Cas12j protein having improved editing activity and use thereof

Abstract

The present application relates to the field of nucleic acid editing, in particular to the technical field of clustered regularly interspaced short palindromic repeats (CRISPR). Specifically, provided are a Cas12j mutant protein having improved editing activity and the use thereof. The present application has wide prospects of application.

Description

Cas12j protein with improved editing activity and its application

[0001] This application claims priority to Chinese patent application CN2023110952452, filed on August 29, 2023, and cites the full text of the aforementioned Chinese patent application. Technical Field

[0002] The present invention relates to the field of gene editing, in particular to the field of clustered regularly interspaced short palindromic repeats (CRISPR). Specifically, the present invention relates to a mutant Cas12j protein with enhanced activity and its application. Background Art

[0003] CRISPR / Cas technology is a widely used gene editing technology that uses RNA to guide specific binding to target sequences on the genome and cut DNA to produce double-strand breaks, and uses biological non-homologous end joining or homologous recombination to perform site-directed gene editing.

[0004] The CRISPR / Cas9 system is the most commonly used Type II CRISPR system, which recognizes a 3'-NGG PAM motif and performs blunt-end cleavage on target sequences. A newly discovered class of CRISPR / Cas systems, Type V, utilizes a 5'-TTN motif and performs sticky-end cleavage on target sequences. Examples include Cpf1, C2c1, CasX, and CasY. However, the various CRISPR / Cas systems currently available have varying advantages and disadvantages. For example, Cas9, C2c1, and CasX all require two guide RNAs, while Cpf1 requires only a single guide RNA and can be used for multiplexed gene editing. CasX is 980 amino acids long, while the more common Cas9, C2c1, CasY, and Cpf1 are typically around 1300 amino acids. Furthermore, the PAM sequences of Cas9, Cpf1, CasX, and CasY are more complex and diverse, while C2c1 recognizes a strict 5'-TTN motif, making its target site more predictable than other systems, thereby reducing potential off-target effects.

[0005] Chinese invention patent CN111770992B discloses a Cas protein Cas12j.19, which also discloses that the protein can perform gene editing in eukaryotic cells. However, its editing activity is not high. In order to improve the editing efficiency of the protein, this application optimizes the protein and improves its editing efficiency in eukaryotic cells.

[0006] Summary of the Invention

[0007] After a large number of experiments and repeated explorations, the inventors of this application improved the editing activity of Cas12j.19 (referred to as Cas12j19 in this application) protein and expanded its application range by site-directed mutagenesis.

[0008] Cas effector proteins

[0009] On the one hand, the present invention provides a Cas mutant protein with improved or enhanced activity, wherein the mutant protein has a mutation at any one or several of the following amino acid positions corresponding to the amino acid sequence shown in SEQ ID No. 1: position 3, position 4, position 99, position 154, position 228, position 231, position 400, position 411, position 647, position 649 or position 658 compared with the amino acid sequence of the parent Cas protein.

[0010] In one embodiment, the mutant protein has a mutation at any one or several of the following amino acid positions corresponding to the amino acid sequence shown in SEQ ID No. 1 compared to the amino acid sequence of the parent Cas protein: position 3, position 4, position 99, position 154, position 228, position 231, position 400, position 411, position 647, position 649 or position 658; any several are selected from: any 2, any 3, any 4, any 5, any 6, any 7, any 8, any 9, any 10 or any 11.

[0011] In one embodiment, the Cas mutant protein further comprises a mutation at the following amino acid site in the amino acid sequence corresponding to SEQ ID No. 1, compared with the amino acid sequence of the parent Cas protein: position 100.

[0012] In one embodiment, the Cas mutant protein has a mutation at the following amino acid position corresponding to the amino acid sequence shown in SEQ ID No. 1 compared to the amino acid sequence of the parent Cas protein:

[0013] The amino acid at position 100 and the amino acid at position 3 were mutated simultaneously;

[0014] or, amino acid position 100 and amino acid position 99 are mutated simultaneously;

[0015] or, amino acid position 100 and amino acid position 154 are mutated simultaneously;

[0016] or, amino acid 100 and amino acid 231 are mutated simultaneously;

[0017] or, amino acid positions 100 and 400 are mutated simultaneously;

[0018] or, amino acid 100 and amino acid 411 are mutated simultaneously;

[0019] or, amino acid position 100 and amino acid position 647 are mutated simultaneously;

[0020] or, amino acid 100 and amino acid 649 are mutated simultaneously;

[0021] or, amino acid 100 and amino acid 658 are mutated simultaneously;

[0022] or, amino acid position 100, amino acid position 400, and amino acid position 763 are mutated simultaneously;

[0023] Alternatively, the 100th amino acid, the 400th amino acid, the 763rd amino acid and the 45th amino acid are mutated simultaneously.

[0024] In one embodiment, the amino acid at position 3 is mutated to a non-S amino acid, for example, A, V, G, L, D, F, W, Y, N, E, Q, K, M, T, C, P, H, R, I; preferably, it is mutated to R.

[0025] In one embodiment, the amino acid at position 4 is mutated to an amino acid other than Y, for example, A, V, G, L, D, F, W, S, N, E, Q, K, M, T, C, P, H, R, or I; preferably, it is mutated to R.

[0026] In one embodiment, the amino acid at position 99 is mutated to a non-L amino acid, for example, A, V, G, Y, D, F, W, S, N, E, Q, K, M, T, C, P, H, R, I; preferably, it is mutated to R.

[0027] In one embodiment, the amino acid at position 154 or the amino acid at position 231 is mutated to a non-A amino acid, for example, L, V, G, Y, D, F, W, S, N, E, Q, K, M, T, C, P, H, R, or I; preferably, it is mutated to R.

[0028] In one embodiment, the amino acid at position 228 or the amino acid at position 400 is mutated to a non-S amino acid, for example, A, V, G, Y, D, F, W, L, N, E, Q, K, M, T, C, P, H, R, I; preferably, it is mutated to R.

[0029] In one embodiment, the amino acid at position 411 or the amino acid at position 647 is mutated to a non-T amino acid, for example, A, V, G, L, D, F, W, Y, N, E, Q, K, M, S, C, P, H, R, or I; preferably, it is mutated to R.

[0030] In one embodiment, the amino acid at position 649 or the amino acid at position 658 is mutated to a non-D amino acid, for example, A, V, G, L, S, F, W, Y, N, E, Q, K, M, T, C, P, H, R, I; preferably, it is mutated to R.

[0031] In one embodiment, the amino acid at position 100 is mutated to an amino acid other than E, for example, A, V, G, L, S, F, W, Y, N, D, Q, K, M, T, C, P, H, R, I; preferably, it is mutated to K.

[0032] In one embodiment, the amino acid at position 763 is mutated to a non-L amino acid, for example, A, V, G, Y, D, F, W, S, N, E, Q, K, M, T, C, P, H, R, I; preferably, it is mutated to R.

[0033] In one embodiment, the amino acid at position 45 is mutated to a non-S amino acid, for example, A, V, G, Y, D, F, W, L, N, E, Q, K, M, T, C, P, H, R, I; preferably, it is mutated to T.

[0034] In one embodiment, the amino acid sequence of the parent Cas protein has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, 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 SEQ ID No. 1.

[0035] In one embodiment, the parent Cas protein is a Cas protein of the Cas12 family, preferably, a Cas protein of the Cas12j family, for example, Cas12j.3-Cas12j.22 disclosed in CN111770992B; in a preferred embodiment, the parent Cas protein is Cas12j19; the amino acid sequence of Cas12j19 has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, 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% or 100% sequence identity compared to SEQ ID No.1.

[0036] Cas proteins or Cas12j proteins from a variety of organisms can be used as parent Cas proteins, and in some embodiments, the parent Cas proteins or Cas12j proteins have nuclease activity. In some embodiments, the parent Cas protein is a nuclease, i.e., it cuts two chains of a target double-helical nucleic acid (e.g., double-helical DNA). In some embodiments, the parent Cas protein is a nickase, i.e., it cuts a single strand of a target double-helical nucleic acid (e.g., double-helical DNA).

[0037] In some embodiments, the parent Cas protein is a natural wild-type Cas protein; in other embodiments, the parent Cas protein is an engineered Cas protein.

[0038] Those skilled in the art will appreciate that protein structure can be altered without adversely affecting its activity and functionality. For example, one or more conservative amino acid substitutions can be introduced into a protein's amino acid sequence without adversely affecting the activity and / or three-dimensional structure of the protein molecule. Examples and implementations of conservative amino acid substitutions will be apparent to those skilled in the art. Specifically, an amino acid residue can be substituted with another amino acid residue belonging to the same group as the substituted residue, i.e., a non-polar amino acid residue can be substituted for another non-polar amino acid residue, a polar uncharged amino acid residue can be substituted for another polar uncharged amino acid residue, a basic amino acid residue can be substituted for another basic amino acid residue, and an acidic amino acid residue can be substituted for another acidic amino acid residue. Such substituted amino acid residues may or may not be encoded by the genetic code. Conservative substitutions, where one amino acid is replaced with another amino acid belonging to the same group, fall within the scope of the present invention, as long as the substitution does not inactivate the biological activity of the protein. Therefore, the proteins of the present invention may contain one or more conservative substitutions in their amino acid sequences, preferably generated by substitutions according to Table 1. Furthermore, the present invention also encompasses proteins containing one or more other non-conservative substitutions, as long as such non-conservative substitutions do not significantly affect the desired function and biological activity of the proteins of the present invention.

