Novel CRISPR-Casdelta enzymes and systems

Abstract

The invention relates to the field of nucleic acid editing, in particular to the technical field of regularly clustered interval short palindromic repeat (CRISPR). In particular, the present invention relates to Cas effector proteins, fusion proteins comprising such proteins, and nucleic acid molecules encoding them. The invention also relates to complexes and compositions for nucleic acid editing (e.g., gene or genome editing) comprising the proteins or fusion proteins of the invention, or nucleic acid molecules encoding them. The invention also relates to methods for nucleic acid editing (e.g., gene or genome editing) using the proteins or fusion proteins comprising the 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 for site-specific gene editing.

[0003] The CRISPR / Cas9 system is the most commonly used Type II CRISPR system, which recognizes the PAM motif of 3'-NGG and performs blunt-end cutting on the target sequence. The CRISPR / Cas Type V system is a newly discovered CRISPR system in the past two years. It has a 5'-TTN motif and performs sticky-end cutting on the target sequence, such as Cpf1, C2c1, CasX, and CasY. However, the different CRISPR / Cas currently available have different advantages and disadvantages. For example, Cas9, C2c1, and CasX all require two RNAs for guide RNA, while Cpf1 only requires one guide RNA and can be used for multiple gene editing. CasX has a size of 980 amino acids, while the common Cas9, C2c1, CasY, and Cpf1 are usually around 1,300 amino acids in size. In addition, the PAM sequences of Cas9, Cpf1, CasX, and CasY are relatively complex and diverse, while C2c1 recognizes the rigorous 5'-TTN, so its target site is easier to predict 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 more robust new CRISPR / Cas system with multi-faceted good performance is of great significance to the development of biotechnology. Summary of the invention

[0005] After a lot of experiments 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 the system.

[0006] Cas effector proteins

[0007] Therefore, in the first aspect, the present invention provides a protein having an amino acid sequence shown in any one of SEQ ID NOs: 1, 2, 3 or its orthologs, homologs, variants or functional fragments; wherein the orthologs, homologs, variants or functional fragments substantially retain the biological function of the sequence from which they are 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, the endonuclease activity, and the activity of binding to and cutting a specific site of the target sequence under the guidance of the guide RNA.

[0009] In certain embodiments, the ortholog, homolog, variant has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity compared to the sequence from which it is derived.

[0010] In certain embodiments, the orthologs, homologs, variants have at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity compared to the sequence shown in any one of SEQ ID NOs: 1, 2, and 3, 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 invention comprises a sequence selected from the following, or consists of a sequence selected from the following:

[0013] (i) a sequence shown in any one of SEQ ID NOs: 1, 2, and 3;

[0014] (ii) a sequence having one or more amino acid substitutions, deletions or additions compared to the sequence shown in any one of SEQ ID NOs: 1, 2, or 3 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, and 40 amino acid substitutions, deletions, or additions); or

[0015] (iii) a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the sequence shown in any one of SEQ ID NOs: 1, 2, and 3.

[0016] Protein truncations

[0017] In a second aspect, the present invention provides protein truncations, which are truncated by one or more amino acids (e.g., 1-10, 11-20, 21-30, 31-40, 41-50, 51-60 or more amino acids) at the N-terminus and / or C-terminus compared to the protein described in the first aspect.

[0018] In certain embodiments, the protein truncation is truncated by 31 amino acids at the N-terminus compared to the protein described in the first aspect.

[0019] In certain embodiments, the protein truncation is truncated by 31 amino acids at the N-terminus compared to the sequence shown in any one of SEQ ID NOs: 1 and 2.

[0020] In certain embodiments, the term "truncated by 31 amino acids at the N-terminus" refers to a continuous truncation of 31 amino acids starting from the starting amino acid at the N-terminus.

[0021] In certain embodiments, the protein truncation comprises or consists of a sequence selected from the following:

[0022] (i) the sequence shown in SEQ ID NO: 3;

[0023] (ii) a sequence having one or more amino acid substitutions, deletions or additions compared to the sequence shown in SEQ ID NO: 3 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 and 40 amino acid substitutions, deletions or additions); or

[0024] (iii) a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the sequence set forth in SEQ ID NO:3.

[0025] In certain embodiments, the protein truncation has the amino acid sequence shown in SEQ ID NO:3.

