efficiency calculation formula: cutting efficiency = cutting peak area/(cutting peak area + non-cutting peak area)

In this example, the target nucleic acid was selected to be double-stranded DNA (PCR product), and the primer:

XQ0001-5FAM：GTATGTTGTGTGGAATTGTG 5'6-FAM；

XQ0002-5ROX：GCTGCGCGTAACCACCACAC 5'ROX

the sequence of the amplified product is as follows:

GTATGTTGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGCTATGACCATGATTACGCCAAGCTGCCCTTCATTAGATCTGTGTGGCCAATTCTCCCCTACGTGCTGCTGAAGTTGCAAGGGCAGCTTCAATTCGCCCTATAGTGAGTCGTATTACAATTCACTGGCCGTCGTTTTACAACGTCGTGACTGGGAAAACCCTGGCGTTACCCAACTTAATCGCCTTGCAGCACATCCCCCTTTCGCCAGCTGGCGTAATAGCGAAGAGGCCCGCACCGATCGCCCTTCCCAACAGTTGCGCAGCCTGAATGGCGAATGGACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGC; the italic portion is PAM sequence and the underlined region is the targeting region.

Cas-sf4274-5spacer1:

GUGGGAACCCUUCCUGAUGGCUCGAUCCGUCGAGACUCCCCUACGUGCUGCUGAAG(underlined regions are targetedZone(s)

The following reaction system is adopted: 20. Mu.L of system, cas-sf4274, 50nM, gRNA100nM, 1. Mu.L of double-stranded target nucleic acid (PCR product). Incubation is carried out at 37 ℃ for 5min, 15min, 30min and 60min, and protease K1 ng/. Mu.l is incubated at room temperature for 20min. Capillary electrophoresis detection (ABI 3730xl genetic analyzer) FAM, ROX. The software Gene mapper 4.1 performs data analysis to calculate the NTS/TS cleavage efficiency.

The results are shown in FIG. 15, where Cas-sf4274 preferentially cleaves NTS.

EXAMPLE 11 cleavage Properties of Cas-sf2201, cas-sf2771, cas-sf2586 proteins-NTS/TS cleavage Effect Rate of

The cleavage efficiency of the proteins Cas-sf2201, cas-sf2771, cas-sf2586 on the target nucleotide double-stranded DNA complementary strand (TS), non-complementary strand (NTS) was examined in vitro using the same method as in example 10.

Results for the Cas-sf2201 protein are shown in figure 16, with Cas-sf2201 simultaneously cleaving TS/NTS.

The results for the Cas-sf2771 protein are shown in figure 17, with Cas-sf2771 preferentially cleaving NTS.

The results for the Cas-sf2586 protein are shown in figure 18, with Cas-sf2586 preferentially cleaving NTS.

EXAMPLE 12 editing efficiency of Cas-sf4274, cas-sf2771 protein in animal cells

Verifying the activity of Cas-sf4274, cas-sf2771 gene editing in animal cells, designing target gR3 for chinese hamster ovary Cells (CHO) FUT8 genes: CAGCCAAGGTTGTGGACGGATCA. The vector pcDNA3.3 is transformed to carry ECFP fluorescent protein genes. Insertion of SV40 NLS-Cas-sf4274-NLS fusion protein through cleavage site BsmB 1; the U6 promoter and the gRNA sequence are inserted through the enzyme cutting site Mfe 1. The CMV promoter initiates expression of the fusion protein SV40 NLS-Cas-sf 4274-NLS-ECFP. The protein Cas-sf4274-NLS is linked to the protein ECFP with the linking peptide T2A. After pUC19 vector is modified, promoter EF-1 alpha starts tdTomato-T2A-GF (gR 3) FP gene expression. After the Cas-sf4274 protein recognition target gR3 is edited, the proportion of GFP positive cells analyzed in CFP (circulating fluid plasma) and tdTomato double-positive cells is Cas-sf4274 protein editing efficiency.

And (3) paving: 293T cells were plated at a confluency of 70-80% and the number of seeded cells in 12 well plates was 1.5 x 10≡5 cells/well.

Transfection: transfection was performed by plating for 12-24h, and 2. Mu.l Hieff Trans was added to 100. Mu.l opti-MEM ^TM A liposome nucleic acid transfection reagent, uniformly mixing and standing for 5 minutes at room temperature; mu.l opti-MEM was added with 1ug of plasmid (pcDNA3.3: pUC19=1:1) and mixed well. Diluted Hieff Trans ^TM The liposome nucleic acid transfection reagent is mixed with the diluted plasmid uniformly and incubated for 20min at room temperature. And adding the incubated mixed solution into a culture medium paved with cells for transfection, and changing the transfection into a normal culture medium for 24 hours for continuous culture for 24 hours. Flow cytometry was used for analysis.