[0039] Conservative amino acid replacement can be carried out at one or more predicted non-essential amino acid residues.A "non-essential" amino acid residue is an amino acid residue that can be changed (deleted, substituted or replaced) without changing biological activity, while an "essential" amino acid residue is required for biological activity.A "conservative amino acid replacement" is a replacement in which an amino acid residue is replaced by an amino acid residue with a similar side chain.Amino acid replacement can be carried out in the non-conserved region of the above-mentioned Cas mutant protein.In general, such replacement is not carried out for conserved amino acid residues, or is not carried out for amino acid residues located 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 can have less conservative or non-conservative changes in conserved regions.

[0040] Table 1

[0041] It is well known in the art that one or more amino acid residues can be changed (replaced, deleted, truncated or inserted) from the N and / or C terminus of a protein while still retaining its functional activity. Therefore, proteins in which one or more amino acid residues are changed from the N and / or C terminus of a Cas protein while retaining its desired functional activity are also within the scope of the present invention. These changes may include changes introduced by modern molecular methods such as PCR, which includes PCR amplification of a protein coding sequence by means of including an amino acid coding sequence among the oligonucleotides used in the PCR amplification.

[0042] It will be appreciated that proteins can be altered in various ways, including amino acid substitutions, deletions, truncations, and insertions, and methods for such manipulations are generally known in the art. For example, amino acid sequence variants of the above-described proteins can be prepared by mutations in the DNA. Other forms of mutagenesis and / or directed evolution can also be accomplished, for example, using known mutagenesis, recombination, and / or shuffling methods, in combination with relevant screening methods, to perform single or multiple amino acid substitutions, deletions, and / or insertions.

[0043] Those skilled in the art will appreciate that these minor amino acid changes in the Cas proteins of the present invention can occur (e.g., naturally occurring mutations) or be 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 domains of the protein, the properties of the polypeptide may be changed, but the polypeptide may retain its activity. If the mutations present are not close to the catalytic domain, active site, or other functional domains, a smaller effect can be expected.

[0044] Those skilled in the art can identify the essential amino acids of the Cas mutant protein of the present invention according to methods known in the art, such as site-directed mutagenesis or protein evolution or analysis of bioinformatics systems. The catalytic domain, active site or other functional domain of the protein can also be determined by physical analysis of the structure, such as by the following techniques: such as nuclear magnetic resonance, crystallography, electron diffraction or photoaffinity labeling, combined with mutations of amino acids at putative key sites.

[0045] In the present invention, amino acid residues can be represented by single letters or 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).

[0046] The term "AxxB" indicates that the amino acid A at position xx is changed to amino acid B. Unless otherwise specified, this refers to the change from amino acid A at position xx from the N-terminus to amino acid B. For example, E100K indicates that E at position 100 is mutated to K. When multiple amino acid positions are mutated simultaneously, a format similar to E100K-S3R can be used. For example, E100K-S3R indicates that E at position 100 is mutated to K and S at position 3 is mutated to R.

[0047] Specific amino acid positions (numbers) within the proteins of the present invention are determined by aligning the amino acid sequence of the target protein with SEQ ID No. 1 using standard sequence alignment tools, such as the Smith-Waterman algorithm or the CLUSTALW2 algorithm, wherein the sequences are considered aligned when the alignment score is the highest. The alignment score can be calculated according to the method described in Wilbur, WJ and Lipman, DJ (1983) Rapid similarity searches of nucleic acid and protein data banks. Proc. Natl. Acad. Sci. USA, 80: 726-730. The default parameters for the ClustalW2 (1.82) algorithm are preferably used: protein gap open penalty = 10.0; protein gap extension penalty = 0.2; protein matrix = Gonnet; protein / DNA end gap = -1; protein / DNAGAPDIST = 4. Preferably, the AlignX program (part of the vectorNTI group) is used to determine the position of specific amino acids in the protein of the present invention by aligning the amino acid sequence of the protein with SEQ ID No. 1 using default parameters suitable for multiple alignment (gap opening penalty: 10 log gap extension penalty 0.05).

[0048] In one embodiment, the Cas mutant protein is selected from any one of the following groups I-III:

[0049] I. A Cas mutant protein obtained by generating a mutation in the amino acid sequence of SEQ ID No. 1 at any one or more of the following amino acid positions: position 3, position 4, position 99, position 154, position 228, position 231, position 400, position 411, position 647, position 649 or position 658;

[0050] II. Compared with the Cas mutant protein described in I, it has the mutation site described in I; and, compared with the Cas mutant protein described in I, it 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%, or at least 99% sequence identity;

[0051] III. Compared with the Cas mutant protein described in I, it has the mutation site described in I; and, compared with the Cas mutant protein described in I, it has a sequence of one or more amino acid substitutions, deletions or additions; the one or more amino acids include 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, deletions or additions.

[0052] In one embodiment, the Cas mutant protein is selected from any one of the following groups I-III:

[0053] 1. A Cas mutant protein obtained by generating a mutation at any one or several of the following amino acid positions of the amino acid sequence shown in SEQ ID No. 1: position 3, position 4, position 99, position 154, position 228, position 231, position 400, position 411, position 647, position 649 or position 658; and the Cas mutant protein further has a mutation at any one or several of the amino acid positions corresponding to position 100, position 763 or position 43 of the amino acid sequence shown in SEQ ID No. 1;

[0054] II. Compared with the Cas mutant protein described in I, it has the mutation site described in I; and, compared with the Cas mutant protein described in I, it 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%, or at least 99% sequence identity;

[0055] III. Compared with the Cas mutant protein described in I, it has the mutation site described in I; and, compared with the Cas mutant protein described in I, it has a sequence of one or more amino acid substitutions, deletions or additions; the one or more amino acids include 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, deletions or additions.

[0056] In one embodiment, the Cas mutant protein is selected from any one of the following groups I-III:

[0057] I. A Cas mutant protein obtained by generating a mutation at any one or more of the following amino acid positions in the amino acid sequence shown in SEQ ID No. 1: position 3, position 4, position 99, position 154, position 228, position 231, position 400, position 411, position 647, position 649 or position 658; and the Cas mutant protein has a K at the amino acid position 100 corresponding to the amino acid sequence shown in SEQ ID No. 1;

[0058] II. Compared with the Cas mutant protein described in I, it has the mutation site described in I; and, compared with the Cas mutant protein described in I, it 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%, or at least 99% sequence identity;

[0059] III. Compared with the Cas mutant protein described in I, it has the mutation site described in I; and compared with the Cas mutant protein described in I, it has a sequence with one or more amino acid substitutions, deletions or additions; the one or more amino acids include 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, deletions or additions. The biological functions of the Cas protein include, but are not limited to, activity binding to guide RNA, endonuclease activity, and activity of binding to and cleaving a specific site of the target sequence under the guidance of the guide RNA, including but not limited to Cis cleavage activity and Trans cleavage activity.

[0060] In the present invention, "Cas mutant protein" can also be referred to as a mutated Cas protein, or a Cas protein variant.

[0061] The present invention also provides a fusion protein, which includes the Cas mutant protein as described above and other modified parts.

[0062] In one embodiment, the modifying moiety is selected from another protein or polypeptide, a detectable label, or any combination thereof.

[0063] In one embodiment, the modifying portion is selected from an epitope tag, a reporter gene sequence, a nuclear localization signal (NLS) sequence, a targeting portion, a transcriptional activation domain (e.g., VP64), a transcriptional repression domain (e.g., a KRAB domain or a SID domain), a nuclease domain (e.g., Fok1), and a domain having an activity selected from the group consisting of nucleotide deaminase, adenosine deaminase, cytidine 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 sequence is well known to those skilled in the art, and examples thereof include, but are not limited to, the SV40 large T antigen, EGL-13, c-Myc, and TUS protein.

[0064] In one embodiment, the NLS sequence is located at, near, or proximal to a terminus (e.g., the N-terminus, the C-terminus, or both) of the Cas protein of the invention.

[0065] The epitope tag is well known to those skilled in the art, including but not limited to His, V5, FLAG, HA, Myc, VSV-G, Trx, etc., and those skilled in the art can select other suitable epitope tags (for example, purification, detection or tracing).

[0066] The reporter gene sequence is well known to those skilled in the art, and examples thereof include but are not limited to GST, HRP, CAT, GFP, HcRed, DsRed, CFP, YFP, BFP, etc.

[0067] In one embodiment, the fusion protein of the present invention comprises a domain capable of binding to a DNA molecule or an intracellular molecule, such as maltose binding protein (MBP), the DNA binding domain (DBD) of Lex A, the DBD of GAL4, and the like.

[0068] In one embodiment, the fusion protein of the invention comprises a detectable label, such as a fluorescent dye, eg, FITC or DAPI.

[0069] In one embodiment, the Cas protein of the present invention is optionally coupled, conjugated or fused to the modifying portion via a linker.

[0070] In one embodiment, the modification portion is directly linked to the N-terminus or C-terminus of the Cas protein of the present invention.

[0071] In one embodiment, the modified portion is connected to the N-terminus or C-terminus of the Cas protein of the present invention via a linker. Such linkers are well known in the art, and examples thereof 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.

[0072] The Cas protein, protein derivative or fusion protein of the present invention is not limited by the method of its production. For example, it can be produced by genetic engineering methods (recombinant technology) or by chemical synthesis methods.

[0073] Cas protein nucleic acid

[0074] In another aspect, the present invention provides an isolated polynucleotide comprising:

[0075] (a) a polynucleotide sequence encoding a Cas mutant protein or fusion protein of the present invention;

[0076] Alternatively, a polynucleotide complementary to the polynucleotide described in (a).