[0026] Derivatized protein

[0027] The protein or protein truncation of the present invention can be derivatized, for example, connected 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 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 connected (by chemical coupling, gene fusion, non-covalent connection or other means) to one or more other molecular groups, such as another protein or polypeptide, a detection reagent, a pharmaceutical agent, etc.

[0028] 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 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 marker 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.

[0029] Conjugate

[0030] Therefore, in a third aspect, the present invention provides a conjugate comprising a protein or protein truncation as described above and a modifying moiety.

[0031] In certain embodiments, the modifying moiety is selected from another protein or polypeptide, a detectable label, or any combination thereof.

[0032] 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 following: 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.

[0033] In certain embodiments, the conjugates of the invention comprise one or more NLS sequences, such as the NLS of the large T antigen of the SV40 virus. In certain exemplary embodiments, the NLS sequence is as shown in SEQ ID NO: 27. In certain embodiments, the NLS sequence is located at, near or close to the end (e.g., N-terminus or C-terminus) of the protein or protein truncation of the invention. In certain exemplary embodiments, the NLS sequence is located at, near or close to the C-terminus of the protein or protein truncation of the invention.

[0034] In certain embodiments, the conjugates of the present invention include 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 a suitable epitope tag according to the desired purpose (e.g., purification, detection, or tracing).

[0035] In certain embodiments, the conjugate of the present invention comprises 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, etc.

[0036] 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.

[0037] In certain embodiments, the conjugates of the invention comprise a detectable label, such as a fluorescent dye, eg, FITC or DAPI.

[0038] In certain embodiments, the protein or protein truncation of the present invention is coupled, conjugated or fused to the modifying moiety, optionally via a linker.

[0039] In certain embodiments, the modifying moiety is directly linked to the N-terminus or C-terminus of the protein or protein truncation of the invention.

[0040] In certain embodiments, the modified portion is connected to the N-terminus or C-terminus of the protein or protein truncation of the present invention via a linker. Such linkers are well known in the art, and examples thereof include but are not limited to linkers comprising one or more (e.g., 1, 2, 3, 4 or 5) amino acids (e.g., Glu or Ser) or amino acid derivatives (e.g., Ahx, β-Ala, GABA or Ava), or PEG, etc.

[0041] Fusion Protein

[0042] In a fourth aspect, the present invention provides a fusion protein comprising the protein of the first aspect or the protein truncation of the second aspect and another protein or polypeptide.

[0043] 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 following: 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.

[0044] In certain embodiments, the fusion proteins of the present invention comprise one or more NLS sequences, such as the NLS of the large T antigen of the SV40 virus. In certain embodiments, the NLS sequence is located at, near or close to the end (e.g., N-terminus or C-terminus) of the protein or protein truncation of the present invention. In certain exemplary embodiments, the NLS sequence is located at, near or close to the C-terminus of the protein or protein truncation of the present invention.

[0045] In certain embodiments, the fusion proteins of the invention comprise an epitope tag.

[0046] In certain embodiments, the fusion proteins of the invention comprise a reporter gene sequence.

[0047] In certain embodiments, the fusion proteins of the invention comprise a domain capable of binding to a DNA molecule or an intracellular molecule.

[0048] In certain embodiments, the protein or protein truncation of the present invention is fused to the additional protein or polypeptide, optionally via a linker.

[0049] In certain embodiments, the additional protein or polypeptide is directly linked to the N-terminus or C-terminus of the protein or protein truncation of the invention.

[0050] In certain embodiments, the additional protein or polypeptide is linked to the N-terminus or C-terminus of the protein or protein truncation of the invention via a linker.

[0051] In certain exemplary embodiments, the fusion protein of the present invention has an amino acid sequence as shown in any one of SEQ ID NOs: 28-30.

[0052] The protein, conjugate or fusion protein of the present invention is not limited by the production method, for example, it can be produced by genetic engineering methods (recombinant technology) or by chemical synthesis methods.

[0053] Direct repeat sequence

[0054] In a fifth aspect, the present invention provides an isolated nucleic acid molecule comprising or consisting of a sequence selected from the following:

[0055] (i) a sequence shown in any one of SEQ ID NO: 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16;

[0056] (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 any one of SEQ ID NOs: 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16;

[0057] (iii) a sequence having at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% sequence identity to the sequence shown in any one of SEQ ID NOs: 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16;

[0058] (iv) a sequence that hybridizes to the sequence described in any one of (i) to (iii) under stringent conditions; or

[0059] (v) a complementary sequence of the sequence described in any one of (i) to (iii);

[0060] Furthermore, the sequence described in any one of (ii) to (v) substantially retains the biological function of the sequence from which it is derived, and the biological function of the sequence refers to the activity as a direct repeat sequence in the CRISPR-Cas system.