The analysis results show that: the editing efficiency of Cas-sf4274 is 5.05%, and the editing efficiency of Cas-sf2771 is 0.04%.

EXAMPLE 13 editing efficiency of Cas-sf2201 protein in animal cells

The activity of Cas-sf2201 protein gene editing was verified in animal cells, and targets were designed for Chinese Hamster Ovary (CHO) FUT8 genes. The vector pcDNA3.3 is transformed to carry EGFP fluorescent protein. Insertion of SV40 NLS-Cas-sf2201-NLS fusion protein through cleavage site BsmB 1; the U6 promoter and the gRNA sequence are inserted through the enzyme cutting site Mfe 1. The CMV promoter initiates expression of the fusion protein SV40 NLS-Cas-sf 2201-NLS-GFP. The protein Cas-sf2201-NLS is linked to the protein GFP with the linker peptide T2A.

And (3) paving: CHO cells were plated at a confluence of 70-80% and the number of cells seeded in 12 well plates was 8 x 10 x 4 cells/well.

Transfection: plating for 12-24h for transfection, adding 2ug plasmid into 100 mu L opti-MEM, and mixing well; 4. Mu.L of diluted plasmid was addedEL Transfection Reagent (TRAN), incubated at room temperature for 15-20min. The incubated mixture is added to the cell-plated medium for transfection. The normal medium is replaced after 24h of transfection, and GFP positive cells are separated after 48h of flow transfection.

Extracting DNA, PCR amplifying the vicinity of the editing region, and carrying out hiTOM sequencing: the collected GFP positive cells were subjected to genomic DNA extraction by a cell/tissue genomic DNA extraction kit (Baitaike). Genomic DNA was amplified by primer PQ0106-FUT8-HiTom-F1: ggagtgagtacggtgtgCGAGTTCTGTTGCATGGTAGG; PQ0106-FUT8-HiTom-R1: GAGTTGGATGCTGGATGGGCCAAGCTTCTTGGTGGTTTC amplified region near the target. The PCR products were subjected to hiTOM sequencing (http:// 121.40.237.174/HiTOM/sample_acceptance_sang. Php).

Sequencing data analysis, and statistics of sequence types and proportions in a target range to obtain editing efficiency of the Cas-sf2201 protein on target positions.

CHO cell FUT8 gene target sequence: gR3-FUT8:

TTCCAGCCAAGGTTGTGGACGGATCAthe italic portion is PAM sequence and the underlined region is the targeting region.

gRNA sequence

GUUGCAACGGCUGAGAAUUGCGUCUUCCGUUGACGCCAGCCAAGGUUGUGGACGGAUCAThe underlined region is the targeting region.

Analysis results show that the editing efficiency of the Cas-sf2201 in a target gR3-Cas12i3-target-FUT8 of CHO cells is 5.18%, the editing type is InDel, and the partial sequencing result of the edited target nucleic acid is shown in FIG. 19.

Although specific embodiments of the invention have been described in detail, those skilled in the art will appreciate that: many modifications and variations of details may be made to adapt to a particular situation and the invention is intended to be within the scope of the invention. The full scope of the invention is given by the appended claims together with any equivalents thereof.

Claims

1. A Cas protein, characterized in that the Cas protein is any one of the following I-III:

I. the amino acid sequence of the Cas protein has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% sequence identity compared to any of SEQ ID nos. 1-4, and substantially retains the biological function of the sequence from which it is derived;

II. The amino acid sequence of the Cas protein has a sequence of substitution, deletion, or addition of one or more amino acids as compared to any one of SEQ ID nos. 1-4, and substantially retains the biological function of the sequence from which it is derived;

III, the Cas protein comprises an amino acid sequence shown in any one of SEQ ID No. 1-4.

2. A fusion protein comprising the Cas protein of claim 1 and other modifying moieties.

3. An isolated polynucleotide, wherein the polynucleotide is a polynucleotide sequence encoding the Cas protein of claim 1 or a polynucleotide sequence encoding the fusion protein of claim 2.