[0077] In one embodiment, the nucleotide sequence is codon optimized for expression in prokaryotes. In one embodiment, the nucleotide sequence is codon optimized for expression in eukaryotic cells.

[0078] In one embodiment, the cell is an animal cell, eg, a mammalian cell.

[0079] In one embodiment, the cell is a human cell.

[0080] In one embodiment, the cell is a plant cell, such as a cell from a cultivated plant (such as cassava, corn, sorghum, wheat, or rice), algae, tree, or vegetable.

[0081] In one embodiment, the polynucleotide is preferably single-stranded or double-stranded.

[0082] Guide RNA (gRNA)

[0083] On the other hand, the present invention provides a gRNA, which includes a first segment and a second segment; the first segment is also called a "skeleton region", "protein binding segment", "protein binding sequence", or "direct repeat sequence"; the second segment is also called a "targeting sequence for targeting nucleic acid" or "targeting segment for targeting nucleic acid", or "guide sequence for targeting target sequence".

[0084] The first segment of the gRNA is capable of interacting with the Cas protein of the present invention, thereby forming a complex between the Cas protein and the gRNA.

[0085] In a preferred embodiment, the first segment is a direct repeat sequence as described above.

[0086] The targeting sequence of the targeting nucleic acid of the present invention or the targeting section of the targeting nucleic acid comprises a nucleotide sequence complementary to the sequence in the target nucleic acid. In other words, the targeting sequence of the targeting nucleic acid of the present invention or the targeting section of the targeting nucleic acid interacts with the target nucleic acid in a sequence-specific manner through hybridization (i.e., base pairing). Therefore, the targeting sequence of the targeting nucleic acid or the targeting section of the targeting nucleic acid can be changed, or can be modified to hybridize any desired sequence in the target nucleic acid. The nucleic acid is selected from DNA or RNA.

[0087] The percent complementarity between the targeting sequence of a targeting nucleic acid or the targeting segment of a targeting nucleic acid and the target sequence of a 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%).

[0088] The "backbone region", "protein binding segment", "protein binding sequence", or "direct repeat sequence" of the gRNA of the present invention can interact with the CRISPR protein (or Cas protein). The gRNA of the present invention guides the interacting Cas protein to a specific nucleotide sequence within the target nucleic acid through the action of the targeting sequence of the target nucleic acid.

[0089] Preferably, the guide RNA comprises a first segment and a second segment from the 5' to the 3' direction.

[0090] In the present invention, the second segment can also be understood as a guide sequence that hybridizes with the target sequence.

[0091] The gRNA of the present invention is capable of forming a complex with the Cas protein.

[0092] carrier

[0093] The present invention also provides a vector comprising the Cas mutant protein, isolated nucleic acid molecule or polynucleotide as described above; preferably, it further comprises a regulatory element operably linked thereto.

[0094] In one embodiment, the regulatory element is selected from one or more of the following groups: enhancer, transposon, promoter, terminator, leader sequence, polyadenylation sequence, marker gene.

[0095] In one embodiment, the vector includes a cloning vector, an expression vector, a shuttle vector, and an integration vector.

[0096] In some embodiments, the vector included in the system is a viral vector (e.g., a retroviral vector, a lentiviral vector, an adenoviral vector, an adeno-associated vector, and a herpes simplex vector), and can also be a plasmid, a virus, a cosmid, a phage, etc., which are well known to those skilled in the art.

[0097] CRISPR system

[0098] The present invention provides an engineered non-naturally occurring vector system, or a CRISPR-Cas system, which includes a Cas mutant protein or a nucleic acid sequence encoding the Cas mutant protein and a nucleic acid encoding one or more guide RNAs.

[0099] In one embodiment, the nucleic acid sequence encoding the Cas mutant protein and the nucleic acid encoding one or more guide RNAs are artificially synthesized.

[0100] In one embodiment, the nucleic acid sequence encoding the Cas mutant protein and the nucleic acid encoding one or more guide RNAs do not naturally co-occur.

[0101] The one or more guide RNAs target one or more target sequences in the cell. The one or more target sequences hybridize to the genomic loci of the DNA molecules encoding the one or more gene products, and guide the Cas protein to the genomic loci of the DNA molecules encoding the one or more gene products. After the Cas protein reaches the target sequence position, it modifies, edits or cuts the target sequence, thereby changing or modifying the expression of the one or more gene products.

[0102] The cells of the present invention include one or more of animals, plants, or microorganisms.

[0103] In some embodiments, the Cas protein is codon-optimized for expression in a cell.

[0104] In some embodiments, the Cas protein directs cleavage of one or both strands at the location of the target sequence.

[0105] The present invention also provides an engineered non-naturally occurring vector system, which may include one or more vectors, wherein the one or more vectors include:

[0106] a) a first regulatory element, which is operably linked to the gRNA,

[0107] b) a second regulatory element, which is operably linked to the Cas protein;

[0108] Components (a) and (b) are located on the same or different carriers of the system.

[0109] The first and second regulatory elements include a promoter (e.g., a constitutive promoter or an inducible promoter), an enhancer (e.g., a 35S promoter or a 35S enhanced promoter), an internal ribosome entry site (IRES), and other expression control elements (e.g., transcription termination signals, such as polyadenylation signals and poly-U sequences).

[0110] In some embodiments, the vector in the system is a viral vector (e.g., a retroviral vector, a lentiviral vector, an adenoviral vector, an adeno-associated vector, and a herpes simplex vector), and can also be a plasmid, a virus, a cosmid, a phage, etc., which are well known to those skilled in the art.

[0111] In some embodiments, the systems provided herein are in a delivery system. In some embodiments, the delivery system is a nanoparticle, a liposome, an exosome, a microbubble, and a gene gun.

[0112] 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.

[0113] 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., an organelle). In one embodiment, the cell is a eukaryotic cell. In other embodiments, the cell is a prokaryotic cell.

[0114] In one embodiment, the Cas protein is connected to one or more NLS sequences. In one embodiment, the fusion protein comprises one or more NLS sequences. In one embodiment, the NLS sequence is connected 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.

[0115] On the other hand, the present invention relates to an engineered CRISPR system, comprising the above-mentioned Cas protein and one or more guide RNAs, wherein the guide RNA includes a direct repeat sequence and a spacer sequence capable of hybridizing with a target nucleic acid, and the Cas protein is capable of binding to the guide RNA and targeting a target nucleic acid sequence complementary to the spacer sequence.

[0116] Protein-nucleic acid complexes / compositions

[0117] In another aspect, the present invention provides a compound or composition comprising:

[0118] (i) a protein component selected from the group consisting of: the above-mentioned Cas proteins, derivatized proteins or fusion proteins, and any combination thereof; and

[0119] (ii) a nucleic acid component comprising (a) a guide sequence capable of hybridizing to a target sequence; and (b) a direct repeat sequence capable of binding to a Cas protein of the present invention.

[0120] The protein component and the nucleic acid component combine with each other to form a complex.

[0121] In one embodiment, the nucleic acid component is a guide RNA in a CRISPR-Cas system.

[0122] 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.

[0123] Activated CRISPR complex

[0124] On the other hand, the present invention also provides an activated CRISPR complex, the activated CRISPR complex comprising: (1) a protein component selected from: a Cas protein, a derivatized protein or a fusion protein of the present invention, and any combination thereof; (2) a gRNA comprising (a) a guide sequence capable of hybridizing with a target sequence; and (b) a direct repeat sequence capable of binding to the Cas protein of the present invention; and (3) a target sequence bound to the gRNA. Preferably, the binding is carried out by binding of the targeting sequence of the targeting nucleic acid on the gRNA to the target nucleic acid.

[0125] As used herein, the terms "activated CRISPR complex," "activated complex," or "ternary complex" refer to the complex formed after the Cas protein, gRNA, and target nucleic acid in the CRISPR system are bound or modified.

[0126] The Cas protein and gRNA of the present invention can form a binary complex that is activated when bound to a nucleic acid substrate to form an activated CRISPR complex. The nucleic acid substrate is complementary to the spacer sequence in the gRNA (or referred to as a guide sequence that hybridizes with the target nucleic acid). In some embodiments, the spacer sequence of the gRNA fully matches the target substrate. In other embodiments, the spacer sequence of the gRNA matches a portion (continuous or discontinuous) of the target substrate.

[0127] In a preferred embodiment, the activated CRISPR complex can exhibit collateral nuclease cleavage activity, which refers to the non-specific cleavage activity or random cleavage activity of the activated CRISPR complex on single-stranded nucleic acids, also known as trans cleavage activity in the art.

[0128] Delivery and delivery compositions

[0129] The Cas proteins, gRNAs, fusion proteins, nucleic acid molecules, vectors, systems, complexes and compositions of the present invention can be delivered by any method known in the art. Such methods include, but are not limited to, electroporation, lipofection, nucleofection, microinjection, sonoporation, gene guns, calcium phosphate-mediated transfection, cationic transfection, lipofection, dendritic transfection, heat shock transfection, nucleofection, magnetofection, lipofection, puncture transfection, optical transfection, reagent-enhanced nucleic acid uptake, and delivery via liposomes, immunoliposomes, viral particles, artificial virions, etc.

[0130] Therefore, in another aspect, the present invention provides a delivery composition comprising a delivery vector and one or more selected from the following: the Cas protein, fusion protein, nucleic acid molecule, vector, system, complex and composition of the present invention.

[0131] In one embodiment, the delivery vehicle is a particle.

