Novel CRISPR-Cas12j.23 enzyme and system

Abstract

The present invention relates to the field of nucleic acid editing, in particular to the technical field of regularly clustered interspaced short palindromic repeats (CRISPR). Specifically, the present invention relates to Cas effector proteins, fusion proteins comprising such proteins, and nucleic acid molecules encoding them. The present invention also relates to complexes and compositions for nucleic acid editing (e.g., gene or genome editing), comprising proteins or fusion proteins of the present invention, or nucleic acid molecules encoding them. The present invention also relates to methods for nucleic acid editing (e.g., gene or genome editing), which use proteins or fusion proteins comprising the present invention.

Description

Technical Field

[0001] The present invention relates to the field of nucleic acid editing, in particular to the technical field of regularly clustered interspaced short palindromic repeats (CRISPR). Specifically, the present invention relates to Cas effector proteins, fusion proteins comprising such proteins, and nucleic acid molecules encoding them. The present invention also relates to complexes and compositions for nucleic acid editing (e.g., gene or genome editing), comprising proteins or fusion proteins of the present invention, or nucleic acid molecules encoding them. The present invention also relates to methods for nucleic acid editing (e.g., gene or genome editing), which use proteins or fusion proteins comprising the present invention. Background Art

[0002] 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.

[0003] 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. The CRISPR / Cas Type V system, a newly discovered CRISPR system in the past two years, 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 relatively 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.

[0004] In summary, given that the currently available CRISPR / Cas systems are limited by some defects, the development of a new CRISPR / Cas system that is more robust and has good performance in many aspects is of great significance to the development of biotechnology. Summary of the Invention

[0005] After extensive experimentation and repeated exploration, the inventors of this application unexpectedly discovered a new type of RNA-guided nuclease. Based on this discovery, the inventors developed a new CRISPR / Cas system and a gene editing method based on this system.

[0006] Cas effector proteins

[0007] Therefore, in a first aspect, the present invention provides a protein having an amino acid sequence as shown in SEQ ID NO: 1 or a direct homologue, homologue, variant or functional fragment thereof; wherein the direct homologue, homologue, variant or functional fragment substantially retains the biological function of the sequence from which it is derived.

[0008] In the present invention, the biological functions of the above sequences include, but are not limited to, the activity of binding to guide RNA, endonuclease activity, and the activity of binding to and cutting specific sites of the target sequence under the guidance of the guide RNA.

[0009] In certain embodiments, the ortholog, homolog, variant has at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the sequence from which it was derived.

[0010] In certain embodiments, the orthologs, homologs, and variants have at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence shown in SEQ ID NO: 1, and substantially retain the biological function of the sequence from which it is derived (e.g., activity of binding to a guide RNA, endonuclease activity, activity of binding to and cutting a specific site of a target sequence under the guidance of a guide RNA).

[0011] In certain embodiments, the protein is an effector protein in a CRISPR / Cas system.

[0012] In certain embodiments, the protein of the present invention comprises or consists of a sequence selected from the group consisting of:

[0013] (i) the sequence shown in SEQ ID NO: 1;

[0014] (ii) a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, deletions or additions) compared to the sequence shown in SEQ ID NO: 1; or

[0015] (iii) a sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the sequence shown in SEQ ID NO: 1.

[0016] In certain embodiments, the protein of the present invention has the amino acid sequence shown in SEQ ID NO:1.

[0017] Derivatized proteins

[0018] The protein of the present invention can be derivatized, for example, by being linked to another molecule (e.g., another polypeptide or protein). Generally, the derivatization (e.g., labeling) of the protein will not adversely affect the desired activity of the protein (e.g., activity binding to a guide RNA, endonuclease activity, activity binding to and cutting a specific site of a target sequence under the guidance of a guide RNA). Therefore, the protein of the present invention is also intended to include such derivatized forms. For example, the protein of the present invention can be functionally linked (by chemical coupling, gene fusion, non-covalent linkage or other means) to one or more other molecular groups, such as another protein or polypeptide, a detection reagent, a pharmaceutical agent, etc.

[0019] In particular, the protein of the present invention can be linked to other functional units. For example, it can be linked to a nuclear localization signal (NLS) sequence to improve the ability of the protein of the present invention to enter the cell nucleus. For example, it can be linked to a targeting moiety to make the protein of the present invention targeted. For example, it can be linked to a detectable label to facilitate detection of the protein of the present invention. For example, it can be linked to an epitope tag to facilitate expression, detection, tracing and / or purification of the protein of the present invention.

[0020] Conjugate

[0021] Thus, in a second aspect, the present invention provides a conjugate comprising a protein as described above and a modifying moiety.

[0022] In certain embodiments, the modifying moiety is selected from another protein or polypeptide, a detectable label, or any combination thereof.

[0023] In certain embodiments, the additional protein or polypeptide is selected from an epitope tag, a reporter gene sequence, a nuclear localization signal (NLS) sequence, a targeting moiety, 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), a domain having an activity selected from the group consisting of nucleotide deaminase, methylase activity, demethylase, transcriptional activation activity, transcriptional repression activity, transcriptional release factor activity, histone modification activity, nuclease activity, single-stranded RNA cleavage activity, double-stranded RNA cleavage activity, single-stranded DNA cleavage activity, double-stranded DNA cleavage activity, and nucleic acid binding activity; and any combination thereof.

[0024] In certain embodiments, the conjugates of the present invention comprise one or more NLS sequences, such as the NLS of the SV40 virus large T antigen. In certain exemplary embodiments, the NLS sequence is as shown in SEQ ID NO: 7. In certain embodiments, the NLS sequence is located at, near, or close to a terminus (e.g., N-terminus or C-terminus) of the protein of the present invention. In certain exemplary embodiments, the NLS sequence is located at, near, or close to the C-terminus of the protein of the present invention.

[0025] In certain embodiments, the conjugates of the present invention comprise an epitope tag. Such epitope tags are well known to those skilled in the art, and examples thereof include, but are not limited to, His, V5, FLAG, HA, Myc, VSV-G, Trx, etc., and those skilled in the art know how to select an appropriate epitope tag according to the desired purpose (e.g., purification, detection, or tracing).

[0026] In certain embodiments, the conjugates of the present invention comprise a reporter gene sequence. Such reporter genes are well known to those skilled in the art, and examples thereof include, but are not limited to, GST, HRP, CAT, GFP, HcRed, DsRed, CFP, YFP, BFP, and the like.

[0027] In certain embodiments, the conjugates of the present invention comprise a domain capable of binding to a DNA molecule or an intracellular molecule, such as maltose binding protein (MBP), the DNA binding domain (DBD) of Lex A, the DBD of GAL4, and the like.

[0028] In certain embodiments, the conjugates of the invention comprise a detectable label, such as a fluorescent dye, eg, FITC or DAPI.

[0029] In certain embodiments, the protein of the present invention is coupled, conjugated or fused to the modifying moiety, optionally via a linker.

[0030] In certain embodiments, the modifying moiety is directly linked to the N-terminus or C-terminus of a protein of the invention.

[0031] In certain embodiments, the modified portion is connected to the N-terminus or C-terminus of the protein of the present invention via a linker. Such linkers are well known in the art, and examples 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.

[0032] Fusion protein

[0033] In a third aspect, the present invention provides a fusion protein comprising the protein of the present invention and another protein or polypeptide.

[0034] In certain embodiments, the additional protein or polypeptide is selected from an epitope tag, a reporter gene sequence, a nuclear localization signal (NLS) sequence, a targeting moiety, 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), a domain having an activity selected from the group consisting of nucleotide deaminase, methylase activity, demethylase, transcriptional activation activity, transcriptional repression activity, transcriptional release factor activity, histone modification activity, nuclease activity, single-stranded RNA cleavage activity, double-stranded RNA cleavage activity, single-stranded DNA cleavage activity, double-stranded DNA cleavage activity, and nucleic acid binding activity; and any combination thereof.

[0035] In certain embodiments, the fusion proteins of the present invention comprise one or more NLS sequences, such as the NLS of the SV40 virus large T antigen. In certain embodiments, the NLS sequence is located at, near, or close to the terminus (e.g., N-terminus or C-terminus) of the protein of the present invention. In certain exemplary embodiments, the NLS sequence is located at, near, or close to the C-terminus of the protein of the present invention.

[0036] In certain embodiments, the fusion proteins of the invention comprise an epitope tag.

[0037] In certain embodiments, the fusion proteins of the present invention comprise a reporter gene sequence.

[0038] In certain embodiments, the fusion proteins of the present invention comprise a domain capable of binding to a DNA molecule or an intracellular molecule.

[0039] In certain embodiments, the protein of the present invention is fused to the additional protein or polypeptide, optionally via a linker.

[0040] In certain embodiments, the additional protein or polypeptide is directly linked to the N-terminus or C-terminus of the protein of the invention.

[0041] In certain embodiments, the additional protein or polypeptide is linked to the N-terminus or C-terminus of the protein of the invention via a linker.

[0042] In certain exemplary embodiments, the fusion protein of the present invention has the amino acid sequence shown in SEQ ID NO:8.

[0043] The protein, conjugate or fusion protein of the present invention is not limited by the method of production. For example, it can be produced by genetic engineering methods (recombinant technology) or by chemical synthesis methods.