[0061] In certain embodiments, the isolated nucleic acid molecule is a direct repeat sequence in a CRISPR-Cas system.

[0062] In certain embodiments, the nucleic acid molecule comprises or consists of a sequence selected from the group consisting of:

[0063] (a) the nucleotide sequence shown in any one of SEQ ID NO: 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16;

[0064] (b) a sequence that hybridizes under stringent conditions to the sequence described in (a); or

[0065] (c) The complementary sequence of the sequence described in (a).

[0066] In certain embodiments, the isolated nucleic acid molecule is RNA.In certain embodiments, the isolated nucleic acid molecule is a direct repeat sequence in a CRISPR / Cas system.

[0067] CRISPR / Cas complex

[0068] In a sixth aspect, the present invention provides a composite comprising:

[0069] (i) a protein component selected from the group consisting of a protein, a protein truncate, a conjugate or a fusion protein of the present invention, and any combination thereof; and

[0070] (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,

[0071] Wherein, the protein component and the nucleic acid component are combined with each other to form a complex.

[0072] In certain embodiments, the targeting sequence is linked to the 3' end of the nucleic acid molecule.

[0073] In certain embodiments, the guide sequence comprises the complement of the target sequence.

[0074] In certain embodiments, the nucleic acid component is a guide RNA in a CRISPR-Cas system.

[0075] In certain embodiments, the nucleic acid molecule is RNA.

[0076] In certain embodiments, the complex does not comprise a trans-acting crRNA (tracrRNA).

[0077] In certain embodiments, the guide sequence is at least 5, at least 10, at least 15, at least 20, at least 25, 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.

[0078] In certain embodiments, the nucleic acid molecule of the separation is 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 molecule of the separation is 15-30 nucleotides in length, for example 15-25 nucleotides, for example 20-25 nucleotides, for example 22-24 nucleotides, for example 23 nucleotides.

[0079] Encoding nucleic acid, vector and host cell

[0080] In a seventh aspect, the present invention provides an isolated nucleic acid molecule comprising:

[0081] (i) a nucleotide sequence encoding a protein, protein truncation or fusion protein of the present invention;

[0082] (ii) a nucleotide sequence encoding the isolated nucleic acid molecule as described in the fifth aspect; or

[0083] (iii) a nucleotide sequence comprising (i) and (ii).

[0084] 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.

[0085] In an eighth aspect, the present invention further provides a vector comprising the isolated nucleic acid molecule as described in the seventh aspect. The vector of the present invention may 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, etc. In certain embodiments, the vector is capable of expressing the protein of the present invention, a protein truncate, a fusion protein, the isolated nucleic acid molecule as described in the fifth aspect, or the complex as described in the sixth aspect in a subject (e.g., a mammal, such as a human).

[0086] In the ninth aspect, the present invention also provides a host cell comprising an 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 (e.g., cassava, corn, sorghum, soybean, wheat, oat or rice cells) and animal cells (e.g., mammalian cells, such as mouse cells, human cells, etc.). The cell of the present invention can also be a cell line, such as 293T cells.

[0087] Compositions and carrier compositions

[0088] In the tenth aspect, the present invention also provides a composition comprising:

[0089] (i) a first component selected from the group consisting of: a protein, a protein truncation, a conjugate, a fusion protein, a nucleotide sequence encoding the protein, a protein truncation or a fusion protein of the present invention, and any combination thereof; and

[0090] (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;

[0091] Wherein, the guide RNA comprises a direct repeat sequence and a guide sequence from the 5' to the 3' direction, and the guide sequence can hybridize with the target sequence;

[0092] The guide RNA is capable of forming a complex with the protein, protein truncation, conjugate or fusion protein described in (i).

[0093] In certain embodiments, the direct repeat sequence is an isolated nucleic acid molecule as defined in the fifth aspect.

[0094] 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.

[0095] In certain embodiments, the composition does not comprise crRNA (tracrRNA).