[0132] In one embodiment, the delivery vehicle is selected from lipid particles, sugar particles, metal particles, protein particles, liposomes, exosomes, microvesicles, gene guns or viral vectors (e.g., replication-defective retroviruses, lentiviruses, adenoviruses or adeno-associated viruses).

[0133] host cells

[0134] The present invention also relates to an in vitro, ex vivo or in vivo cell or cell line or their progeny, wherein the cell or cell line or their progeny comprises: the Cas protein, fusion protein, nucleic acid molecule, protein-nucleic acid complex, activated CRISPR complex, vector, and delivery composition of the present invention.

[0135] In certain embodiments, the cell is a prokaryotic cell.

[0136] 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, such as a cell of a non-human primate, cattle, sheep, pig, dog, monkey, rabbit, rodent (such as rat or mouse). In certain embodiments, the cell is a non-mammalian eukaryotic cell, such as a cell of poultry (such as chicken), fish or crustacean (such as clams, shrimp). In certain embodiments, the cell is a plant cell, such as a cell or cultivated plant or food crop such as cassava, corn, sorghum, soybean, wheat, oat or rice that a monocot or dicot has, such as an algae, tree or production plant, fruit or vegetable (for example, trees such as citrus trees, nut trees; Solanaceae, cotton, tobacco, tomato, grape, coffee, cocoa, etc.).

[0137] In certain embodiments, the cell is a stem cell or a stem cell line.

[0138] In certain cases, the host cells of the invention comprise genetic or genomic modifications that are not present in their wild-type form.

[0139] Gene Editing Methods and Applications

[0140] The Cas mutant protein, nucleic acid, composition, CIRSPR / Cas system, vector system, delivery composition, activated CRISPR complex, or host cell of the present invention can be used for any one or more of the following purposes: targeting and / or editing target nucleic acid; cutting double-stranded DNA, single-stranded DNA, or single-stranded RNA; non-specific cutting and / or degradation of side branch nucleic acid; non-specific cutting of single-stranded nucleic acid; nucleic acid detection; detection of nucleic acid in target sample; specific editing of double-stranded nucleic acid; base editing of double-stranded nucleic acid; base editing of single-stranded nucleic acid. In other embodiments, it can also be used to prepare reagents or kits for any one or more of the above purposes.

[0141] The present invention also provides the use of the above-mentioned Cas protein, nucleic acid, composition, CIRSPR / Cas system, vector system, delivery composition or activated CRISPR complex in gene editing, gene targeting or gene cleavage; or, use in the preparation of reagents or kits for gene editing, gene targeting or gene cleavage.

[0142] In one embodiment, the gene editing, gene targeting or gene cleavage is performed inside and / or outside the cell.

[0143] The present invention also provides a method for editing a target nucleic acid, targeting a target nucleic acid, or cutting a target nucleic acid, the method comprising contacting the target nucleic acid with the above-mentioned Cas protein, nucleic acid, composition, CIRSPR / Cas system, vector system, delivery composition, or activated CRISPR complex. In one embodiment, the method is to edit, target, or cut a target nucleic acid inside or outside a cell.

[0144] The gene editing or editing of target nucleic acid includes modifying genes, knocking out genes, changing the expression of gene products, repairing mutations, and / or inserting polynucleotides, gene mutations.

[0145] The editing can be performed in prokaryotic cells and / or eukaryotic cells.

[0146] On the other hand, the present invention also provides the use of the above-mentioned Cas protein, nucleic acid, composition, CIRSPR / Cas system, vector system, delivery composition or activated CRISPR complex in nucleic acid detection, or in the preparation of reagents or kits for nucleic acid detection.

[0147] On the other hand, the present invention also provides a method for cutting single-stranded nucleic acids, the method comprising contacting a nucleic acid population with the above-mentioned Cas protein and gRNA, wherein the nucleic acid population comprises a target nucleic acid and a plurality of non-target single-stranded nucleic acids, and the Cas protein cuts the plurality of non-target single-stranded nucleic acids.

[0148] The gRNA is capable of binding to the Cas protein.

[0149] The gRNA is capable of targeting the target nucleic acid.

[0150] The contacting can be inside the cell in vitro, ex vivo or in vivo.

[0151] Preferably, the cleavage of the single-stranded nucleic acid is non-specific cleavage.

[0152] On the other hand, the present invention also provides the use of the above-mentioned Cas protein, nucleic acid, composition, CIRSPR / Cas system, vector system, delivery composition or activated CRISPR complex in non-specific cleavage of single-stranded nucleic acid, or in the preparation of a reagent or kit for non-specific cleavage of single-stranded nucleic acid.

[0153] On the other hand, the present invention also provides a kit for gene editing, gene targeting or gene cleavage, which comprises the above-mentioned Cas protein, gRNA, nucleic acid, the above-mentioned composition, the above-mentioned CIRSPR / Cas system, the above-mentioned vector system, the above-mentioned delivery composition, the above-mentioned activated CRISPR complex or the above-mentioned host cell.

[0154] On the other hand, the present 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 with the guide RNA.

[0155] It is known in the art that precursor RNA can be cleaved or processed into the mature guide RNA described above.

[0156] In another aspect, the invention provides the use of the above-mentioned Cas protein, nucleic acid, composition, CIRSPR / Cas system, vector system, delivery composition, activated CRISPR complex or host cell in preparing a preparation or kit, wherein the preparation or kit is used for:

[0157] (i) gene or genome editing;

[0158] (ii) target nucleic acid detection and / or diagnosis;

[0159] (iii) editing a target sequence in a target locus to modify an organism or non-human organism;

[0160] (iv) treatment of disease;

[0161] (iv) Targeting target genes.

[0162] Preferably, the above-mentioned gene or genome editing is performed inside or outside the cell.

[0163] Preferably, the target nucleic acid detection and / or diagnosis is performed in vitro.

[0164] Preferably, the treatment of the disease is the treatment of a condition caused by a defect in the target sequence in the target locus.

[0165] In another aspect, the present invention provides a method for detecting a target nucleic acid in a sample, the method comprising contacting the sample with the Cas protein, gRNA (guide RNA) and a single-stranded nucleic acid detector, the gRNA comprising a region that binds to the Cas protein and a guide sequence that hybridizes with the target nucleic acid; detecting a detectable signal generated by the Cas protein cleaving the single-stranded nucleic acid detector, thereby detecting the target nucleic acid; the single-stranded nucleic acid detector does not hybridize with the gRNA.

[0166] Method for specifically modifying target nucleic acid

[0167] On the other hand, the present invention also provides a method for specifically modifying a target nucleic acid, the method comprising: contacting the target nucleic acid with the above-mentioned Cas protein, nucleic acid, the above-mentioned composition, the above-mentioned CIRSPR / Cas system, the above-mentioned vector system, the above-mentioned delivery composition or the above-mentioned activated CRISPR complex.

[0168] The specific modification can occur in vivo or in vitro.

[0169] The specific modification can occur inside or outside the cell.

[0170] In some cases, the cell is selected from a prokaryotic cell or a eukaryotic cell, eg, an animal cell, a plant cell, or a microbial cell.

[0171] In one embodiment, the modification refers to a break in the target sequence, such as a single-strand / double-strand break in DNA, or a single-strand break in RNA.

[0172] 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.

[0173] In one embodiment, the modification further comprises inserting an editing template (eg, an exogenous nucleic acid) into the break.

[0174] In one embodiment, the method further comprises: contacting the target nucleic acid with an editing template, or delivering the editing template to a cell containing the target nucleic acid. In this embodiment, the method repairs the broken target gene by homologous recombination with an exogenous template polynucleotide; in some embodiments, the repair results in a mutation comprising an insertion, deletion, or substitution of one or more nucleotides in the target gene; in other embodiments, the mutation results in one or more amino acid changes in a protein expressed from a gene containing the target sequence.

[0175] Detection (non-specific cleavage)

[0176] On the other hand, the present invention provides a method for detecting a target nucleic acid in a sample, the method comprising contacting the sample with the above-mentioned Cas protein, nucleic acid, the above-mentioned composition, the above-mentioned CIRSPR / Cas system, the above-mentioned vector system, the above-mentioned delivery composition or the above-mentioned activated CRISPR complex and a single-stranded nucleic acid detector; detecting a detectable signal generated by the Cas protein cleaving the single-stranded nucleic acid detector, thereby detecting the target nucleic acid.

[0177] In the present invention, the target nucleic acid includes ribonucleotides or deoxyribonucleotides; including single-stranded nucleic acids and double-stranded nucleic acids, such as single-stranded DNA, double-stranded DNA, single-stranded RNA, and double-stranded RNA.

[0178] In one embodiment, the target nucleic acid is derived from a sample such as a virus, bacteria, microorganism, soil, water, human body, animal, plant, etc. Preferably, the target nucleic acid is a product enriched or amplified by methods such as PCR, NASBA, RPA, SDA, LAMP, HAD, NEAR, MDA, RCA, LCR, RAM, etc.

[0179] 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, a papillomavirus, a hepadnavirus, a herpes virus, adenovirus, a poxvirus, a parvovirus, a coronavirus; preferably, the virus is a coronavirus, preferably, SARS, SARS-CoV2 (COVID-19), HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1, Mers-Cov.

[0180] In the present invention, the gRNA has at least 50% matching with the target sequence on the target nucleic acid, preferably at least 60%, preferably at least 70%, preferably at least 80%, preferably at least 90%.

[0181] In one embodiment, when the target sequence contains one or more characteristic sites (such as a specific mutation site or SNP), the characteristic sites are completely matched with the gRNA.