[0044] Directly repeated sequences

[0045] In a fourth aspect, the present invention provides an isolated nucleic acid molecule comprising or consisting of a sequence selected from the group consisting of:

[0046] (i) the sequence shown in SEQ ID NO: 4 or 6;

[0047] (ii) a sequence having one or more base substitutions, deletions or additions (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 base substitutions, deletions or additions) compared to the sequence shown in SEQ ID NO: 4 or 6;

[0048] (iii) a sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the sequence set forth in SEQ ID NO: 4 or 6;

[0049] (iv) a sequence that hybridizes under stringent conditions to the sequence described in any one of (i) to (iii); or

[0050] (v) a complementary sequence of the sequence described in any one of (i) to (iii);

[0051] Furthermore, the sequence described in any one of (ii) to (v) substantially retains the biological function of the sequence from which it is derived, wherein the biological function of the sequence refers to the activity as a direct repeat sequence in the CRISPR-Cas system.

[0052] In certain embodiments, the isolated nucleic acid molecule is a direct repeat sequence in a CRISPR-Cas system.

[0053] In certain embodiments, the nucleic acid molecule comprises or consists of a sequence selected from the group consisting of:

[0054] (a) the nucleotide sequence shown in SEQ ID NO: 4 or 6;

[0055] (b) a sequence that hybridizes under stringent conditions to the sequence described in (a); or

[0056] (c) The complementary sequence of the sequence described in (a).

[0057] In certain embodiments, the isolated nucleic acid molecule is RNA.

[0058] CRISPR / Cas complex

[0059] In a fifth aspect, the present invention provides a composite comprising:

[0060] (i) a protein component selected from the group consisting of a protein, a conjugate or a fusion protein of the present invention, and any combination thereof; and

[0061] (ii) a nucleic acid component comprising, from 5' to 3' direction, an isolated nucleic acid molecule as described above and a guide sequence capable of hybridizing to a target sequence,

[0062] Wherein, the protein component and the nucleic acid component combine with each other to form a complex.

[0063] In certain embodiments, the targeting sequence is linked to the 3' end of the nucleic acid molecule.

[0064] In certain embodiments, the guide sequence comprises the complement of the target sequence.

[0065] In certain embodiments, the nucleic acid component is a guide RNA in a CRISPR-Cas system.

[0066] In certain embodiments, the nucleic acid molecule is RNA.

[0067] In certain embodiments, the complex does not comprise a trans-acting crRNA (tracrRNA).

[0068] In certain embodiments, the complex is targeted to a third component, which is a double-stranded polynucleotide comprising a target sequence adjacent to the motif sequence.

[0069] In certain embodiments, the target sequence is located 3' to the motif sequence.

[0070] In certain embodiments, the target sequence is less than 40, 35, 30, 25, 20, 15, 10, 5, 4, 3, 2, or 1 nucleotides away from the motif sequence.

[0071] In certain embodiments, the guide sequence is at least 5, at least 10, at least 15, at least 20, at least 25, or at least 30 nucleotides in length. In certain embodiments, the guide sequence is 10-30, or 15-25, or 15-22, or 19-25, or 19-22 nucleotides in length.

[0072] In certain embodiments, the nucleic acid molecules of the separation are 55-70 nucleotides in length, for example 55-65 nucleotides, for example 60-65 nucleotides, for example 62-65 nucleotides, for example 63-64 nucleotides. In certain embodiments, the nucleic acid molecules of the separation are 15-30 nucleotides in length, for example 15-25 nucleotides, for example 20-25 nucleotides, for example 22-24 nucleotides, for example 23 nucleotides.

[0073] Encoding nucleic acid, vector and host cell

[0074] In a sixth aspect, the present invention provides an isolated nucleic acid molecule comprising:

[0075] (i) a nucleotide sequence encoding a protein or fusion protein of the present invention;

[0076] (ii) a nucleotide sequence encoding the isolated nucleic acid molecule according to the fourth aspect; or

[0077] (iii) a nucleotide sequence comprising (i) and (ii).

[0078] In certain embodiments, the nucleotide sequence described in any one of (i)-(iii) is codon-optimized for expression in prokaryotes. In certain embodiments, the nucleotide sequence described in any one of (i)-(iii) is codon-optimized for expression in eukaryotic cells.

[0079] In a seventh aspect, the present invention further provides a vector comprising the isolated nucleic acid molecule described in the sixth aspect. The vector of the present invention can be a cloning vector or an expression vector. In certain embodiments, the vector of the present invention is, for example, a plasmid, a cosmid, a phage, a cosmid, or the like. In certain embodiments, the vector is capable of expressing a protein of the present invention, a fusion protein, the isolated nucleic acid molecule described in the fourth aspect, or the complex described in the fifth aspect in a subject (e.g., a mammal, such as a human).

[0080] In an eighth aspect, the present invention further provides a host cell comprising the isolated nucleic acid molecule or vector as described above. Such host cells include, but are not limited to, prokaryotic cells such as Escherichia coli cells, and eukaryotic cells such as yeast cells, insect cells, plant cells, and animal cells (e.g., mammalian cells, such as mouse cells, human cells, etc.). The cells of the present invention can also be cell lines, such as 293T cells.

[0081] Compositions and carrier compositions

[0082] In a ninth aspect, the present invention further provides a composition comprising:

[0083] (i) a first component selected from the group consisting of a protein, a conjugate, a fusion protein, a nucleotide sequence encoding the protein or fusion protein of the present invention, and any combination thereof; and

[0084] (ii) a second component, which is a nucleotide sequence comprising a guide RNA, or a nucleotide sequence encoding the nucleotide sequence comprising a guide RNA;

[0085] Wherein, the guide RNA comprises a direct repeat sequence and a guide sequence from the 5' to the 3' direction, and the guide sequence is capable of hybridizing with the target sequence;

[0086] The guide RNA is capable of forming a complex with the protein, conjugate or fusion protein described in (i).

[0087] In certain embodiments, the direct repeat sequence is an isolated nucleic acid molecule as defined in the fourth aspect.

[0088] In certain embodiments, the guide sequence is linked to the 3' end of the direct repeat sequence.In certain embodiments, the guide sequence comprises a complementary sequence to the target sequence.

[0089] In certain embodiments, the composition does not comprise crRNA (tracrRNA).

[0090] In certain embodiments, the composition is non-naturally occurring or modified. In certain embodiments, at least one component of the composition is non-naturally occurring or modified. In certain embodiments, the first component is non-naturally occurring or modified; and / or, the second component is non-naturally occurring or modified.

[0091] In certain embodiments, when the target sequence is DNA, the target sequence is located 3' to the protospacer adjacent motif (PAM).

[0092] In certain embodiments, when the target sequence is RNA, the target sequence is not restricted by a PAM domain.

[0093] In certain embodiments, the target sequence is a DNA or RNA sequence from a prokaryotic or eukaryotic cell. In certain embodiments, the target sequence is a non-naturally occurring DNA or RNA sequence.

[0094] In certain embodiments, the target sequence is present in a cell. In certain embodiments, the target sequence is present in the nucleus or in the cytoplasm (e.g., an organelle). In certain embodiments, the cell is a eukaryotic cell. In certain embodiments, the cell is a prokaryotic cell.

[0095] In certain embodiments, the protein is linked to one or more NLS sequences. In certain embodiments, the conjugate or fusion protein comprises one or more NLS sequences. In certain embodiments, the NLS sequence is linked to the N-terminus or C-terminus of the protein. In certain embodiments, the NLS sequence is fused to the N-terminus or C-terminus of the protein.

[0096] In a tenth aspect, the present invention further provides a composition comprising one or more carriers, wherein the one or more carriers comprise:

[0097] (i) a first nucleic acid comprising a nucleotide sequence encoding a protein or fusion protein of the present invention; optionally, the first nucleic acid is operably linked to a first regulatory element; and

[0098] (ii) a second nucleic acid comprising a nucleotide sequence encoding a guide RNA; optionally, the second nucleic acid is operably linked to a second regulatory element;

[0099] in:

[0100] The first nucleic acid and the second nucleic acid are present on the same or different vectors;

[0101] The guide RNA comprises a direct repeat sequence and a guide sequence from the 5' to the 3' direction, and the guide sequence is capable of hybridizing with the target sequence;

[0102] The guide RNA is capable of forming a complex with the effector protein or fusion protein described in (i).

[0103] In certain embodiments, the direct repeat sequence is an isolated nucleic acid molecule as defined in the fourth aspect.

[0104] In certain embodiments, the guide sequence is linked to the 3' end of the direct repeat sequence.In certain embodiments, the guide sequence comprises a complementary sequence to the target sequence.

[0105] In certain embodiments, the composition does not comprise trans-acting crRNA (tracrRNA).

[0106] In certain embodiments, the composition is non-naturally occurring or modified. In certain embodiments, at least one component in the composition is non-naturally occurring or modified.

[0107] In certain embodiments, the first regulatory element is a promoter, such as an inducible promoter.

[0108] In certain embodiments, the second regulatory element is a promoter, such as an inducible promoter.

[0109] In certain embodiments, when the target sequence is DNA, the target sequence is located 3' to the protospacer adjacent motif (PAM).

[0110] In certain embodiments, when the target sequence is RNA, the target sequence is not restricted by a PAM domain.

[0111] In certain embodiments, the target sequence is a DNA or RNA sequence from a prokaryotic or eukaryotic cell. In certain embodiments, the target sequence is a non-naturally occurring DNA or RNA sequence.

[0112] In certain embodiments, the target sequence is present in a cell. In certain embodiments, the target sequence is present in the nucleus or in the cytoplasm (e.g., an organelle). In certain embodiments, the cell is a eukaryotic cell. In certain embodiments, the cell is a prokaryotic cell.

[0113] In certain embodiments, the protein is linked to one or more NLS sequences. In certain embodiments, the conjugate or fusion protein comprises one or more NLS sequences. In certain embodiments, the NLS sequence is linked to the N-terminus or C-terminus of the protein. In certain embodiments, the NLS sequence is fused to the N-terminus or C-terminus of the protein.