4. A gRNA capable of binding to the Cas protein of claim 1.

5. A homeotropic sequence comprising any of SEQ ID nos. 13 to 17.

6. A vector comprising the polynucleotide of claim 3 operably linked to regulatory elements.

7. A CRISPR-Cas system, characterized in that it comprises the Cas protein of claim 1 and at least one gRNA of claim 4.

8. A carrier system comprising one or more carriers, the one or more carriers comprising:

a) A first regulatory element operably linked to the gRNA of claim 4,

b) A second regulatory element operably linked to the Cas protein of claim 1;

9. A composition, characterized in that it comprises:

(i) A protein component selected from the group consisting of: the Cas protein of claim 1 or the fusion protein of claim 2;

(ii) A nucleic acid component selected from the group consisting of: the gRNA of claim 4, or a nucleic acid encoding the gRNA of claim 4, or a precursor RNA of the gRNA of claim 4, or a precursor RNA nucleic acid encoding the gRNA of claim 4;

10. An activated CRISPR complex, said activated CRISPR complex comprising:

(iii) A target sequence bound to the gRNA according to claim 4.

11. An engineered host cell comprising the Cas protein of claim 1, or the fusion protein of claim 2, or the polynucleotide of claim 3, or the vector of claim 6, or the CRISPR-Cas system of claim 7, or the vector system of claim 8, or the composition of claim 9, or the activated CRISPR complex of claim 10.

12. Use of the Cas protein of claim 1, or the fusion protein of claim 2, or the polynucleotide of claim 3, or the vector of claim 6, or the CRISPR-Cas system of claim 7, or the vector system of claim 8, or the composition of claim 9, or the activated CRISPR complex of claim 10, or the host cell of claim 11 in gene editing, gene targeting, or gene cleavage; alternatively, use in the preparation of a reagent or kit for gene editing, gene targeting or gene cleavage.

13. Use of the Cas protein of claim 1, or the fusion protein of claim 2, or the polynucleotide of claim 3, or the vector of claim 6, or the CRISPR-Cas system of claim 7, or the vector system of claim 8, or the composition of claim 9, or the activated CRISPR complex of claim 10, or the host cell of claim 11 in a system selected from any one or any of the following:

Targeting and/or editing a target nucleic acid; cleaving double-stranded DNA, single-stranded DNA, or single-stranded RNA; nonspecific cleavage and/or degradation of collateral nucleic acids; nonspecifically cleaving the single-stranded nucleic acid; detecting nucleic acid; editing the double-stranded nucleic acid specifically; base editing double-stranded nucleic acid; base editing single stranded nucleic acids.

14. A method of editing, targeting, or cleaving a target nucleic acid, the method comprising contacting the target nucleic acid with the Cas protein of claim 1, or the fusion protein of claim 2, or the polynucleotide of claim 3, or the vector of claim 6, or the CRISPR-Cas system of claim 7, or the vector system of claim 8, or the composition of claim 9, or the activated CRISPR complex of claim 10, or the host cell of claim 11.

15. A method of cleaving a single-stranded nucleic acid, the method comprising contacting a nucleic acid population with the Cas protein of claim 1 and the gRNA of claim 4, wherein the nucleic acid population comprises a target nucleic acid and at least one non-target single-stranded nucleic acid, the gRNA is capable of targeting the target nucleic acid, the Cas protein cleaves the non-target single-stranded nucleic acid.

16. A kit for gene editing, gene targeting or gene cleavage, the kit comprising the Cas protein of claim 1, or the fusion protein of claim 2, or the polynucleotide of claim 3, or the vector of claim 6, or the CRISPR-Cas system of claim 7, or the vector system of claim 8, or the composition of claim 9, or the activated CRISPR complex of claim 10, or the host cell of claim 11.

17. A kit for detecting a target nucleic acid in a sample, the kit comprising: (a) The Cas protein of claim 1, or a nucleic acid encoding the Cas protein; (b) The gRNA of claim 4, or a nucleic acid encoding the gRNA, or a precursor RNA comprising the gRNA, or a nucleic acid encoding the precursor RNA; and (c) a single stranded nucleic acid detector that is single stranded and does not hybridize to the gRNA.

18. Use of the Cas protein of claim 1, or the fusion protein of claim 2, or the polynucleotide of claim 3, or the vector of claim 6, or the CRISPR-Cas system of claim 7, or the vector system of claim 8, or the composition of claim 9, or the activated CRISPR complex of claim 10, or the host cell of claim 11 in the preparation of a formulation or kit for:

(i) Gene or genome editing;

(ii) Target nucleic acid detection and/or diagnosis;

(iv) Treatment of disease;

(v) Targeting a target gene;

(vi) Cutting the target gene.

19. A method of detecting a target nucleic acid in a sample, the method comprising contacting the sample with the Cas protein of claim 1, a gRNA (guide RNA) comprising a region that binds to the Cas protein and a guide sequence that hybridizes to the target nucleic acid, and a single stranded nucleic acid detector; detecting a detectable signal generated by the Cas protein cleaving single-stranded nucleic acid detector, thereby detecting a target nucleic acid; the single stranded nucleic acid detector does not hybridize to the gRNA.