[0182] In one embodiment, the detection method may comprise one or more gRNAs with different guide sequences, which target different target sequences.

[0183] 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 modifiers, and single-stranded nucleic acid detectors containing a base-free spacer, etc.; "nucleic acid analogs" include but are not limited to: locked nucleic acid, bridge nucleic acid, morpholino nucleic acid, ethylene glycol nucleic acid, hexitol nucleic acid, threose nucleic acid, arabinose nucleic acid, 2'oxymethyl RNA, 2'methoxyacetyl RNA, 2'fluoro RNA, 2'amino RNA, 4'thio RNA and combinations thereof, including optional ribonucleotides or deoxyribonucleotide residues.

[0184] In the present invention, the detectable signal is achieved by the following means: vision-based detection, sensor-based detection, color detection, fluorescence signal-based detection, gold nanoparticle-based detection, fluorescence polarization, colloidal phase transition / dispersion, electrochemical detection and semiconductor-based detection.

[0185] In the present invention, preferably, a fluorescent group and a quenching group are provided at both ends of the single-stranded nucleic acid detector, respectively, so that when the single-stranded nucleic acid detector is cleaved, a detectable fluorescent signal can be exhibited. The fluorescent group is selected from one or any combination of FAM, FITC, VIC, JOE, TET, CY3, CY5, ROX, Texas Red, or LC RED460; and the quenching group is selected from one or any combination of BHQ1, BHQ2, BHQ3, Dabcy1, or Tamra.

[0186] In other embodiments, different labeling molecules are respectively set at the 5' end and the 3' end of the single-stranded nucleic acid detector, and the colloidal gold test results of the single-stranded nucleic acid detector before and after being cut by the Cas protein are detected by colloidal gold detection; the single-stranded nucleic acid detector will show different color development results on the colloidal gold detection line and the quality control line before and after being cut by the Cas protein.

[0187] In some embodiments, the method of detecting a target nucleic acid may further include 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.

[0188] In some embodiments, the method of detecting a target nucleic acid can also include using RNA reporter nucleic acid and DNA reporter nucleic acid on different channels (e.g., fluorescent colors), and determining the level of detectable signal by measuring the signal levels of the RNA and DNA reporter molecules, and by measuring the amount of target nucleic acid in the RNA and DNA reporter molecules, and sampling based on the level of the combined (e.g., using a minimum or product) detectable signal.

[0189] In one embodiment, the target gene is present in a cell.

[0190] In one embodiment, the cell is a prokaryotic cell.

[0191] In one embodiment, the cell is a eukaryotic cell.

[0192] In one embodiment, the cell is an animal cell.

[0193] In one embodiment, the cell is a human cell.

[0194] In one embodiment, the cell is a plant cell, such as a cell from a cultivated plant (such as cassava, corn, sorghum, wheat, or rice), algae, tree, or vegetable.

[0195] In one embodiment, the target gene is present in a nucleic acid molecule (eg, a plasmid) in vitro.

[0196] In one embodiment, the target gene is present in a plasmid.

[0197] Definition of terms

[0198] Unless otherwise indicated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, procedures in molecular genetics, nucleic acid chemistry, chemistry, molecular biology, biochemistry, cell culture, microbiology, cell biology, genomics, and recombinant DNA used herein are conventional procedures widely used in the relevant fields. To facilitate a better understanding of the present invention, definitions and explanations of relevant terms are provided below.

[0199] Nucleic acid cleavage or nucleic acid cleavage herein includes: DNA or RNA breakage (Cis cleavage) in the target nucleic acid produced by the Cas enzyme described herein, DNA or RNA breakage in the side branch nucleic acid substrate (single-stranded nucleic acid substrate) (i.e., non-specific or non-targeted, Trans cleavage). In some embodiments, the cleavage is a double-stranded DNA break. In some embodiments, the cleavage is a single-stranded DNA break or a single-stranded RNA break.

[0200] CRISPR system

[0201] As used herein, the terms "clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated (Cas) (CRISPR-Cas) system" or "CRISPR system" are used interchangeably and have the meaning commonly understood by those skilled in the art, which generally includes transcripts or other elements associated with the expression of CRISPR-associated ("Cas") genes, or transcripts or other elements capable of directing the activity of the Cas genes.

[0202] CRISPR / Cas complex

[0203] As used herein, the term "CRISPR / Cas complex" refers to a complex formed by the binding of guide RNA or mature crRNA to the Cas protein, which comprises a direct repeat sequence that hybridizes to the guide sequence of the target sequence and binds to the Cas protein, and the complex is capable of recognizing and cleaving a polynucleotide that can hybridize to the guide RNA or mature crRNA.

[0204] Guide RNA (gRNA)

[0205] As used herein, the terms "guide RNA (gRNA)", "mature crRNA", and "guide sequence" are used interchangeably and have meanings generally understood by those skilled in the art. Generally speaking, a guide RNA can comprise a direct repeat sequence and a guide sequence, or consist essentially of or consist of a direct repeat sequence and a guide sequence.

[0206] In some cases, a guide sequence is any polynucleotide sequence that has sufficient complementarity to a target sequence to hybridize with the target sequence and guide specific binding of the CRISPR / Cas complex to the target sequence. In one embodiment, when optimally aligned, 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%. Determining optimal alignment is within the capabilities of one of ordinary skill in the art. For example, there are publicly available and commercially available alignment algorithms and programs such as, but not limited to, ClustalW, Smith-Waterman in matlab, Bowtie, Geneious, Biopython, and SeqMan.

[0207] Target sequence

[0208] "Target sequence" refers to a polynucleotide targeted by a guide sequence in a gRNA, such as a sequence having complementarity with the guide sequence, wherein hybridization between the target sequence and the guide sequence promotes the formation of a CRISPR / Cas complex (including Cas protein and gRNA). Complete complementarity is not required, as long as there is sufficient complementarity to cause hybridization and promote the formation of a CRISPR / Cas complex.

[0209] The target sequence can comprise any polynucleotide, such as DNA or RNA. In some cases, the target sequence is located 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 in an organelle of a eukaryotic cell, such as a mitochondria or chloroplast. A sequence or template that can be used to recombine into a target locus comprising the target sequence is referred to as an "editing template" or "editing polynucleotide" or "editing sequence". In one embodiment, the editing template is an exogenous nucleic acid. In one embodiment, the recombination is homologous recombination.

[0210] In the present invention, a "target sequence" or "target polynucleotide" or "target nucleic acid" can be any polynucleotide that is endogenous or exogenous to a cell (e.g., a eukaryotic cell). For example, the target polynucleotide can be a polynucleotide that is present in the nucleus of a eukaryotic cell. The target polynucleotide can be a sequence encoding a gene product (e.g., a protein) or a non-coding sequence (e.g., a regulatory polynucleotide or junk DNA). In some cases, the target sequence should be associated with a protospacer adjacent motif (PAM).

[0211] Single-stranded nucleic acid detector

[0212] The single-stranded nucleic acid detector of the present invention refers to a sequence containing 2-200 nucleotides, preferably 2-150 nucleotides, preferably 3-100 nucleotides, preferably 3-30 nucleotides, preferably 4-20 nucleotides, and more preferably 5-15 nucleotides. It is preferably a single-stranded DNA molecule, a single-stranded RNA molecule, or a single-stranded DNA-RNA hybrid.

[0213] The single-stranded nucleic acid detector includes different reporter groups or labeling molecules at both ends. When it is in the initial state (i.e., uncleaved state), it does not present a reporter signal. When the single-stranded nucleic acid detector is cleaved, it presents a detectable signal, i.e., a detectable difference is shown after cleavage and before cleavage.

[0214] In one embodiment, the reporter group or labeling molecule includes a fluorescent group and a quencher group, wherein the fluorescent group is selected from one or any combination of FAM, FITC, VIC, JOE, TET, CY3, CY5, ROX, Texas Red or LC RED460; and the quencher group is selected from one or any combination of BHQ1, BHQ2, BHQ3, Dabcy1 or Tamra.

[0215] In one embodiment, the single-stranded nucleic acid detector has a first molecule (such as FAM or FITC) connected to the 5' end and a second molecule (such as biotin) connected to the 3' end. The reaction system containing the single-stranded nucleic acid detector is used in conjunction with a flow strip to detect target nucleic acid (preferably, colloidal gold detection method). The flow strip is designed to have two capture lines, with an antibody that binds to the first molecule (i.e., the first molecule antibody) at the sample contact end (colloidal gold), an antibody that binds to the first molecule antibody at the first line (control line), and an antibody that binds to the second molecule (i.e., the second molecule antibody, such as avidin) at the second line (test line). When the reaction flows along the strip, the first molecule antibody binds to the first molecule and carries the cut or uncut oligonucleotide to the capture line. The cut reporter will bind to the antibody of the first molecule antibody at the first capture line, and the uncut reporter will bind to the second molecule antibody at the second capture line. The binding of the reporter group to each line will result in a strong readout / signal (e.g., color). As more reporters are cut, more signals will accumulate at the first capture line, and less signals will appear at the second line. In certain aspects, the present invention relates to the use of a flow strip as described herein for detecting nucleic acids. In certain aspects, the present invention relates to a method for detecting nucleic acids using a flow strip as defined herein, such as a (lateral) flow test or a (lateral) flow immunochromatographic assay. In certain aspects, the molecules in the single-stranded nucleic acid detector may be interchangeable or their positions may be altered, and any such modifications are encompassed by the present invention as long as the reporting principle is the same or similar to that of the present invention.