[0114] In certain embodiments, a type of vector is a plasmid, which refers to a circular double-stranded DNA loop in which other DNA fragments can be inserted, for example, by standard molecular cloning techniques. Another type of vector is a viral vector, in which virally derived DNA or RNA sequences are present in a vector for packaging viruses (for example, retroviruses, replication-defective retroviruses, adenoviruses, replication-defective adenoviruses, and adeno-associated viruses). Viral vectors also include polynucleotides carried by viruses for transfection into a host cell. Some vectors (for example, bacterial vectors and additional mammalian vectors with bacterial replication origins) can replicate autonomously in the host cell into which they are introduced. Other vectors (for example, non-additional mammalian vectors) are integrated into the genome of the host cell after introducing the host cell, and thus replicate together with the host genome. Moreover, some vectors can instruct the expression of their operably connected genes. Such a vector is referred to as an "expression vector" at this. The common expression vector used in recombinant DNA technology is typically a plasmid form.

[0115] Recombinant expression vectors may contain the nucleic acid molecules of the present invention in a form suitable for nucleic acid expression in a host cell, which means that these recombinant expression vectors contain one or more regulatory elements selected based on the host cell to be used for expression, which are operably linked to the nucleic acid sequence to be expressed.

[0116] Delivery and delivery compositions

[0117] The proteins, conjugates, fusion proteins of the present invention, the isolated nucleic acid molecules of the fourth aspect, the complexes of the present invention, the isolated nucleic acid molecules of the sixth aspect, the vectors of the seventh aspect, and the compositions of the ninth and tenth aspects can be delivered by any method known in the art. Such methods include, but are not limited to, electroporation, lipofection, nucleofection, microinjection, sonoporation, gene gun, calcium phosphate-mediated transfection, cationic transfection, lipofection, dendritic transfection, heat shock transfection, nucleofection, magnetofection, lipofection, puncture transfection, optical transfection, agent-enhanced nucleic acid uptake, and delivery via liposomes, immunoliposomes, viral particles, artificial virions, and the like.

[0118] Therefore, in another aspect, the present invention provides a delivery composition comprising a delivery vector and one or more selected from the following: a protein, a conjugate, a fusion protein of the present invention, an isolated nucleic acid molecule as described in the fourth aspect, a complex of the present invention, an isolated nucleic acid molecule as described in the sixth aspect, a vector as described in the seventh aspect, a composition as described in the ninth aspect and the tenth aspect.

[0119] In certain embodiments, the delivery vehicle is a particle.

[0120] In certain embodiments, the delivery vehicle is selected from lipid particles, sugar particles, metal particles, protein particles, liposomes, exosomes, microvesicles, a gene gun, or a viral vector (e.g., a replication-defective retrovirus, a lentivirus, an adenovirus, or an adeno-associated virus).

[0121] Reagent test kit

[0122] In another aspect, the present invention provides a kit comprising one or more of the components described above. In certain embodiments, the kit comprises one or more components selected from the group consisting of a protein, conjugate, fusion protein of the present invention, an isolated nucleic acid molecule as described in the fourth aspect, a complex of the present invention, an isolated nucleic acid molecule as described in the sixth aspect, a vector as described in the seventh aspect, and a composition as described in the ninth and tenth aspects.

[0123] In certain embodiments, the kit of the present invention comprises the composition of the ninth aspect. In certain embodiments, the kit further comprises instructions for using the composition.

[0124] In certain embodiments, the kit of the present invention comprises the composition of the tenth aspect. In certain embodiments, the kit further comprises instructions for using the composition.

[0125] In certain embodiments, the components included in the kits of the present invention may be provided in any suitable container.

[0126] In certain embodiments, the kit further comprises one or more buffers. The buffer can be any buffer, including but not limited to sodium carbonate buffer, sodium bicarbonate buffer, borate buffer, Tris buffer, MOPS buffer, HEPES buffer and combinations thereof. In certain embodiments, the buffer is alkaline. In certain embodiments, the buffer has a pH from about 7 to about 10.

[0127] In certain embodiments, the kit further comprises one or more oligonucleotides corresponding to a targeting sequence for insertion into a vector so as to operably link the targeting sequence and regulatory elements. In certain embodiments, the kit comprises a homologous recombination template polynucleotide.

[0128] Methods and uses

[0129] In another aspect, the present invention provides a method for modifying a target gene, comprising: contacting the complex described in the fifth aspect, the composition described in the ninth aspect, or the composition described in the tenth aspect with the target gene, or delivering it to a cell containing the target gene; the target sequence is present in the target gene.

[0130] In certain embodiments, the target gene is present in a cell. In certain embodiments, the cell is a prokaryotic cell. In certain embodiments, the cell is a eukaryotic cell. In certain embodiments, the cell is a mammalian cell. In certain embodiments, the cell is a human cell. In certain embodiments, the cell is selected from non-human primate, cattle, pig or rodent cells. In certain embodiments, the cell is a non-mammalian eukaryotic cell, such as poultry or fish. In certain embodiments, the cell is a plant cell, such as a cell of a cultivated plant (such as cassava, corn, sorghum, wheat or rice), algae, tree or vegetable.

[0131] In certain embodiments, the target gene is present in a nucleic acid molecule (e.g., a plasmid) in vitro. In certain embodiments, the target gene is present in a plasmid.

[0132] In certain embodiments, the methods result in breakage of the target sequence (e.g., double-strand breaks in DNA or single-strand breaks in RNA).

[0133] In certain embodiments, the disruption results in decreased transcription of the target gene.

[0134] In certain embodiments, the method further comprises: contacting an editing template (e.g., an exogenous nucleic acid) with the target gene, or delivering it to a cell comprising the target gene. In such embodiments, the method repairs the broken target gene by homologous recombination with an editing template (e.g., an exogenous nucleic acid), wherein the repair results in a mutation comprising an insertion, deletion, or substitution of one or more nucleotides of the target gene. In certain embodiments, the mutation results in one or more amino acid changes in a protein expressed from a gene comprising the target sequence.

[0135] Thus, in certain embodiments, the modification further comprises inserting an editing template (eg, an exogenous nucleic acid) into the break.

[0136] In certain embodiments, the protein, conjugate, fusion protein, isolated nucleic acid molecule, complex, vector or composition is contained in a delivery vehicle.

[0137] In certain embodiments, the delivery vehicle is selected from lipid particles, sugar particles, metal particles, protein particles, liposomes, exosomes, viral vectors (such as replication-defective retroviruses, lentiviruses, adenoviruses, or adeno-associated viruses).

[0138] In certain embodiments, the methods are used to modify a cell, cell line, or organism by altering one or more target sequences in a target gene or a nucleic acid molecule encoding a target gene product.

[0139] In another aspect, the present invention provides a method for altering the expression of a gene product, comprising: contacting the complex described in the fifth aspect, the composition described in the ninth aspect, or the composition described in the tenth aspect with a nucleic acid molecule encoding the gene product, or delivering it to a cell containing the nucleic acid molecule, wherein the target sequence is present in the nucleic acid molecule.

[0140] In certain embodiments, the nucleic acid molecule is present in a cell. In certain embodiments, the cell is a prokaryotic cell. In certain embodiments, the cell is a eukaryotic cell. In certain embodiments, the cell is a mammalian cell. In certain embodiments, the cell is a human cell. In certain embodiments, the cell is selected from non-human primate, cattle, pig or rodent cells. In certain embodiments, the cell is a non-mammalian eukaryotic cell, such as poultry or fish. In certain embodiments, the cell is a plant cell, such as a cell of a cultivated plant (such as cassava, corn, sorghum, wheat or rice), algae, tree or vegetable.

[0141] In certain embodiments, the nucleic acid molecule is present in a nucleic acid molecule (e.g., a plasmid) in vitro. In certain embodiments, the nucleic acid molecule is present in a plasmid.

[0142] In certain embodiments, the expression of the gene product is altered (e.g., enhanced or reduced). In certain embodiments, the expression of the gene product is enhanced. In certain embodiments, the expression of the gene product is reduced.

[0143] In certain embodiments, the gene product is a protein.

[0144] In certain embodiments, the protein, conjugate, fusion protein, isolated nucleic acid molecule, complex, vector or composition is contained in a delivery vehicle.

[0145] In certain embodiments, the delivery vehicle is selected from lipid particles, sugar particles, metal particles, protein particles, liposomes, exosomes, viral vectors (such as replication-defective retroviruses, lentiviruses, adenoviruses, or adeno-associated viruses).

[0146] In certain embodiments, the methods are used to modify a cell, cell line, or organism by altering one or more target sequences in a target gene or a nucleic acid molecule encoding a target gene product.

[0147] In another aspect, the present invention relates to the protein as described in the first aspect, the conjugate as described in the second aspect, the fusion protein as described in the third aspect, the isolated nucleic acid molecule as described in the fourth aspect, the complex as described in the fifth aspect, the isolated nucleic acid molecule as described in the sixth aspect, the vector as described in the seventh aspect, the composition as described in the ninth aspect, the composition as described in the tenth aspect, the kit or delivery composition of the present invention, for use in nucleic acid editing.

[0148] In certain embodiments, the nucleic acid editing comprises gene or genome editing, such as modifying a gene, knocking out a gene, altering the expression of a gene product, repairing a mutation, and / or inserting a polynucleotide.

[0149] In another aspect, the present invention relates to the protein according to the first aspect, the conjugate according to the second aspect, the fusion protein according to the third aspect, the isolated nucleic acid molecule according to the fourth aspect, the complex according to the fifth aspect, the isolated nucleic acid molecule according to the sixth aspect, the vector according to the seventh aspect, the composition according to the ninth aspect, the composition according to the tenth aspect, the kit or delivery composition of the present invention, and use thereof in preparing a preparation for:

[0150] (i) ex vivo gene or genome editing;

[0151] (ii) detection of single-stranded DNA in vitro;

[0152] (iii) editing a target sequence in a target locus to modify an organism or non-human organism;

[0153] (iv) treating a disorder caused by a defect in the target sequence in the target locus.