[0096] 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. In certain embodiments, the first component is non-naturally occurring or modified; and / or, the second component is non-naturally occurring or modified.

[0097] In certain embodiments, when the target sequence is DNA, the target sequence is located at the 3' end of the protospacer adjacent motif (PAM), and the PAM has a sequence shown as 5'-RYR, wherein R is selected from A or G, and Y is selected from T or C. In certain embodiments, the sequence of the PAM is selected from ATG, ACG, GTG, ATA, ACA, GCA, GTA and / or GCG.

[0098] In certain embodiments, when the target sequence is RNA, the target sequence has no PAM domain restriction.

[0099] In certain embodiments, the target sequence is a DNA or RNA sequence from a prokaryotic cell or a eukaryotic cell. In certain embodiments, the target sequence is a non-naturally occurring DNA or RNA sequence.

[0100] In certain embodiments, the target sequence is present in a cell. In certain embodiments, the target sequence is present in a cell 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.

[0101] In certain embodiments, the protein or protein truncation is connected 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 connected 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.

[0102] In the eleventh aspect, the present invention further provides a composition comprising one or more carriers, wherein the one or more carriers comprise:

[0103] (i) a first nucleic acid comprising a nucleotide sequence encoding a protein, a protein truncate or a fusion protein of the present invention; optionally, the first nucleic acid is operably linked to a first regulatory element; and

[0104] (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;

[0105] in:

[0106] The first nucleic acid and the second nucleic acid are present on the same or different vectors;

[0107] 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;

[0108] The guide RNA is capable of forming a complex with the protein, protein truncation or fusion protein described in (i).

[0109] In certain embodiments, the direct repeat sequence is an isolated nucleic acid molecule as defined in the fifth aspect.

[0110] 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.

[0111] In certain embodiments, the composition does not comprise trans-acting crRNA (tracrRNA).

[0112] 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.

[0113] In certain embodiments, the first regulatory element is a promoter, such as an inducible promoter.

[0114] In certain embodiments, the second regulatory element is a promoter, such as an inducible promoter.

[0115] In certain embodiments, when the target sequence is DNA, the target sequence is located at the 3' end of the protospacer adjacent motif (PAM), and the PAM has a sequence represented by 5'-RYR, wherein R is A or G, and Y is T or C.

[0116] In certain embodiments, the sequence of the PAM is selected from ATG, ACG, GTG, ATA, ACA, GCA, GTA and / or GCG.

[0117] In certain embodiments, when the target sequence is RNA, the target sequence has no PAM domain restriction.

[0118] In certain embodiments, the target sequence is a DNA or RNA sequence from a prokaryotic cell or a eukaryotic cell. In certain embodiments, the target sequence is a non-naturally occurring DNA or RNA sequence.

[0119] In certain embodiments, the target sequence is present in a cell. In certain embodiments, the target sequence is present in a cell 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.

[0120] In certain embodiments, the protein is connected 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 connected 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.

[0121] 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 a virally derived DNA or RNA sequence is present in a vector for packaging a virus (e.g., a retrovirus, a replication-defective retrovirus, an adenovirus, a replication-defective adenovirus, and an adeno-associated virus). The viral vector also includes a polynucleotide carried by a virus for transfection into a host cell. Some vectors (e.g., bacterial vectors and additional mammalian vectors with a bacterial origin of replication) can replicate autonomously in the host cell into which they are introduced. Other vectors (e.g., non-additional mammalian vectors) are integrated into the genome of the host cell after being introduced into the host cell, and thus replicated together with the host genome. Moreover, some vectors can direct the expression of the gene that they are operably connected. Such a vector is referred to as an "expression vector" herein. The common expression vector used in recombinant DNA technology is typically a plasmid form.

[0122] 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.

[0123] Delivery and delivery compositions

[0124] The protein, protein truncation, conjugate, fusion protein of the present invention, the isolated nucleic acid molecule as described in the fifth aspect, the complex of the present invention, the isolated nucleic acid molecule as described in the seventh aspect, the vector as described in the eighth aspect, and the composition as described in the tenth aspect and the eleventh aspect can be delivered by any method known in the art. Such methods include, but are not limited to, electroporation, lipofection, nuclear transfection, microinjection, sonoporation, gene gun, calcium phosphate-mediated transfection, cationic transfection, liposome transfection, dendritic transfection, heat shock transfection, nuclear transfection, magnetofection, lipofection, puncture transfection, optical transfection, agent-enhanced nucleic acid uptake, and delivery via liposomes, immunoliposomes, viral particles, artificial virions, etc.