[0216] The detection method of the present invention can be used for quantitative detection of target nucleic acids. The quantitative detection index can be quantified based on the signal strength of the reporter group, such as the luminescence intensity of the fluorescent group, or the width of the color band.

[0217] wild type

[0218] As used herein, the term "wild type" has the meaning generally understood by those skilled in the art to refer to the typical form of an organism, strain, gene, or characteristic as it exists in nature, as distinguished from mutant or variant forms, which can be isolated from a source in nature and has not been intentionally modified by man.

[0219] Derivatization

[0220] As used herein, the term "derivatization" refers to the chemical modification of an amino acid, polypeptide, or protein wherein one or more substituents have been covalently attached to the amino acid, polypeptide, or protein. The substituents may also be referred to as side chains.

[0221] A derivatized protein is a derivative of the protein. Generally, the derivatization of the protein does not adversely affect the desired activity of the protein (e.g., the activity of binding to the guide RNA, the endonuclease activity, the activity of binding to and cutting a specific site of the target sequence under the guidance of the guide RNA), that is, the derivative of the protein has the same activity as the protein.

[0222] Derivatized proteins

[0223] Also known as "protein derivatives", refers to modified forms of proteins, for example, wherein one or more amino acids of the protein may be deleted, inserted, modified and / or substituted.

[0224] Non-naturally occurring

[0225] As used herein, the terms "non-naturally occurring" or "engineered" are used interchangeably and indicate the involvement of human effort. When these terms are used to describe a nucleic acid molecule or polypeptide, they indicate that the nucleic acid molecule or polypeptide is at least substantially free from at least one other component with which it is associated in nature or as found in nature.

[0226] Orthologue (ortholog)

[0227] As used herein, the term "orthologue" has the meaning commonly understood by those skilled in the art. As a further guide, an "orthologue" of a protein as described herein refers to a protein belonging to a different species that performs the same or similar function as the protein to which it is an orthologue.

[0228] Identity

[0229] As used herein, the term "identity" refers to the match 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 the two DNA molecules is occupied by adenine, or a position in each of the 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 matching positions shared by the two sequences divided by the number of positions compared x 100. For example, if 6 out of 10 positions in 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 match). Typically, two sequences are compared when they are aligned for maximum identity. Such an alignment can be achieved, for example, by using the method of Needleman et al. (1970) J. Mol. Biol. 48:443-453, which can be conveniently performed using a computer program such as the Align program (DNAstar, Inc.). The percent identity between two amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl Biosci., 4:11-17 (1988)), which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. In addition, the percent identity between two amino acid sequences can be determined using the Needleman and Wunsch (J Mol Biol. 48:444-453 (1970)) algorithm, which has been incorporated into the GAP program in the GCG software package (available at www.gcg.com), using a Blossum 62 matrix or a PAM250 matrix and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.

[0230] carrier

[0231] The term "vector" refers to a nucleic acid molecule that is capable of transporting another nucleic acid molecule to which it is attached. Vectors include, but are not limited to, single-stranded, double-stranded, or partially double-stranded nucleic acid molecules; nucleic acid molecules comprising one or more free ends, or no free ends (e.g., circular); nucleic acid molecules comprising DNA, RNA, or both; and other various polynucleotides known in the art. A vector can be introduced into a host cell by transformation, transduction, or transfection so that the genetic material elements it carries are expressed in the host cell. A vector can be introduced into a host cell to produce transcripts, proteins, or peptides, including proteins, fusion proteins, isolated nucleic acid molecules, etc. as described herein (e.g., CRISPR transcripts, such as nucleic acid transcripts, proteins, or enzymes). A vector can contain a variety of elements that control expression, including, but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. In addition, the vector may also contain a replication initiation site.

[0232] One type of vector is a "plasmid," which refers to a circular double stranded DNA loop into which additional DNA segments can be inserted, eg, by standard molecular cloning techniques.

[0233] Another type of vector is a viral vector, in which a virally derived DNA or RNA sequence is present in a vector for packaging a virus (e.g., a retrovirus, a replication-defective retrovirus, adenovirus, a replication-defective adenovirus, and adeno-associated virus). The viral vector also comprises a polynucleotide carried by a virus for transfection into a host cell. Some vectors (e.g., bacterial vectors and episomal mammalian vectors with a bacterial origin of replication) can replicate autonomously in the host cell into which they are introduced.

[0234] Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of the host cell upon introduction into the host cell and are thereby replicated along with the host genome. Furthermore, some vectors are capable of directing the expression of genes to which they are operably linked. Such vectors are referred to herein as "expression vectors."

[0235] host cells

[0236] As used herein, the term "host cell" refers to cells that can be used to introduce a vector, including but not limited to prokaryotic cells such as Escherichia coli or Bacillus subtilis, and eukaryotic cells such as microbial cells, fungal cells, animal cells, and plant cells.

[0237] Those skilled in the art will appreciate that the design of the expression vector may depend on factors such as the choice of the host cell to be transformed, the level of expression desired, and the like.

[0238] Regulatory elements

[0239] 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), which are described in detail in Goeddel, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, CA (1990). In some cases, regulatory elements include those that direct constitutive expression of a nucleotide sequence in many types of host cells and those that direct expression of the nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences). Tissue-specific promoters can primarily direct expression in a desired tissue of interest, such as muscle, neurons, bone, skin, blood, a specific organ (e.g., liver, pancreas), or a particular cell type (e.g., lymphocytes). In some cases, regulatory elements can 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 some cases, the term "regulatory element" encompasses enhancer elements such as WPRE; the CMV enhancer; the R-U5' segment in the LTR of HTLV-I ((Mol. Cell. Biol., Vol. 8(1), pp. 466-472, 1988); the SV40 enhancer; and the intron sequence between exons 2 and 3 of rabbit β-globin (Proc. Natl. Acad. Sci. USA., Vol. 78(3), pp. 1527-31, 1981).

[0240] promoter

[0241] 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 can initiate expression of a downstream gene. A constitutive promoter is a nucleotide sequence that, when operably linked to a polynucleotide encoding or defining a gene product, results in the production of the gene product in a cell under most or all physiological conditions of the cell. An inducible promoter is a nucleotide sequence that, when operably linked to a polynucleotide encoding or defining a gene product, results in the production of the gene product in the cell substantially only when an inducer corresponding to the promoter is present in the cell. A tissue-specific promoter is a nucleotide sequence that, when operably linked to a polynucleotide encoding or defining a gene product, results in the production of the gene product in the cell substantially only when the cell is a cell of the tissue type corresponding to the promoter.

[0242] NLS

[0243] A "nuclear localization signal" or "nuclear localization sequence" (NLS) is an amino acid sequence that "tags" a protein for import into the cell nucleus via nuclear transport, i.e., proteins with an NLS are transported to the cell nucleus. Typically, an NLS comprises a positively charged Lys or Arg residue exposed on the surface of the protein. Exemplary nuclear localization sequences include, but are not limited to, NLSs from the following: SV40 large T antigen, EGL-13, c-Myc, and TUS protein. In some embodiments, the NLS comprises the 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 sequence KIPIK in the yeast transcription repressor Matα2, and PY-NLS.

[0244] operably connected

[0245] 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).

[0246] Complementarity

[0247] 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 traditional Watson-Crick or other non-traditional types. Percent complementarity represents 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 out of 10 are 50%, 60%, 70%, 80%, 90%, and 100% complementary). "Complete complementarity" means that all consecutive residues of a nucleic acid sequence form hydrogen bonds with the same number of consecutive residues in a second nucleic acid sequence. As used herein, "substantially complementary" refers to a degree of complementarity that is 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.

[0248] Strict conditions

[0249] As used herein, "stringent conditions" for hybridization refer to conditions under which a nucleic acid having complementarity with a target sequence predominantly hybridizes to the target sequence and does not substantially hybridize to non-target sequences. Stringent conditions are generally sequence-dependent and vary depending on many factors. Generally speaking, the longer the sequence, the higher the temperature at which the sequence specifically hybridizes to its target sequence.

[0250] hybridization

[0251] The terms "hybridize" or "complementary" or "substantially complementary" refer to a nucleic acid (e.g., RNA, DNA) comprising a nucleotide sequence that enables it to non-covalently bind, i.e., form base pairs and / or G / U base pairs, "anneal" or "hybridize" with another nucleic acid in a sequence-specific, antiparallel manner (i.e., a nucleic acid specifically binds to a complementary nucleic acid).

[0252] Hybridization requires that the two nucleic acids contain complementary sequences, although there may be mismatches between the bases. Suitable conditions for hybridization between two nucleic acids depend on the length of the nucleic acids and the degree of complementarity, which are variables well known in the art. Typically, the length of a hybridizable nucleic acid is 8 nucleotides or more (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).

[0253] It is understood that the sequence of a polynucleotide need not be 100% complementary to the sequence of its target nucleic acid to hybridize specifically. A polynucleotide 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 the target region in the target nucleic acid sequence with which it hybridizes.

[0254] The hybridization of the target sequence and 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 nucleic acid sequences of the target sequence and the gRNA can hybridize to form a complex; or represents that at least 12, 15, 16, 17, 18, 19, 20, 21, 22 or more bases of the nucleic acid sequences of the target sequence and the gRNA can complement each other and hybridize to form a complex.

[0255] Express

[0256] As used herein, the term "expression" refers to the process by which a polynucleotide is transcribed from a DNA template (e.g., into mRNA or other RNA transcripts) and / or the process by which the transcribed mRNA is subsequently translated into a peptide, polypeptide, or protein. The transcript and the encoded polypeptide may be collectively referred to as a "gene product." If the polynucleotide is derived from genomic DNA, expression may include splicing of the mRNA in a eukaryotic cell.