[0154] Cells and cell progeny

[0155] In some cases, the modifications introduced into the cell by the methods of the present invention can cause the cell and its progeny to be altered to improve the production of its biological product (such as an antibody, starch, ethanol or other desired cellular output). In some cases, the modifications introduced into the cell by the methods of the present invention can cause the cell and its progeny to include changes that cause the produced biological product to change.

[0156] Therefore, in another aspect, the present invention also relates to a cell obtained by the method as described above, or a progeny thereof, wherein the cell contains a modification that is not present in its wild type form.

[0157] The present invention also relates to cell products of the cells described above or their progeny.

[0158] The present invention also relates to an in vitro, ex vivo or in vivo cell or cell line or their progeny, which comprises: the protein as described in the first aspect, the conjugate as described in the second aspect, the fusion protein as described in the third aspect, the isolated nucleic acid molecule as described in the fourth aspect, the complex as described in the fifth aspect, the isolated nucleic acid molecule as described in the sixth aspect, the vector as described in the seventh aspect, the composition as described in the ninth aspect, the composition as described in the tenth aspect, the kit or delivery composition of the present invention.

[0159] In certain embodiments, the cell is a prokaryotic cell.

[0160] 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.).

[0161] In certain embodiments, the cell is a stem cell or a stem cell line.

[0162] Definition of terms

[0163] 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.

[0164] In the present invention, the expression “Cas12j.23” refers to a Cas effector protein first discovered and identified by the present inventors, which has an amino acid sequence selected from the following:

[0165] (i) the sequence shown in SEQ ID NO: 1;

[0166] (ii) a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, deletions or additions) compared to the sequence shown in SEQ ID NO: 1; or

[0167] (iii) a sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the sequence shown in SEQ ID NO: 1.

[0168] Cas12j.23 of the present invention is a nuclease that binds to and cuts a specific site of a target sequence under the guidance of a guide RNA, and has both DNA and RNA endonuclease activity.

[0169] 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 meanings generally understood by those skilled in the art, and typically include transcripts or other elements related to the expression of CRISPR-associated ("Cas") genes, or transcripts or other elements capable of directing the activity of the Cas genes. Such transcripts or other elements may include sequences encoding Cas effector proteins and guide RNAs comprising CRISPR RNA (crRNA), as well as trans-acting crRNA (tracrRNA) sequences contained in the CRISPR-Cas9 system, or other sequences or transcripts from the CRISPR locus. In the CRISPR system based on Cas12j.23 described in the present invention, a tracrRNA sequence is not required.

[0170] As used herein, the terms "Cas effector protein" and "Cas effector enzyme" are used interchangeably and refer to any protein greater than 800 amino acids in length that is present in the CRISPR-Cas system. In some cases, such proteins refer to proteins identified from the Cas locus.

[0171] As used herein, the terms "guide RNA (guide RNA)", "mature crRNA" are used interchangeably and have the meanings generally understood by those skilled in the art. In general, guide RNA can include a direct repeat sequence and a guide sequence (guide sequence), or essentially consist of or consist of a direct repeat sequence and a guide sequence (also referred to as a spacer sequence in the context of an endogenous CRISPR system). In some cases, a guide sequence is any polynucleotide sequence that has sufficient complementarity with a target sequence to hybridize with the target sequence and guide the specific binding of the CRISPR / Cas complex to the target sequence. In certain embodiments, when optimally aligned, the degree of complementarity between the 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%. It is within the capabilities of those of ordinary skill in the art to determine the optimal alignment. For example, there are publicly available and commercially available alignment algorithms and programs, such as, but not limited to, ClustalW, Smith-Waterman algorithm (Smith-Waterman), Bowtie, Geneious, Biopython, and SeqMan in matlab.

[0172] In some cases, the guide sequence is at least 5, at least 10, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 35, at least 40, at least 45, or at least 50 nucleotides in length. In some cases, the guide sequence is no more than 50, 45, 40, 35, 30, 25, 24, 23, 22, 21, 20, 15, 10, or fewer nucleotides in length. In certain embodiments, the guide sequence is 10-30, or 15-25, or 15-22, or 19-25, or 19-22 nucleotides in length.

[0173] In some cases, the direct repeat sequence is at least 10, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 56, at least 57, at least 58, at least 59, at least 60, at least 61, at least 62, at least 63, at least 64, at least 65, or at least 70 nucleotides in length. In some cases, the repeat sequence in the same direction is no more than 70, 65, 64, 63, 62, 61, 60, 59, 58, 57, 56, 55, 50, 45, 40, 35, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 15, 10 or less nucleotides in length. In certain embodiments, the repeat sequence in the same direction is 55-70 nucleotides in length, such as 55-65 nucleotides, such as 60-65 nucleotides, such as 62-65 nucleotides, such as 63-64 nucleotides. In certain embodiments, the repeat sequence in the same direction is 15-30 nucleotides in length, such as 15-25 nucleotides, such as 20-25 nucleotides, such as 22-24 nucleotides, such as 23 nucleotides.

[0174] As used herein, the term "CRISPR / Cas complex" refers to a ribonucleoprotein complex formed by the binding of a guide RNA or mature crRNA to a Cas protein, which comprises a guide sequence that hybridizes to a target sequence and binds to the Cas protein. The ribonucleoprotein complex is capable of recognizing and cleaving a polynucleotide that can hybridize to the guide RNA or mature crRNA.

[0175] Therefore, in the case of forming a CRISPR / Cas complex, a "target sequence" refers to a polynucleotide targeted by a guide sequence designed to be targeted, such as a sequence having complementarity with the guide sequence, wherein hybridization between the target sequence and the guide sequence will promote the formation of a CRISPR / Cas complex. Complete complementarity is not required, as long as there is sufficient complementarity to cause hybridization and promote the formation of a CRISPR / Cas complex. The target sequence can comprise any polynucleotide, such as DNA or RNA. 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 certain embodiments, the editing template is an exogenous nucleic acid. In certain embodiments, the recombination is homologous recombination.

[0176] In the present invention, the expression "target sequence" or "target polynucleotide" can be any endogenous or exogenous polynucleotide for a cell (e.g., a eukaryotic cell). For example, the target polynucleotide can be a polynucleotide 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 useless DNA). In some cases, it is believed that the target sequence should be associated with a protospacer adjacent motif (PAM). The precise sequence and length requirements for PAM vary depending on the Cas effector enzyme used, but PAM is typically a 2-5 base pair sequence adjacent to the protospacer sequence (i.e., target sequence). Those skilled in the art will be able to identify PAM sequences for use with a given Cas effector protein. Herein, "specific motif sequence recognized by Cas protein" or "motif sequence" refers to a PAM sequence.

[0177] In some cases, the target sequence or target polynucleotide may include a plurality of disease-related genes and polynucleotides and signal transduction biochemical pathway-related genes and polynucleotides. Non-limiting examples of such target sequences or target polynucleotides include those listed in U.S. Provisional Patent Applications 61 / 736,527 and 61 / 748,427, filed on December 12, 2012 and January 2, 2013, respectively, and International Application No. PCT / US2013 / 074667, filed on December 12, 2013, all of which are incorporated herein by reference.

[0178] In some cases, examples of target sequences or target polynucleotides include sequences associated with signal transduction biochemical pathways, such as signal transduction biochemical pathway-related genes or polynucleotides. Examples of target polynucleotides include disease-associated genes or polynucleotides. "Disease-associated" genes or polynucleotides refer to any genes or polynucleotides that produce transcriptional or translational products at abnormal levels or in abnormal forms in cells derived from disease-affected tissues compared to tissues or cells of non-disease controls. In cases where the altered expression is associated with the emergence and / or progression of the disease, it can be a gene that is expressed at an abnormally high level; alternatively, it can be a gene that is expressed at an abnormally low level. Disease-associated genes also refer to genes with one or more mutations or genetic variations that are directly responsible for or are disequilibrium with one or more genes responsible for the etiology of the disease. The transcribed or translated products can be known or unknown and can be at normal or abnormal levels.

[0179] 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.

[0180] 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.

[0181] 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.

[0182] 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 either 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.

[0183] As used herein, the term "vector" refers to a nucleic acid delivery vehicle into which a polynucleotide can be inserted. When a vector is capable of expressing a protein encoded by the inserted polynucleotide, it is referred to as an expression vector. A vector can be introduced into a host cell via transformation, transduction, or transfection, allowing the genetic material elements it carries to be expressed in the host cell. Vectors are well known to those skilled in the art and include, but are not limited to, plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), or P1-derived artificial chromosomes (PACs); bacteriophages such as lambda phage or M13 phage; and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillomas (such as SV40). 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, a vector may also contain an initiation of replication site.

[0184] As used herein, the term "host cell" refers to a cell that can be used to introduce a vector, including but not limited to prokaryotic cells such as Escherichia coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, insect cells such as S2 Drosophila cells or Sf9, or animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells or human cells.

[0185] 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 desired expression level, etc. A vector can be introduced into a host cell to thereby produce a transcript, protein, or peptide, including a protein, fusion protein, isolated nucleic acid molecule, etc. as described herein (e.g., a CRISPR transcript, such as a nucleic acid transcript, protein, or enzyme).

[0186] 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, California (1990). In some cases, regulatory elements include those that direct the constitutive expression of a nucleotide sequence in many types of host cells and those that direct the nucleotide sequence to be expressed only in certain host cells (e.g., tissue-specific regulatory sequences). Tissue-specific promoters can primarily direct expression in the desired tissue of interest, such as muscle, neurons, bone, skin, blood, specific organs (e.g., liver, pancreas), or special cell types (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-U 5' 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).