[0125] Therefore, in the twelfth aspect, the present invention provides a delivery composition comprising a delivery vector and one or more selected from the following: a protein, a protein truncate, a conjugate, a fusion protein of the present invention, an isolated nucleic acid molecule as described in the fifth aspect, a complex of the present invention, an isolated nucleic acid molecule as described in the seventh aspect, a vector as described in the eighth aspect, and a composition as described in the tenth aspect and the eleventh aspect.

[0126] In certain embodiments, the delivery vehicle is a particle.

[0127] In certain embodiments, the delivery vehicle is selected from a lipid particle, a sugar particle, a metal particle, a protein particle, a liposome, an exosome, a microvesicle, a gene gun, or a viral vector (e.g., a replication-defective retrovirus, a lentivirus, an adenovirus, or an adeno-associated virus).

[0128] Reagent test kit

[0129] 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 following: a protein, a protein truncate, a conjugate, a fusion protein, an isolated nucleic acid molecule as described in the fifth aspect, a complex of the present invention, an isolated nucleic acid molecule as described in the seventh aspect, a vector as described in the eighth aspect, a composition as described in the tenth aspect and the eleventh aspect.

[0130] In certain embodiments, the kit of the present invention comprises the composition as described in the tenth aspect. In certain embodiments, the kit further comprises instructions for using the composition.

[0131] In certain embodiments, the kit of the present invention comprises the composition as described in the eleventh aspect. In certain embodiments, the kit further comprises instructions for using the composition.

[0132] In certain embodiments, the components included in the kits of the present invention may be provided in any suitable container.

[0133] 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.

[0134] In certain embodiments, the kit further comprises one or more oligonucleotides corresponding to a guide sequence for insertion into a vector so as to operably link the guide sequence and regulatory elements. In certain embodiments, the kit comprises a homologous recombination template polynucleotide.

[0135] Methods and uses

[0136] In another aspect, the present invention provides a method for modifying a target gene, comprising: contacting the complex of the present invention, the composition as described in the tenth aspect and the eleventh aspect with the target gene, or delivering it to a cell containing the target gene; wherein the target sequence is present in the target gene.

[0137] In certain embodiments, the method is used to modify a target gene in vitro or ex vivo. In certain embodiments, the method is not a method for treating a human or animal by therapy. In certain embodiments, the method does not include a step of modifying a human germline genetic trait.

[0138] 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 primates, cattle, pigs 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.

[0139] 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.

[0140] In certain embodiments, the method results in a target sequence break (e.g., a DNA double-strand break or an RNA single-strand break). In certain embodiments, the break results in a reduction in transcription of the target gene.

[0141] In certain embodiments, the method further comprises: contacting the editing template (e.g., 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 the editing template (e.g., exogenous nucleic acid), wherein the repair results in a mutation, including 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.

[0142] Thus, in certain embodiments, the modification further comprises inserting an editing template (eg, an exogenous nucleic acid) into the break.

[0143] In certain embodiments, the protein, protein truncation, conjugate, fusion protein, isolated nucleic acid molecule, complex, vector or composition is contained in a delivery vehicle.

[0144] 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).

[0145] 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.

[0146] In another aspect, the present invention provides a method for changing the expression of a gene product, comprising: contacting the complex of the present invention, the composition as described in the tenth aspect and the eleventh 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.

[0147] In certain embodiments, the method is used to change the expression of a gene product in vitro or in vitro. In certain embodiments, the method is not a method for treating a human or animal by therapy. In certain embodiments, the method does not include the step of modifying human germline genetic characteristics.

[0148] 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 primates, cattle, pigs 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.

[0149] 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.

[0150] 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.

[0151] In certain embodiments, the gene product is a protein.

[0152] In certain embodiments, the protein, protein truncation, conjugate, fusion protein, isolated nucleic acid molecule, complex, vector or composition is contained in a delivery vehicle.

[0153] 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).

[0154] 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.