[0257] connector

[0258] As used herein, the term "linker" refers to a linear polypeptide formed by connecting multiple amino acid residues via peptide bonds. The linker of the present invention can be an artificially synthesized amino acid sequence, or a naturally occurring polypeptide sequence, such as a polypeptide having a hinge region function. Such linker polypeptides are well known in the art (see, for example, Holliger, P. et al. (1993) Proc. Natl. Acad. Sci. USA 90: 6444-6448; Poljak, RJ et al. (1994) Structure 2: 1121-1123).

[0259] treat

[0260] As used herein, the term "treat" refers to treating or curing a disorder, delaying the onset of symptoms of a disorder, and / or delaying the progression of a disorder.

[0261] Subjects

[0262] As used herein, the term "subject" includes, but is not limited to, various animals, plants, and microorganisms.

[0263] animal

[0264] For example, mammals, such as bovines, equines, ovines, porcines, canines, felines, lagomorphs, rodents (e.g., mice or rats), non-human primates (e.g., macaques or cynomolgus monkeys), or humans. In certain embodiments, the subject (e.g., a human) has a disorder (e.g., a disorder caused by a disease-associated gene defect).

[0265] plant

[0266] The term "plant" is to be understood as meaning 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 artichokes, Brussels sprouts, rocket, leeks, asparagus, lettuce (e.g., head lettuce, leaf lettuce, romaine lettuce), bok choy, yellow taro, melons (e.g., cantaloupe, watermelon, Crenshaw melon, honeydew melon, cantaloupe), oilseed crops (e.g., Brussels sprouts, cabbage, cauliflower, broccoli, kale, kale, Chinese cabbage, bok choy), cardoon, carrot, napa, okra, onion, celery, parsley, chickpeas, parsnips, endive, peppers, potatoes, cucurbits (e.g., zucchini, cucumber, courgette, squash, pumpkin), radish, cabbage, Onions, rutabagas, eggplant (also known as eggplant), salsify, lettuce, shallots, endive, garlic, spinach, green onions, squash, greens, beets (sugar beets and fodder beets), sweet potatoes, Swiss chard, horseradish, tomatoes, turnips, and spices; fruits and / or vines such as apples, apricots, cherries, nectarines, peaches, pears, plums, prunes, cherries, quince, almonds, chestnuts, hazelnuts, pecans, pistachios, walnuts, citrus, blueberries, boysenberries, erry), cranberries, currants, loganberries, raspberries, strawberries, blackberries, grapes, avocados, bananas, kiwis, persimmons, pomegranates, pineapples, tropical fruits, pome fruits, melons, mangoes, papayas, and lychees; field crops such as clover, alfalfa, evening primrose, meadowsweet, corn / maize (feed corn, sweet corn, popcorn), hops, jojoba, peanuts, rice, safflower, small grain cereals (barley, oats, rye, wheat, etc.), sorghum, tobacco, kapok, legumes (beans, lentils, peas beans, soybeans), oil plants (rapeseed, mustard, olive, sunflower, coconut, castor oil plant, cocoa bean, peanut), Arabidopsis, fiber plants (cotton, flax, jute), Lauraceae (cinnamon, camphor), or a plant such as coffee, sugar cane, tea, and natural rubber plant; and / or bedding plants, such as flowering plants, cacti, succulents and / or ornamental plants, as well as trees such as forests (broadleaf trees and evergreen trees, such as conifers), fruit trees, ornamental trees, and nut-bearing trees, as well as shrubs and other seedlings.

[0267] Advantageous Effects of the Invention

[0268] The present invention improves the activity of Cas12j19 protein through mutation and has broad application prospects.

[0269] The embodiments of the present invention 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 intended only to illustrate the present invention and are not intended to limit the scope of the invention. Various objects and advantages of the present invention will become apparent to those skilled in the art based on the following detailed description of the accompanying drawings and preferred embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0270] Figure 1. Verification of editing efficiency of Cas proteins with single-site amino acid mutations. The vertical axis represents the ratio of GFP fluorescence in CFP and RFP detected by flow cytometry, i.e., editing efficiency, and the horizontal axis represents different mutants.

[0271] Figure 2. Editing activity of the double spike-in Cas protein combined with E100K. The vertical axis represents the ratio of GFP fluorescence in CFP and RFP detected by flow cytometry, i.e., the editing efficiency, and the horizontal axis represents different mutants.

[0272] Figure 3. Verification of the editing activity of multi-mer Cas proteins. The vertical axis represents the ratio of GFP fluorescence in CFP and RFP detected by flow cytometry, i.e., the editing efficiency, and the horizontal axis represents different mutants.

[0273] Figure 4. Recognition results of multi-spike Cas proteins for different PAMs. The vertical axis represents the ratio of GFP fluorescence in CFP and RFP detected by flow cytometry, that is, the editing efficiency. The horizontal axis represents different mutants. WT is wild-type Cas12j19, and E100K-S400R-L763R-S45T is a combined four-spike protein. DETAILED DESCRIPTION

[0274] The following examples are only used to describe the present invention, but are not intended to limit the present invention. Unless otherwise specified, the experiment and method described in the embodiment are carried out substantially according to conventional methods well known in the art and described in various references. For example, the conventional techniques such as immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics and recombinant DNA used in the present invention can be found in Sambrook, Fritsch and Maniatis, " molecular cloning: laboratory manual " (MOLECULAR CLONING:A LABORATORY MANUAL), 2nd edition (1989); " current protocols in molecular biology " (FM Ausubel et al. edit, (1987)); " methods in enzymes " (METHODS IN ENZYMOLOGY) series (Academic Publishing Company): " PCR 2: practical methods " (PCR 2:A PRACTICAL APPROACH) (MJ MacPherson, BD Hames and GR Taylor, eds. (1995)), Harlow and Lane, eds. (1988) ANTIBODIES, A LABORATORY MANUAL, and ANIMAL CELL CULTURE (RI Freshney, ed. (1987)).

[0275] In addition, if specific conditions are not specified in the examples, the experiments were performed under conventional conditions or the conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments is not specified, they are all conventional products that can be obtained commercially. It is understood that the examples describe the present invention by way of example and are not intended to limit the scope of the present invention. All publications and other references mentioned herein are incorporated herein by reference in their entirety.

[0276] Example 1. Acquisition of Cas mutant proteins

[0277] For known Cas protein (Cas12j.19 in CN111770992B, in the present embodiment, it is referred to as Cas12j19), the applicant predicts the key amino acid sites that may affect its biological function through bioinformatics, and mutates the amino acid sites to obtain Cas mutant proteins with improved editing activity. Specifically, the Cas12j19 coding sequence is codon optimized and synthesized, the amino acid sequence of wild-type Cas12j19 is shown in SEQ ID No.1, and its nucleotide sequence is shown in SEQ ID No.2. The amino acid to which potential Cas12j19 is bound to the target sequence is subjected to site-directed mutagenesis by bioinformatics methods.

[0278] SEQ ID No. 1:

[0279] SEQ ID No. 2:

[0280] Variants of the Cas protein were generated by PCR-based site-directed mutagenesis. The specific method is to divide the DNA sequence of the Cas12j19 protein into two parts with the mutation site as the center, design two pairs of primers to amplify the two parts of the DNA sequence respectively, and introduce the sequence that needs to be mutated on the primers. Finally, the two fragments are loaded into the pcDNA3.3-eGFP vector by Gibson cloning. The combination of mutants is constructed by splitting the DNA of the Cas12j19 protein into multiple segments and using PCR and Gibson clone. Fragment amplification kit: TransStart FastPfu DNA Polymerase (containing 2.5mM dNTPs), please refer to the instructions for the specific experimental process. Gel recovery kit: Gel DNA Extraction Mini Kit. Detailed experimental procedures are provided in the instructions. Vector construction kit: pEASY-Basic Seamless Cloning and Assembly Kit (CU201-03). Detailed experimental procedures are provided in the instructions.

[0281] Based on the above amino acid mutation sites, the wild-type protein (WT) of Cas12j19 and proteins with mutations at different amino acid single sites were obtained.

[0282] Example 2. Verification of the editing activity of Cas mutant proteins

[0283] A fluorescent reporter system suitable for verifying Cas12j19 cutting was constructed with reference to the literature (Yang, Yi, et al. "Highly efficient and rapid detection of the cleavage activity of Cas9 / gRNA via a fluorescent reporter." Applied biochemistry and biotechnology 180.4(2016):655-667.). The fluorescent reporter vector contains RFP and non-luminescent GFFP fluorescent proteins, and the Cas vector contains CFP fluorescent protein. The FUT8-3:ATGGAGGCTGTCTACAATGGGGA target on the GFFP sequence was selected for testing. The underlined position is the PAM sequence, and the DR sequence is GUGCUGCUGUCUCCCAGACGGGAGGCAGAACUGCAC. After transfection of CHO cells for 48 hours, the ratio of GFP fluorescence in CFP and RFP was used to determine the editing efficiency by flow cytometry. Different mutants obtained in Example 1 were selected for testing.