[0187] 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.

[0188] 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).

[0189] 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.

[0190] As used herein, " stringent conditions " for hybridization refer to a nucleic acid with complementarity to a target sequence that primarily hybridizes with the target sequence and does not substantially hybridize to the conditions on a non-target sequence. Stringent conditions are typically sequence-dependent and vary depending on many factors. Generally speaking, the longer the sequence, the higher the temperature at which the sequence-specific hybridization occurs on its target sequence. Non-limiting examples of stringent conditions are described in " Laboratory Techniques In Biochemistry And Molecular Biology - Hybridization With Nucleic Acid Probes " by Tijssen (1993), Part I, Chapter II, " Overview of principles of hybridization and the strategy of nucleic acid probe assay ", Elsevier, New York.

[0191] As used herein, the term "hybridization" refers to a reaction in which one or more polynucleotide reactions form a complex that is stabilized via hydrogen bonding of the bases between the nucleotide residues. Hydrogen bonding can occur by means of Watson-Crick base pairing, Hoogstein binding, or in any other sequence-specific manner. The complex can comprise two chains forming a duplex, three or more chains forming a multi-chain complex, a single self-hybridizing chain, or any combination thereof. A hybridization reaction can constitute a step in a broader process (such as the beginning of PCR or the cutting of a polynucleotide via an enzyme). A sequence that can hybridize with a given sequence is referred to as the "complement" of the given sequence.

[0192] 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.

[0193] 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).

[0194] 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.

[0195] As used herein, the term "subject" includes, but is not limited to, various animals, such as 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., human) suffers from a disorder (e.g., a disorder caused by a disease-associated gene defect).

[0196] Advantageous Effects of the Invention

[0197] Compared to the prior art, the Cas proteins and systems of the present invention offer significant advantages. For example, the Cas effector proteins of the present invention are smaller in molecular size than Cas9, C2c1, CasY, and Cpf1 proteins, and therefore exhibit superior transfection efficiency. For example, the Cas effector proteins of the present invention are capable of DNA cleavage in eukaryotic organisms, and compared to the previously reported FnCpf1 with a 5'-TTN PAM domain, the Cas proteins of the present invention exhibit significantly stronger cleavage activity in human cell lines. For example, the Cas proteins of the present invention utilize a more rigorous PAM recognition method, which can reduce off-target effects.

[0198] Below will be described in detail by example embodiments of the present invention, but those skilled in the art will understand that the following examples are only used to illustrate the present invention, rather than to limit the scope of the invention. According to the following detailed description of preferred embodiments, various objects and advantages of the present invention will become apparent to those skilled in the art.

[0199] Sequence information

[0200] Information on the partial sequences involved in the present invention is provided in Table 1 below.

[0201] Table 1: Description of sequences

[0202]

[0203]

[0204]

[0205]

[0206] DETAILED DESCRIPTION

[0207] The invention will now be described with reference to the following examples which are intended to illustrate the invention rather than to limit it.

[0208] Unless otherwise specified, the experiments and methods described in the embodiments are carried out substantially according to conventional methods well known in the art and described in various references. For example, 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: A LABORATORY MANUAL, 2nd edition (1989); CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (editor F M Ausubel et al., (1987)); METHODS IN ENZYMOLOGY series (Academic Publishing Company): PCR 2: A PRACTICAL METHOD. 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)).

[0209] 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.

[0210] The sources of some of the reagents involved in the following examples are as follows:

[0211] LB liquid medium: 10g tryptone, 5g yeast extract, 10g NaCl, dilute to 1L, and sterilize. If antibiotics are needed, add them after the medium has cooled, to a final concentration of 50μg / ml.

[0212] Chloroform / isoamyl alcohol: Add 10 ml of isoamyl alcohol to 240 ml of chloroform and mix well.

[0213] RNP buffer: 100 mM NaCl, 50 mM Tris-HCl, 10 mM MgCl2, 100 μg / ml BSA, pH 7.9.

[0214] The prokaryotic expression vectors pACYC-Duet-1 and pUC19 were purchased from Beijing Quanshijin Biotechnology Co., Ltd.

[0215] Escherichia coli competent EC100 was purchased from Epicentre.

[0216] Example 1. Acquisition of Cas12j.23 gene and Cas12j.23 guide RNA

[0217] 1. CRISPR and gene annotation: Prodigal was used to annotate the microbial genome and metagenomic data from the NCBI and JGI databases to obtain all proteins. Piler-CR was used to annotate the CRISPR loci. All parameters were default.

[0218] 2. Protein Filtering: We de-redundant annotated proteins by sequence consistency, removing proteins with identical sequences and classifying proteins longer than 800 amino acids as macromolecules. Since all currently discovered effector proteins of the second type of CRISPR / Cas system are greater than 900 amino acids in length, we only consider macromolecules when mining CRISPR effector proteins to reduce computational complexity.

[0219] 3. Acquisition of CRISPR-associated macromolecular proteins: Each CRISPR locus will be extended 10Kb upstream and downstream to identify non-redundant macromolecular proteins in the CRISPR adjacent region.

[0220] 4. Clustering of CRISPR-associated macromolecular proteins: Use BLASTP to perform internal pairwise alignments of non-redundant macromolecular CRISPR-associated proteins, outputting alignments with an E value < 1E-10. Use MCL to perform cluster analysis of the BLASTP output results to identify CRISPR-associated protein families.

[0221] 5. Identification of CRISPR-enriched macromolecular protein families: Use BLASTP to align proteins from CRISPR-associated protein families to a non-redundant macromolecular protein database that has been stripped of CRISPR-associated proteins, outputting alignments with an E-value < 1E-10. If the number of homologous proteins found in a non-CRISPR-associated protein database is less than 100%, this indicates that proteins from this family are enriched in the CRISPR region. This method allows us to identify CRISPR-enriched macromolecular protein families.

[0222] 6. Protein Function and Domain Annotation: We annotated CRISPR-enriched macromolecular protein families using the Pfam database, the NR database, and Cas proteins collected from NCBI, identifying new CRISPR / Cas protein families. We performed multiple sequence alignments of each CRISPR / Cas family protein using Mafft, followed by conserved domain analysis using JPred and HHpred, identifying protein families containing the RuvC domain.

[0223] On this basis, the inventors obtained a new Cas effector protein, Cas12j.23, the protein sequence is shown in SEQ ID NO: 1, and the nucleotide sequence encoding the protein is shown in SEQ ID NO: 2. The prototype direct repeat sequence corresponding to Cas12j.23 (the repeat sequence contained in the pre-crRNA) is shown in SEQ ID NO: 3, and the mature direct repeat sequence corresponding to Cas12j.23 (the repeat sequence contained in the mature crRNA) is shown in SEQ ID NO: 5.

[0224] Example 2. Processing of original crRNA by Cas12j.23 gene

[0225] 1. Artificially synthesize the double-stranded DNA molecule shown in SEQ ID NO: 2, and simultaneously artificially synthesize the double-stranded DNA molecule shown in SEQ ID NO: 4.

[0226] 2. The double-stranded DNA molecule synthesized in step 1 was connected to the prokaryotic expression vector pACYC-Duet-1 to obtain the recombinant plasmid pACYC-Duet-1+CRISPR / Cas12j.23.

[0227] The recombinant plasmid pACYC-Duet-1+CRISPR / Cas12j.23 was sequenced. The sequencing results showed that the recombinant plasmid pACYC-Duet-1+CRISPR / Cas12j.23 contained the sequences shown in SEQ ID NO:2 and SEQ ID NO:4, and expressed the Cas12j.23 protein shown in SEQ ID NO:1 and the prototype direct repeat sequence of Cas12j.23 shown in SEQ ID NO:3. The recombinant plasmid pACYC-Duet-1+CRISPR / Cas12j.23 was introduced into Escherichia coli EC100 to obtain a recombinant bacterium, which was named EC100 / pACYC-Duet-1+CRISPR / Cas12j.23.

[0228] 3. Take a single clone of EC100 / pACYC-Duet-1+CRISPR / Cas12j.23 and inoculate it into 100 mL of LB liquid medium (containing 50 μg / mL ampicillin). Incubate with shaking at 37°C and 200 rpm for 12 h to obtain a culture solution.

[0229] 4. Extract bacterial RNA: Transfer 1.5 mL of bacterial culture to a pre-chilled microcentrifuge tube and centrifuge at 6000 × g for 5 minutes at 4°C. After centrifugation, discard the supernatant and resuspend the cell pellet in 200 μL of Max Bacterial Enhancement Reagent preheated to 95°C. Mix by pipetting. Incubate at 95°C for 4 minutes. Add 1 mL of Mix by pipetting and incubate at room temperature for 5 minutes. Add 0.2 mL of cold chloroform, shake the tube by hand for 15 seconds, and incubate at room temperature for 2-3 minutes. Centrifuge at 12,000 × g for 15 minutes at 4°C. Transfer 600 μL of supernatant to a new tube, add 0.5 mL of cold isopropanol to precipitate RNA, invert to mix, and incubate at room temperature for 10 minutes. Centrifuge at 15,000 × g at 4°C for 10 minutes, discard the supernatant, add 1 mL of 75% ethanol, and vortex to mix. Centrifuge at 7500 × g at 4°C for 5 minutes, discard the supernatant, and air dry. Dissolve the RNA precipitate in 50 μL of RNase-free water and incubate at 60°C for 10 minutes.