[0155] In another aspect, the present invention relates to the protein as described in the first aspect, the protein truncation as described in the second aspect, the conjugate as described in the third aspect, the fusion protein as described in the fourth aspect, the isolated nucleic acid molecule as described in the fifth aspect, the complex as described in the sixth aspect, the isolated nucleic acid molecule as described in the seventh aspect, the vector as described in the eighth aspect, the composition as described in the tenth aspect, the kit of the present invention, and use thereof in the preparation of a preparation for nucleic acid editing (e.g., in vitro or ex vivo nucleic acid editing).

[0156] In certain embodiments, the nucleic acid to be edited is present in a cell. In certain embodiments, the cell is a prokaryotic cell or a eukaryotic cell. In certain embodiments, the nucleic acid to be edited is present in an in vitro nucleic acid molecule (e.g., a plasmid).

[0157] In certain embodiments, the nucleic acid editing includes gene or genome editing, such as modifying a gene, knocking out a gene, changing the expression of a gene product, repairing a mutation, and / or inserting a polynucleotide. In certain embodiments, the gene or genome editing does not include a step of modifying human germline genetic characteristics. In certain embodiments, the use is not a method for treating a human or animal by therapy.

[0158] In certain embodiments, the use further comprises repairing the edited target sequence by homologous recombination with an exogenous template polynucleotide, wherein the repair can produce a mutation of the target sequence, including insertion, deletion or substitution of one or more nucleotides.

[0159] In another aspect, the present invention relates to the protein as described in the first aspect, the protein truncation as described in the second aspect, the conjugate as described in the third aspect, the fusion protein as described in the fourth aspect, the isolated nucleic acid molecule as described in the fifth aspect, the complex as described in the sixth aspect, the isolated nucleic acid molecule as described in the seventh aspect, the vector as described in the eighth aspect, the composition as described in the tenth aspect, the kit of the present invention, and their use in preparing preparations for: (i) in vitro or ex vivo DNA detection; (ii) editing target sequences in target loci to modify organisms or non-human organisms (e.g., prokaryotes).

[0160] In certain embodiments, the preparation is used for the detection of single-stranded DNA or double-stranded DNA (eg, the detection of single-stranded or double-stranded DNA in prokaryotic cells).

[0161] In certain embodiments, the DNA detection is used to detect tumors, viruses or bacteria. Without being limited to theory, it is believed that due to the non-specific cleavage characteristics of Casδ protein on single-stranded DNA after target DNA recognition, when target DNA (e.g., tumor-specific marker, virus or bacteria-specific marker) is present, by adding detectable single-stranded DNA and detecting the non-specific cleavage of the single-stranded DNA, tumors, Ebola, avian influenza, African swine fever and other viruses or bacteria can be detected.

[0162] In another aspect, the present invention also provides a method for detecting whether a target nucleic acid is present in a sample, comprising the following steps:

[0163] (1) contacting the sample with a labeled DNA probe and any of the following components: the complex of the present invention, the composition of the tenth aspect and the eleventh aspect, or the kit of the present invention;

[0164] wherein the guide sequence contained in the complex, composition or kit is capable of hybridizing with the target nucleic acid, and the DNA probe does not hybridize with the guide sequence;

[0165] In certain embodiments, the DNA probe emits a detectable signal after being cleaved;

[0166] (2) detecting a detectable signal generated by the protein or protein truncation contained in the complex, composition or kit cleaving the DNA probe, thereby determining whether the target nucleic acid is present in the sample.

[0167] In certain embodiments, one end (eg, the 5' end) of the DNA probe is labeled with a fluorescent group, and the other end (eg, the 3' end) is labeled with a quencher group.

[0168] In certain embodiments, the sequence of the target nucleic acid is a sequence obtained from a pathogen. In certain embodiments, the pathogen is selected from a virus, a bacterium, a fungus, a protozoan, a parasite or any combination thereof.

[0169] In certain embodiments, the sequence of the target nucleic acid is obtained from the genome of a tumor cell.

[0170] The target nucleic acid detected by the application can be DNA or RNA. Therefore, in certain embodiments, the method also includes the step of contacting the sample with a reagent for reverse transcription. In certain embodiments, the reagent for reverse transcription is selected from reverse transcriptase, oligonucleotide primers, dNTP or any combination thereof.

[0171] In certain embodiments, the target nucleic acid is single-stranded or double-stranded. In certain embodiments, the sequence of the target nucleic acid is a DNA or RNA sequence from a prokaryotic or eukaryotic cell; or, the sequence of the target nucleic acid is a non-naturally occurring DNA or RNA sequence.