[0284] Figure 1 shows the editing efficiency of different Cas proteins, where the editing efficiency of the wild-type Cas12j19 protein (WT, shown in SEQ ID No. 1) is considered to be 1, and different points represent Cas proteins with different single amino acid site mutations. As shown in Figure 1, the dotted line indicates that the editing efficiency is 1.3 times that of the wild-type editing efficiency. After normalization and plotting, the editing activity of most mutant proteins with single amino acid site mutations is greatly reduced relative to the wild-type Cas12j19; however, the editing efficiency of mutant proteins S3R, Y4R, A154R, S228R, A231R, L99R, S400R, T411R, T647R, D649R, and D658R is significantly improved relative to the wild-type, and its editing efficiency is more than 1.3 times that of the wild-type Cas12j19. The above mutant proteins S3R, Y4R, A154R, S228R, A231R, L99R, S400R, T411R, T647R, D649R, and D658R are relative to SEQ ID In the sequence shown in No. 1, the 3rd amino acid, 4th amino acid, 154th amino acid, 228th amino acid, 231st amino acid, 99th amino acid, 400th amino acid, 411th amino acid, 647th amino acid, 649th amino acid, and 658th amino acid from the N-terminus are mutated to R, respectively.

[0285] It can be seen that the 3rd, 4th, 154th, 228th, 231st, 99th, 400th, 411th, 647th, 649th or 658th position from the N-terminus of SEQ ID No. 1 is the key site for the activity of Cas12j19. By mutating the above amino acid sites, the editing efficiency of Cas12j19 can be greatly improved.

[0286] Example 3. Verification of the editing activity of polysporin Cas proteins

[0287] For further optimization of the editing efficiency of Cas12j19, the amino acid site that can improve the editing efficiency screened in Example 2 was combined with the 100th amino acid of SEQ ID No.1 to K for mutation site, and the activity of the mutated Cas protein was verified. The FUT8-3 on the GFFP sequence: ATGGAGGCTGTCTACAATGGGGA target was selected for testing. The underlined position is the PAM sequence, and the DR sequence is GUGCUGCUGUCUCCCAGACGGGAGGCAGAACUGCAC. The mutant protein was obtained and the verification method was referred to Example 1-2 to obtain the double-site mutant proteins E100K-S3R, E100K-L99R, E100K-A154R, E100K-A231R, E100K-S400R, E100K-T411R, E100K-T647R, E100K-D649R, and E100K-D658R.

[0288] The results are shown in Figure 2. The editing efficiency of wild-type Cas12j19 (WT, shown in SEQ ID No. 1) is about 30%. The double-stranded Cas protein greatly improves the editing efficiency, with an increase of about 1-3 times. Among them, the editing efficiency of the E100K-S400R mutant protein is about 90%, which has the best editing effect.

[0289] Further optimization was performed based on the E100K-S400R mutant protein. Given that the fluorescent mutant of FUT8-3 was too efficient, the FUT8-8 sequence: ATGGAAGGCTGCTTCTGTTCCCA was selected as the target for testing. The underlined position is the PAM sequence. The methods for constructing the mutant protein and verifying the editing efficiency were the same as those in the above examples.

[0290] The results are shown in Figure 3. When the target is FUT8-8, the editing efficiency of wild-type Cas12j19 (WT, shown in SEQ ID No. 1) is 6%, and the editing efficiency of the combined three-spiked protein E100K-S400R-L763R (relative to SEQ ID No. 1, the 100th amino acid mutated to K, and at the same time, the 400th amino acid mutated to R, and the 763rd amino acid mutated to R) and the combined four-spiked protein E100K-S400R-L763R-S45T (relative to SEQ ID No. 1, the 100th amino acid mutated to K, and at the same time, the 400th amino acid mutated to R, the 763rd amino acid mutated to R, and the 45th amino acid mutated to T) are both around 90%, which is significantly improved compared to the wild-type Cas12j19.

[0291] The recognition of different PAMs by the combined four-spike protein E100K-S400R-L763R-S45T screened above was detected. The results are shown in Figure 4. The wild-type Cas12j19 showed significant recognition preference when the PAMs were ATA and ATG, while the combined four-spike protein E100K-S400R-L763R-S45T showed significant recognition preference at all PAMs tested. The editing efficiency of the combined four-spike protein E100K-S400R-L763R-S45T at targets with different PAMs was significantly improved compared with the wild-type Cas12j19. The mutant protein E100K-S400R-L763R-S45T has a wider PAM recognition range than the wild-type Cas12j protein and has broader application prospects.

[0292] Example 4. Verification of Cas protein editing efficiency in plants

[0293] In this embodiment, wild-type Cas12j19 (WT, shown in SEQ ID No. 1), E100K mutant Cas12j19 (relative to SEQ ID No. 1, the 100th amino acid mutated to K), E100K-S400R-L763R-S45T mutant Cas12j19 (relative to SEQ ID No. 1, the 100th amino acid mutated to K, and the 400th amino acid mutated to R, the 763rd amino acid mutated to R, and the 45th amino acid mutated to T) were used to verify their gene editing efficiency in rice and soybean. Gene editing vectors were constructed using conventional techniques in the art, and genetic transformation of rice and soybean was performed. Primers were designed on both sides of the gRNA targeting site, and the amplified fragments were subjected to Sanger sequencing to determine their editing type and calculate the editing efficiency.

[0294] The editing efficiency of the above-mentioned different Cas12j19 at different targets in rice and soybean is shown in the following table.

[0295] As can be seen from the above table, the four-spike protein E100K-S400R-L763R-S45T has significantly improved editing efficiency in targets tested in rice and soybeans compared with wild-type Cas12j19 and E100K single-spike protein; even in some targets where wild-type Cas12j19 and E100K single-spike protein have no editing efficiency, the four-spike protein E100K-S400R-L763R-S45T also shows good editing efficiency.

[0296] Although the specific embodiments of the present invention have been described in detail, those skilled in the art will understand that various modifications and changes can be made to the details based on all the teachings published, and these changes are all within the scope of protection of the present invention. The entire invention is given by the appended claims and any equivalents thereof.

Claims

1. A Cas mutant protein, wherein the mutant protein has a mutation at any one or any several (for example, any 2, any 3, any 4, any 5, any 6, any 7, any 8, any 9, any 10 or any 11) amino acid positions corresponding to the amino acid sequence shown in SEQ ID No. 1 compared with the amino acid sequence of the parent Cas protein: position 400, position 3, position 4, position 99, position 154, position 228, position 231, position 411, position 647, position 649 or position 658; Preferably, compared with the amino acid sequence of the parent Cas protein, the Cas mutant protein further includes mutations at the following amino acid sites corresponding to the amino acid sequence shown in SEQ ID No. 1: position 100; More preferably, the Cas mutant protein has a mutation at the following amino acid sites corresponding to the amino acid sequence shown in SEQ ID No. 1 compared to the amino acid sequence of the parent Cas protein: the 400th amino acid, the 100th amino acid and the 763rd amino acid are mutated at the same time, or the 400th amino acid, the 100th amino acid, the 763rd amino acid and the 45th amino acid are mutated at the same time.

2. A fusion protein, comprising the Cas mutant protein according to claim 1 and other modified parts; preferably, the modified part is selected from another protein or polypeptide, a detectable marker or any combination thereof.

3. An isolated polynucleotide, characterized in that The polynucleotide is a polynucleotide sequence encoding the Cas mutant protein according to claim 1, or a polynucleotide sequence encoding the fusion protein according to claim 2.

4. A carrier, characterized in that The vector comprises the polynucleotide according to claim 3 and a regulatory element operably linked thereto.

5. A CRISPR-Cas system, characterized in that: The system comprises the Cas mutant protein of claim 1 and at least one gRNA; The gRNA is capable of binding to the Cas mutant protein described in claim 1.

6. A composition, characterized in that The composition comprises: (i) a protein component selected from: the Cas mutant protein according to claim 1 or the fusion protein according to claim 2; (ii) a nucleic acid component, which is a gRNA, wherein the gRNA is capable of binding to the Cas mutant protein according to claim 1; The protein component and the nucleic acid component are combined with each other to form a complex.

7. An engineered host cell, characterized in that The host cell comprises the Cas mutant protein of claim 1, or the fusion protein of claim 2, or the polynucleotide of claim 3, or the vector of claim 4, or the CRISPR-Cas system of claim 5, or the composition of claim 6.

8. Use of the Cas mutant protein according to claim 1, or the fusion protein according to claim 2, or the polynucleotide according to claim 3, or the vector according to claim 4, or the CRISPR-Cas system according to claim 5, or the composition according to claim 6, or the host cell according to claim 7 in any one or more of the following: Gene editing, gene targeting, gene cleavage, targeting and / or editing target nucleic acid, cleavage of double-stranded DNA, single-stranded DNA or single-stranded RNA, non-specific cleavage and / or degradation of collateral nucleic acid, non-specific cleavage of single-stranded nucleic acid, nucleic acid detection, specific editing of double-stranded nucleic acid, base editing of double-stranded nucleic acid, base editing of single-stranded nucleic acid; Alternatively, the use in preparing a reagent or a kit, wherein the reagent or the kit is used for: gene or genome editing, target nucleic acid detection and / or diagnosis, editing the target sequence in the target locus to modify the organism, treatment of disease, targeting the target gene, cutting the target gene.

9. A method for editing, targeting or cutting a target nucleic acid, the method comprising the step of contacting the target nucleic acid with the Cas mutant protein of claim 1, or the fusion protein of claim 2, or the polynucleotide of claim 3, or the vector of claim 4, or the CRISPR-Cas system of claim 5, or the composition of claim 6, or the host cell of claim 7.

10. A kit for gene editing, gene targeting or gene cleavage, the kit comprising the Cas mutant protein of claim 1, or the fusion protein of claim 2, or the polynucleotide of claim 3, or the vector of claim 4, or the CRISPR-Cas system of claim 5, or the composition of claim 6, or the host cell of claim 7.

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