[0230] 5. DNA Digestion: Dissolve 20 μg RNA in 39.5 μL ddH2O and incubate at 65°C for 5 minutes. Incubate on ice for 5 minutes. Add 0.5 μL RNAI, 5 μL buffer, and 5 μL DNaseI and incubate at 37°C for 45 minutes (50 μL system). Adjust the volume to 100 μL with 50 μL ddH2O. Centrifuge the tube at 16,000 g for 30 seconds in a 2 mL Phase-Lock tube. Add 100 μL of phenol:chloroform:isoamyl alcohol (25:24:1) and 100 μL of digested RNA. Shake for 15 seconds. Centrifuge at 16,000 g for 12 minutes at 15°C. Remove the supernatant and transfer it to a new 1.5 mL centrifuge tube. Add an equal volume of 1 / 10 NaoAC isopropanol to the supernatant. Incubate for 1 hour or overnight at -20°C. Centrifuge at 16,000 g for 30 minutes at 4°C and discard the supernatant. Wash the pellet with 350 μL of 75% ethanol and centrifuge at 16,000 g for 10 min at 4°C. Discard the supernatant. Allow to dry. Dissolve the pellet in 20 μL of RNase-free water at 65°C for 5 min. Measure the concentration using a NanoDrop gel and run the gel.

[0231] 6. 3' Dephosphorylation and 5' Phosphorylation: Add 20 μg of digested RNA to 42.5 μL of each volume with water and incubate at 90°C for 2 min. Chill on ice for 5 min. Add 5 μL of 10× T4 PNK buffer, 0.5 μL of RNaI, and 2 μL of T4 PNK (50 μL) and incubate at 37°C for 6 h. Add 1 μL of T4 PNK and 1.25 μL of 100 mM ATP and incubate at 37°C for 1 h. Add 47.75 μL of ddH2O to adjust the volume to 100 μL. Centrifuge at 16,000 g in a 2mL Phase-Lock tube for 30 seconds. Then, add 100 μL of phenol:chloroform:isoamyl alcohol (25:24:1) and 100 μL of digested RNA. Shake for 15 seconds and centrifuge at 16,000 g at 15°C for 12 min. Remove the supernatant to a new 1.5 mL centrifuge tube and add an equal volume of isopropanol (1 / 10 NaoAC) to the supernatant. Incubate for 1 hour or overnight at -20°C. Centrifuge at 16,000 g for 30 minutes at 4°C and discard the supernatant. Wash the pellet with 350 μL of 75% ethanol and centrifuge at 16,000 g for 10 minutes at 4°C. Discard the supernatant. Allow to air dry. Dissolve the pellet in 21 μL of RNase-free water and incubate at 65°C for 5 minutes. Measure the concentration using a NanoDrop.

[0232] 7. RNA Monophosphorylation: Incubate 20 μL RNA at 90°C for 1 minute, then cool on ice for 5 minutes. Add 2 μL RNA 5'Polphosphatase 10× Reaction Buffer, 0.5 μL Inhibitor, 1 μL RNA 5'Polphosphatase (20 units), and RNase-free water to 20 μL. Incubate at 37°C for 60 minutes. Add 80 μL ddH2O to adjust the volume to 100 μL. Centrifuge a 2 mL Phase-Lock tube at 16,000 g for 30 seconds, then add 100 μL phenol:chloroform:isoamyl alcohol (25:24:1) and 100 μL of digested RNA. Shake for 15 seconds, then centrifuge at 16,000 g for 12 minutes at 15°C. Transfer the supernatant to a new 1.5 mL centrifuge tube and add an equal volume of isopropanol to the supernatant, resulting in a total volume of 1 / 10 NaoAC. Incubate for 1 hour or overnight at -20°C. Centrifuge at 16,000 g for 30 min at 4°C, discard the supernatant, wash the pellet with 350 μL of 75% ethanol, centrifuge at 16,000 g for 10 min at 4°C, discard the supernatant. Allow to air dry, then add 21 μL of RNase-free water and dissolve the pellet at 65°C for 5 min. Measure the concentration using a NanoDrop.

[0233] 8. Preparation of cDNA library: 16.5 μL RNase-free water; 5 μL Poly(A) Polymerase 10× Reaction buffer; 5 μL 10 mM ATP; 1.5 μL RiboGuard RNase Inhibitor; 20 μL RNA substrate. 2 μL Poly(A) Polymerase (4 units). 50 μL total volume. Incubate at 37°C for 20 min. Add 50 μL dH2O to adjust the volume to 100 μL. Centrifuge at 16,000 g for 30 seconds in a 2 mL Phase-Lock tube. Add 100 μL phenol:chloroform:isoamyl alcohol (25:24:1) and 100 μL digested RNA. Shake for 15 seconds. Centrifuge at 16,000 g for 12 minutes at 15°C. Transfer the supernatant to a new 1.5 mL centrifuge tube and add an equal volume of isopropanol (1 / 10 NaoAC) to the supernatant. Incubate for 1 hour or overnight at -20°C. Centrifuge at 16,000 g for 30 minutes at 4°C, discard the supernatant, air dry, and dissolve the precipitate in 11 μL of RNase-free water at 65°C for 5 minutes. Measure the concentration using a NanoDrop.

[0234] 9. After adding sequencing adapters to the cDNA library, send it to Beijing Berry Biosciences for sequencing.

[0235] 10. Perform quality filtering on the raw data and remove sequences with an average base quality value lower than 30. After removing the adapters, retain the RNA sequence of 25 nt to 50 nt and align it to the reference sequence of the CRISPR array using bowtie.

[0236] 11. Through comparison, we found that the pre-crRNA of Cas12j.23 can be successfully processed into mature crRNA in Escherichia coli. The mature crRNA is composed of Repeat sequence and guide sequence.

[0237] 12. Using ViennaRNA and VARNA for structure prediction and visualization analysis of mature crRNA, we found that the 3' end of the Repeat sequence of Cas12j.23 crRNA can form a neck loop.

[0238] Example 3. Identification of the PAM domain of Cas12j.23 gene

[0239] 1. Construct and sequence the recombinant plasmid pACYC-Duet-1+CRISPR / Cas12j.23. Based on the sequencing results, the structure of the recombinant plasmid pACYC-Duet-1+CRISPR / Cas12j.23 was described as follows: the small fragment between the restriction endonuclease Pml I and Kpn I recognition sequences of the vector pACYC-Duet-1 was replaced with the double-stranded DNA molecule shown in positions 1 to 2436 from the 5' end of the sequence shown in SEQ ID NO: 2. The recombinant plasmid pACYC-Duet-1+CRISPR / Cas12j.23 expresses the Cas12j.23 protein shown in SEQ ID NO: 1, the prototype direct repeat sequence of Cas12j.23 shown in SEQ ID NO: 3, and the guide sequence identified by the PAM domain shown in SEQ ID NO: 11.

[0240] 2. The recombinant plasmid pACYC-Duet-1+CRISPR / Cas12j.23 contains an expression cassette, the nucleotide sequence of which is shown in SEQ ID NO:9. In the sequence shown in SEQ ID NO:9, positions 1 to 44 from the 5' end are the nucleotide sequence of the pLacZ promoter, positions 45 to 3248 are the nucleotide sequence of the Cas12j.23 gene, and positions 3249 to 3335 are the nucleotide sequence of the terminator (for terminating transcription). Positions 3336 to 3370 from the 5' end are the nucleotide sequence of the J23119 promoter, positions 3371 to 3436 are the nucleotide sequence of the CRISPR array, and positions 3437 to 3463 are the nucleotide sequence of the rrnB-T1 terminator (for terminating transcription).

[0241] 3. Obtaining recombinant E. coli: The recombinant plasmid pACYC-Duet-1+CRISPR / Cas12j.23 was introduced into E. coli EC100 to obtain recombinant E. coli, named EC100 / pACYC-Duet-1+CRISPR / Cas12j.23. The recombinant plasmid pACYC-Duet-1 was introduced into E. coli EC100 to obtain recombinant E. coli, named EC100 / pACYC-Duet-1.

[0242] 4. Construction of the PAM library: The sequence shown in SEQ ID NO: 10 was artificially synthesized and ligated into the pUC19 vector, wherein the sequence shown in SEQ ID NO: 10 includes eight random bases at the 5' end and the target sequence. Eight random bases were designed in front of the 5' end of the target sequence of the PAM library to construct a plasmid library. The plasmids were respectively transformed into E. coli containing the CRISPR / Cas12j.23 locus and E. coli without the CRISPR / Cas12j.23 locus. After treatment at 37°C for 1 hour, we extracted the plasmids and PCR amplified and sequenced the PAM region sequence.

[0243] 5. Obtaining the PAM library domain: The number of occurrences of the combined PAM sequences in the experimental and control groups was counted and normalized using the total number of PAM sequences in each group. For any PAM sequence, when the log2 (normalized value of the control group / normalized value of the experimental group) was greater than 3.5, we considered this PAM to be significantly depleted. We then obtained the significantly depleted PAM sequences from all PAM sequences. Furthermore, we used Weblogo to predict the significantly depleted PAM sequences and ultimately obtained the PAM domain of Cas12j.23.

[0244] 6. Verification of the PAM library domain: Through the PAM library depletion experiment, we obtained the PAM domain of Cas12j.23. In order to verify the rigor of this domain, we set up 10 groups of PAMs for in vivo experiments and sequenced the editing activity of Cas12j.23 on these PAMs. First, we integrated the 30nt target and PAM sequence into the non-conserved position of the kanamycin gene of the plasmid, and then mixed it with the complex formed by CRSPR / Cas12j.23 and guide RNA for 8 hours. By plating and counting the number of colonies, we can determine the depletion activity of Cas12j.23 on different PAM sequences. Through the experimental results, we can see that the CRISPR / Cas12j.23 system can only effectively edit target sequences with specific PAM domains, while it has no editing activity on the remaining target sequences, thereby verifying the accuracy of Cas12j.23's PAM domain recognition.