[0172] In certain embodiments, the detectable signal is determined by one or more methods selected from the group consisting of imaging-based detection, sensor-based detection, color detection, gold nanoparticle-based detection, fluorescence polarization, colloidal phase transition / dispersion, electrochemical detection, and semiconductor-based sensing.

[0173] In certain embodiments, the method further comprises the step of amplifying the target nucleic acid in the sample.

[0174] Cells and cell progeny

[0175] In some cases, the modification introduced into the cell by the method of the present invention can cause the cell and its progeny to be changed to improve the production of its biological product (such as antibody, starch, ethanol or other desired cell output). In some cases, the modification introduced into the cell by the method of the present invention can cause the cell and its progeny to include changes that cause the produced biological product to change.

[0176] 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 not present in its wild type form.

[0177] The present invention also relates to cell products of the cells as described above or their progeny.

[0178] The present invention also relates to an in vitro, ex vivo or in vivo cell or cell line or their progeny, wherein the cell or cell line or their progeny comprises: the protein as described in the first aspect, the protein truncation as described in the second aspect, the conjugate as described in the third aspect, the fusion protein as described in the fourth aspect, the isolated nucleic acid molecule as described in the fifth aspect, the complex as described in the sixth aspect, the isolated nucleic acid molecule as described in the seventh aspect, the vector as described in the eighth aspect, the composition as described in the tenth aspect, and the composition as described in the eleventh aspect.

[0179] In certain embodiments, the cell is a prokaryotic cell.

[0180] 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, a cow, a sheep, a pig, a dog, a monkey, a rabbit, a rodent (such as a rat or a mouse). In certain embodiments, the cell is a non-mammalian eukaryotic cell, such as a cell of a poultry bird (such as a chicken), a fish or a crustacean (such as a clam, a shrimp). In certain embodiments, the cell is a plant cell, such as a cell or a cultivated plant or a food crop such as cassava, corn, sorghum, soybean, wheat, oat or rice, such as algae, a tree or a production plant, a fruit or a vegetable (for example, a tree such as a citrus tree, a nut tree; Solanum, cotton, tobacco, tomato, grape, coffee, cocoa, etc.).

[0181] In certain embodiments, the cell is a stem cell or a stem cell line.

[0182] Definition of terms

[0183] In the present invention, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. In addition, the molecular genetics, nucleic acid chemistry, chemistry, molecular biology, biochemistry, cell culture, microbiology, cell biology, genomics and recombinant DNA procedures used herein are conventional procedures widely used in the corresponding fields. At the same time, in order to better understand the present invention, the definitions and explanations of the relevant terms are provided below.

[0184] In the present invention, the expression “Casδ” refers to a Cas effector protein first discovered and identified by the inventors, which has an amino acid sequence selected from the following:

[0185] (i) a sequence shown in any one of SEQ ID NOs: 1, 2, and 3;

[0186] (ii) a sequence having one or more amino acid substitutions, deletions or additions compared to the sequence shown in any one of SEQ ID NOs: 1, 2, or 3 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, and 40 amino acid substitutions, deletions, or additions); or

[0187] (iii) a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the sequence shown in any one of SEQ ID NOs: 1, 2, and 3.

[0188] The Casδ 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.

[0189] As used herein, the term "Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR-Associated (Cas) (CRISPR-Cas) System" or "CRISPR System" is used interchangeably and has a meaning generally understood by those skilled in the art, which generally includes a transcript or other element related to the expression of a CRISPR-associated ("Cas") gene, or a transcript or other element capable of directing the activity of the Cas gene. 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 Casδ-based CRISPR system described in the present invention, a tracrRNA sequence is not required.

[0190] As used herein, the terms "Cas effector protein", "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.

[0191] As used herein, the terms "guide RNA", "mature crRNA" are used interchangeably and have the meanings commonly understood by those skilled in the art. In general, guide RNA may include a direct repeat sequence and a guide sequence, or may be substantially composed of or composed of a direct repeat sequence and a guide sequence (also referred to as a spacer 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 a CRISPR / Cas complex to the target sequence. In certain embodiments, when optimally aligned, the degree of complementarity between a guide sequence and its corresponding target sequence is at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99%. 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.

[0192] 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.

[0193] 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 same direction repeat sequence 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 same direction repeat sequence 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 same direction repeat sequence 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.

[0194] 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.