[0245] Example 4. Identification of DNA cleavage patterns in the CRISPR / Cas12j.23 system

[0246] 1. In vitro expression and purification of Cas12j.23 protein

[0247] The steps for in vitro expression and purification of Cas12j.23 protein are as follows:

[0248] 1. Artificially synthesize the nucleotide sequence shown in SEQ ID NO: 2.

[0249] 2. The double-stranded DNA molecule synthesized in step 1 was ligated with the prokaryotic expression vector pET-30a(+) to obtain the recombinant plasmid pET-30a-CRISPR / Cas12j.23. The recombinant plasmid pET-30a-CRISPR / Cas12j.23 was sequenced. The sequencing results showed that the recombinant plasmid pET-30a-CRISPR / Cas12j.23 expressed the Cas12j.23 protein with a nuclear localization signal as shown in SEQ ID NO: 8.

[0250] 3. Introduce the recombinant plasmid pET-30a-CRISPR / Cas12j.23 into Escherichia coli EC100 to obtain a recombinant bacterium, which is named EC100-CRISPR / Cas12j.23. Pick a single colony of EC100-CRISPR / Cas12j.23 and inoculate it into 100 mL of LB liquid medium (containing 50 μg / mL ampicillin). Incubate with shaking at 37°C and 200 rpm for 12 h to obtain a culture solution.

[0251] 4. Take the culture solution and inoculate it into 50mL LB liquid medium (containing 50μg / mL ampicillin) at a volume ratio of 1:100. Cultivate at 37℃ and 200rpm with shaking until OD 600nm The value was 0.6, and then IPTG was added to a concentration of 1 mM. The culture was shaken at 28°C and 220 rpm for 4 h, and the cells were centrifuged at 4°C and 10,000 rpm for 10 min to collect the bacterial precipitate.

[0252] 5. Take the bacterial pellet, add 100 mL of pH 8.0, 100 mM Tris-HCl buffer, resuspend and ultrasonically disrupt (ultrasonic power 600 W, cycle program: disruption 4 s, pause 6 s, total 20 min), then centrifuge at 4°C, 10000 rpm for 10 min, and collect supernatant A.

[0253] 6. Take supernatant A, centrifuge at 4°C, 12000 rpm for 10 min, and collect supernatant B.

[0254] 7. Supernatant B was purified using a nickel column produced by GE (refer to the instructions of the nickel column for the specific purification steps), and then the Cas12j.23 protein was quantified using a protein quantification kit produced by Thermo Fisher Scientific.

[0255] 2. Transcription and purification of Cas12j.23 protein guide RNA:

[0256] 1. Design the templates for guide RNA transcription. The structure of the transcription template is: (1) T7 promoter + mature direct repeat sequence of Cas12j.23 (SEQ ID NO: 5) + guide sequence (SEQ ID NO: 6). Primers were designed using Primer 5.0 software to ensure that the forward primer and reward primer had at least 18 bp of overlapping sequence.

[0257] 2. Prepare the following reaction system, mix gently by pipetting, centrifuge briefly, and place in a PCR instrument for slow annealing. The PCR system is as follows:

[0258]

[0259] 3. Use MinElute PCR Purification Kit to purify the template. The steps are as follows:

[0260] 1) Add 5 volumes of PB to the PCR product, place a MinElute column on a 2 ml collection tube, incubate at room temperature for 2 min, and centrifuge at 12,000 g for 2 min.

[0261] 2) Discard the waste solution and add 750 μl Buffer PE (remember to add ethanol before use) and centrifuge at 12,000 g / 2 min.

[0262] 3) Discard the waste liquid, add 350 μl Buffer PE, centrifuge at 12000 g / 2 min, discard the waste liquid, centrifuge at 12000 g for 2 min;

[0263] 4) Transfer the MinElute column to a new 1.5 ml centrifuge tube, open the tube lid, and incubate at 65°C for 2 minutes.

[0264] 5) Add 20 μl of preheated EB solution, let it stand for 2 minutes, and then centrifuge at 12000g / 2min. To improve the recovery rate, pass the contents of the centrifuge tube 2-3 times through the MinElute centrifuge column;

[0265] 6) Measure the concentration using Nanodrop and store at -20°C until use.

[0266] 4. Purification of guide RNA: Phenol: chloroform: isoamyl alcohol (25:24:1) extraction to remove DNAseI in the system;

[0267] 1) Add 80 μl RNA-free H2O to the post-transcription reaction system to adjust the volume to 100 μl;

[0268] 2) Remove 2 ml of Phase Lock Gel (PLG) Heavy and centrifuge at 15,000 g for 2 min. Add 100 μl of phenol:chloroform:isoamyl alcohol (25:24:1) and 100 μl of DNAseI-digested RNA. Gently flick the Phase-Lock tube 5-10 times to mix thoroughly. Centrifuge at 15°C / 16,000 g for 12 min.

[0269] 3) Take a new RNA-free 1.5ml centrifuge tube and aspirate the supernatant from the previous step into the tube, being careful not to aspirate the gel. Add an equal volume of isopropanol and one-tenth the volume of sodium acetate solution to the supernatant. Mix thoroughly by pipetting with a pipette and place in a -20°C refrigerator for 1 hour or overnight.

[0270] 4) Centrifuge at 4°C / 16,000 g for 30 min, discard the supernatant, add 75% pre-chilled ethanol, pipette and mix the precipitate, centrifuge at 4°C / 16,000 g for 12 min, discard the supernatant, let stand in a fume hood for 2-3 min to dry the ethanol on the RNA surface, add 100 μl of RNA-free H2O, and pipette and mix.

[0271] 5. Use Nanodrop to determine the concentration of purified crRNA, and uniformly dilute to 250 ng / μl, dispense into 200 μl PCR centrifuge tubes, and freeze at -80°C for later use.

[0272] 6. Establishment of double-stranded DNA enzyme digestion system:

[0273] (1) Prepare the following reaction system, gently pipette to mix, and briefly centrifuge. Incubate at 37°C for 15 minutes. The DNA cleavage reaction system is as follows:

[0274]

[0275] (2) Add 300 ng of substrate DNA (100 ng / μl) to 3 μl, gently pipette to mix, and briefly centrifuge. Incubate at 37°C for 8 hours.

[0276] (3) Add RNAse and incubate at 37°C for 15 min to fully digest RNA impurities in the system;

[0277] (4) Add proteinase K and incubate at 58°C for 15 min to digest the Cas12j.23 protein;

[0278] (5) Agarose gel testing.

[0279] The gel running results showed that Cas12j.23 can effectively cut double-stranded DNA.

[0280] Example 5. Cleavage of Cas12j.23 in human cell lines

[0281] The eukaryotic expression vector containing the Cas12j.23 gene and the PCR product containing the U6 promoter and guide RNA (containing the prototype direct repeat sequence shown in SEQ ID NO:3 and the guide sequence of the eukaryotic editing shown in SEQ ID NO:5) were transferred into human HEK293T cells by lipofectamine transfection and cultured at 37 degrees Celsius for 72 hours at a concentration of 5% carbon dioxide. The DNA of all cells was extracted, and the sequence containing 700bp of the target site was amplified. The PCR product was connected to the B-simple vector for generation sequencing. Sequencing was completed by Thermo Fisher Scientific. The sequencing results were compared to the VEGFA gene of the human genome. The cutting mode of Cas12j.23 for the target target site was identified, and its editing efficiency for VEGFA was identified. The PCR product was subjected to second-generation sequencing library construction by Tn5. Sequencing was completed by Beijing Annouda Gene Technology Co., Ltd., and the editing efficiency of Cas12j.23 for VEGFA was identified. At the same time, the cutting of DNMT1 gene by Cas12j.23 was also identified.

[0282] 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. An effector protein in a CRISPR / Cas system, whose amino acid sequence is shown in SEQ ID NO:

1.

2. A conjugate comprising the protein of claim 1 and a modified portion; wherein: The modified portion is selected from another protein or polypeptide, a detectable label, and any combination thereof; wherein the other protein or polypeptide is selected from an epitope tag, a reporter gene sequence, a nuclear localization signal sequence, a transcription activation domain, a transcription inhibition domain, a nuclease domain, and any combination thereof.

3. The conjugate of claim 2, wherein the modification portion is linked to the N-terminus or C-terminus of the protein via a linker.

4. The conjugate of claim 2, which has one or more of the following features: (i) the transcriptional activation domain is VP64; (ii) the transcriptional repression domain is a KRAB domain or a SID domain; (iii) the nuclease domain is Fok1.

5. The conjugate according to claim 2, wherein The conjugate comprises an epitope tag.

6. The conjugate according to claim 2, wherein The conjugate comprises an NLS sequence.

7. The conjugate according to claim 6, wherein The NLS sequence is shown in SEQ ID NO:

7.

8. The conjugate according to claim 7, wherein The NLS sequence is located at the N-terminus or C-terminus of the protein.

9. A fusion protein comprising the protein of claim 1 and another protein or polypeptide; wherein: The additional protein or polypeptide is selected from an epitope tag, a reporter gene sequence, a nuclear localization signal sequence, a transcription activation domain, a transcription repression domain, a nuclease domain, and any combination thereof.

10. The fusion protein according to claim 9, wherein The additional protein or polypeptide is connected to the N-terminus or C-terminus of the protein via a linker.

11. The fusion protein of claim 9, which has one or more of the following features: (i) the transcriptional activation domain is VP64; (ii) the transcriptional repression domain is a KRAB domain or a SID domain; (iii) the nuclease domain is Fok1.

12. The fusion protein according to claim 9, wherein The fusion protein comprises an epitope tag.

13. The fusion protein according to claim 9, wherein The fusion protein comprises an NLS sequence.

14. The fusion protein according to claim 13, wherein The NLS sequence is shown in SEQ ID NO:

7.

15. The fusion protein according to claim 14, wherein The NLS sequence is located at the N-terminus or C-terminus of the protein.

16. The fusion protein according to claim 9, wherein The fusion protein has an amino acid sequence as shown in SEQ ID NO:

8.

17. An isolated nucleic acid molecule, the sequence of which is shown in SEQ ID NO:

4.

18. A composite comprising: (i) a protein component selected from the group consisting of: a protein according to claim 1, a conjugate according to any one of claims 2 to 8, or a fusion protein according to any one of claims 9 to 16; and (ii) a nucleic acid component comprising, from 5' to 3' direction, the isolated nucleic acid molecule of claim 17 and a guide sequence capable of hybridizing to a target sequence, in, The protein component and the nucleic acid component are combined with each other to form a complex.

19. The complex according to claim 18, wherein The guide sequence is linked to the 3' end of the nucleic acid molecule.

20. The complex according to claim 18, wherein The guide sequence comprises a complementary sequence to the target sequence.

21. The complex according to claim 18, wherein The nucleic acid component is the guide RNA in the CRISPR / Cas system.

22. The complex of claim 18, wherein The nucleic acid molecule is RNA.

23. The complex of claim 18, wherein The complex does not contain a trans-acting crRNA.

24. An isolated nucleic acid molecule having the sequence: (i) a nucleotide sequence encoding the protein of claim 1, or the fusion protein of any one of claims 9 to 16; (ii) the nucleotide sequence of the isolated nucleic acid molecule of claim 17; and / or, (iii) consists of the nucleotide sequences of (i) and (ii).

25. The isolated nucleic acid molecule of claim 24, wherein The nucleotide sequence described in any one of (i) to (iii) is codon-optimized for expression in prokaryotic cells or eukaryotic cells.

26. A vector comprising the isolated nucleic acid molecule of claim 24 or 25.

27. A host cell comprising the isolated nucleic acid molecule of claim 24 or 25 or the vector of claim 26.

28. A composition comprising: (i) a first component selected from: a protein according to claim 1, a conjugate according to any one of claims 2 to 8, a fusion protein according to any one of claims 9 to 16, or a nucleotide sequence encoding the protein or fusion protein; and (ii) a second component, which is a nucleotide sequence comprising a guide RNA; in, The guide RNA comprises a direct repeat sequence and a guide sequence from 5' to 3' direction, and the guide sequence can hybridize with the target sequence; The guide RNA is capable of forming a complex with the protein, conjugate or fusion protein described in (i).

29. The composition of claim 28, wherein The direct repeat sequence is an isolated nucleic acid molecule as defined in claim 17.

30. The composition of claim 28, wherein The targeting sequence is linked to the 3' end of the direct repeat sequence.

31. The composition of claim 28, wherein The guide sequence comprises a complementary sequence to the target sequence.

32. The composition of claim 28, wherein The composition does not comprise a trans-acting crRNA.

33. A composition comprising one or more carriers comprising: (i) a first nucleic acid, which is a nucleotide sequence encoding the protein of claim 1 or the fusion protein of any one of claims 9 to 16; wherein, The first nucleic acid is operably linked to a first regulatory element; and (ii) a second nucleic acid, which is a nucleotide sequence encoding a guide RNA; wherein the second nucleic acid is operably linked to a second regulatory element; in: The first nucleic acid and the second nucleic acid are present on the same or different vectors; The guide RNA comprises a direct repeat sequence and a guide sequence from 5' to 3' direction, and the guide sequence can hybridize with the target sequence; The guide RNA is capable of forming a complex with the protein or fusion protein described in (i).

34. The composition of claim 33, wherein The direct repeat sequence is an isolated nucleic acid molecule as defined in claim 17.

35. The composition of claim 33, wherein The targeting sequence is linked to the 3' end of the direct repeat sequence.

36. The composition of claim 33, wherein The guide sequence comprises a complementary sequence to the target sequence.

37. The composition of claim 33, wherein The composition does not comprise a trans-acting crRNA.

38. The composition of claim 33, wherein The first regulatory element and / or the second first regulatory element is a promoter.

39. The composition of claim 38, wherein The promoter is an inducible promoter.

40. The composition of any one of claims 28 to 38, wherein When the target sequence is DNA, the target sequence is located at the 3' end of the protospacer adjacent to the motif.

41. The composition of claim 40, wherein The target sequence is a DNA sequence from a prokaryotic cell or a eukaryotic cell; alternatively, the target sequence is a non-naturally occurring DNA sequence.

42. The composition of any one of claims 28-39, 41, wherein The protein is linked to one or more NLS sequences, or the conjugate or fusion protein comprises one or more NLS sequences.

43. A kit comprising one or more components selected from the following: the protein of claim 1, the conjugate of any one of claims 2-8, the fusion protein of any one of claims 9-16, the isolated nucleic acid molecule of claim 17, the complex of any one of claims 18-23, the isolated nucleic acid molecule of claim 24 or 25, the vector of claim 26, the host cell of claim 27, and the composition of any one of claims 28-42.

44. A delivery composition comprising a delivery vector and one or more selected from the following: the protein of claim 1, the conjugate of any one of claims 2-8, the fusion protein of any one of claims 9-16, the isolated nucleic acid molecule of claim 17, the complex of any one of claims 18-23, the isolated nucleic acid molecule of claim 24 or 25, the vector of claim 26, the host cell of claim 27, and the composition of any one of claims 28-42.

45. The composition of claim 44, wherein the delivery vehicle is a particle.

46. ​​The composition of claim 44, wherein the delivery vehicle is selected from lipid particles, metal particles, protein particles, exosomes, gene guns, or viral vectors.

47. A method for modifying a target gene for purposes other than disease diagnosis and treatment, comprising: The complex according to any one of claims 18 to 23 or the composition according to any one of claims 28 to 42 is contacted with the target gene, or delivered to a cell comprising the target gene; the target sequence is present in the target gene.

48. The method of claim 47, wherein the target gene is present in a cell.

49. The method of claim 47, wherein the cell is a prokaryotic cell.

50. The method of claim 47, wherein the cell is a eukaryotic cell.

51. The method of claim 47, wherein the cell is selected from the group consisting of an animal cell and a plant cell.

52. The method of claim 47, wherein the target gene is present in a nucleic acid molecule in vitro.

53. The method of claim 47, which results in DNA double-strand breaks.

54. A method for altering the expression of a gene product for purposes other than disease diagnosis and treatment, comprising: The complex of any one of claims 18 to 23 or the composition of any one of claims 28 to 42 is contacted with a nucleic acid molecule encoding the gene product, or delivered to a cell comprising the nucleic acid molecule, wherein the target sequence is present in the nucleic acid molecule.

55. The method of claim 54, wherein the nucleic acid molecule is present in a cell.

56. The method of claim 54, wherein the cell is a prokaryotic cell.

57. The method of claim 54, wherein the cell is a eukaryotic cell.

58. The method of claim 54, wherein the cell is selected from the group consisting of an animal cell and a plant cell.

59. The method of claim 54, wherein the nucleic acid molecule is present in a nucleic acid molecule in vitro.

60. The method of claim 54, wherein expression of the gene product is altered.

61. The method of claim 54, wherein the gene product is a protein.

62. The method of any one of claims 47-61, wherein the protein, conjugate, fusion protein, complex, vector or composition is contained in a delivery vehicle.

63. The method of claim 62, wherein the delivery vehicle is selected from lipid particles, metal particles, protein particles, exosomes, and viral vectors.

64. The method of any one of claims 47-61, which is used to modify a cell, a cell line or an organism by altering one or more target sequences in a target gene or a nucleic acid molecule encoding a target gene product.

65. An ex vivo cell or cell line or progeny thereof, comprising: the protein of claim 1, the conjugate of any one of claims 2-8, the fusion protein of any one of claims 9-16, the isolated nucleic acid molecule of claim 17, the complex of any one of claims 18-23, the isolated nucleic acid molecule of claim 24 or 25, the vector of claim 30, or the composition of any one of claims 28-42.

66. The cell or cell line or progeny thereof of claim 65, wherein the cell is a eukaryotic cell.

67. The cell or cell line or progeny thereof of claim 65, wherein the cell is an animal cell or a plant cell.

68. The cell or cell line of claim 65, or progeny thereof, wherein the cell is a stem cell or a stem cell line.

69. Use of the protein of claim 1, the conjugate of any one of claims 2-8, the fusion protein of any one of claims 9-16, the isolated nucleic acid molecule of claim 17, the complex of any one of claims 18-23, the isolated nucleic acid molecule of claim 24 or 25, the vector of claim 26, the composition of any one of claims 28-42 or the kit of claim 43 in the preparation of a reagent for use in nucleic acid editing; wherein, The nucleic acid editing is gene editing.

70. The use of claim 69, wherein the gene editing comprises modifying a gene, knocking out a gene, changing the expression of a gene product, repairing a mutation, and / or inserting a polynucleotide.

71. Use of the protein of claim 1, the conjugate of any one of claims 2 to 8, the fusion protein of any one of claims 9 to 16, the isolated nucleic acid molecule of claim 17, the complex of any one of claims 18 to 23, the isolated nucleic acid molecule of claim 24 or 25, the vector of claim 26, the composition of any one of claims 28 to 42, or the kit of claim 43 in the preparation of a preparation for: (i) ex vivo gene or genome editing; (ii) Detection of single-stranded DNA in vitro; (iii) editing a target sequence in a target locus to modify an organism; and / or (iv) treating a disorder caused by a defect in the target sequence in the target locus.

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