[0195] 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 complementary to the guide sequence, wherein the 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 enough 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 a cell. In some cases, the target sequence may be located in an organelle of a eukaryotic cell, such as a mitochondria or a 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.

[0196] 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 are able to identify the PAM sequence used with a given Cas effector protein. Herein, "a specific motif sequence recognized by the Cas protein" or "motif sequence" refers to the PAM sequence.

[0197] In some cases, the target sequence or target polynucleotide may include multiple 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 PCT / US2013 / 074667, filed on December 12, 2013, all of which are incorporated herein by reference.

[0198] 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-related genes or polynucleotides. "Disease-related" genes or polynucleotides refer to any gene or polynucleotide that produces transcription or translation 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 the case where the altered expression is associated with the appearance and / or progression of the disease, it can be a gene expressed at an abnormally high level; alternatively, it can be a gene expressed at an abnormally low level. Disease-related 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.

[0199] As used herein, the term "wild type" has the meaning generally understood by those skilled in the art, which refers to the typical form of an organism, strain, gene, or the characteristics that distinguish it from mutant or variant forms when it exists in nature, which can be isolated from a source in nature and has not been intentionally modified by man.

[0200] 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, it means 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.

[0201] As used herein, the term "orthologue" has the meaning commonly understood by those skilled in the art. As a further guide, an "orthologue" of a protein as described herein refers to a protein belonging to a different species that performs the same or similar function as its orthologue.

[0202] As used herein, the term "identity" is used to refer to the matching of sequences between two polypeptides or between two nucleic acids. When a position in both sequences being compared is occupied by the same base or amino acid monomer subunit (e.g., a position in each of 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 a total of 6 positions match). Typically, the comparison is made when the two sequences are aligned to produce maximum identity. Such an alignment can be achieved by using, for example, the method of Needleman et al. (1970) J. Mol. Biol. 48: 443-453, which can be conveniently performed by a computer program such as the Align program (DNAstar, Inc.). The percent identity between two amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl Biosci., 4: 11-17 (1988)), which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. In addition, the percent identity between two amino acid sequences can be determined using the Needleman and Wunsch (J Mol. Biol. 48: 444-453 (1970)) algorithm, which has been incorporated into the GAP program in the GCG software package (available at www.gcg.com), using a Blossum 62 matrix or a PAM250 matrix and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.

[0203] As used herein, the term "vector" refers to a nucleic acid delivery vehicle into which a polynucleotide can be inserted. When a vector can express the protein encoded by the inserted polynucleotide, the vector is called an expression vector. The vector can be introduced into a host cell by transformation, transduction or transfection so that the genetic material elements it carries are expressed in the host cell. 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 (YAC), bacterial artificial chromosomes (BAC) or artificial chromosomes (PAC) derived from P1; bacteriophages such as lambda phage or M13 phage and animal viruses, etc. 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 viruses), poxviruses, baculoviruses, papillomaviruses, papillomaviruses (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, the vector may also contain a replication initiation site.

[0204] 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.

[0205] 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).

[0206] 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 sequences that direct the constitutive expression of a nucleotide sequence in many types of host cells and those sequences 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 desired tissues of interest, such as muscle, neurons, bone, skin, blood, specific organs (e.g., liver, pancreas), or special cell types (e.g., lymphocytes). In some cases, regulatory elements can also direct expression in a timing-dependent manner (e.g., in a cell cycle-dependent or developmental stage-dependent manner), which may or may not be tissue or cell type specific. In some cases, the term "regulatory element" encompasses enhancer elements such as WPRE; CMV enhancer; R-U5' fragment in the LTR of HTLV-I ((Mol. Cell. Biol., Vol. 8(1), pp. 466-472, 1988); SV40 enhancer; and intron sequences between exons 2 and 3 of rabbit β-globin (Proc. Natl. Acad. Sci. USA., Vol. 78(3), pp. 1527-31, 1981).

[0207] 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 a 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 essentially 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 a gene product in the cell essentially only when the cell is a cell of the tissue type corresponding to the promoter.

[0208] 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).

[0209] 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. The percentage of 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 of at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% over a region of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50 or more nucleotides, or to two nucleic acids that hybridize under stringent conditions.

[0210] As used herein, " stringent conditions " for hybridization refer to a nucleic acid with complementarity to a target sequence that mainly hybridizes with the target sequence and does not hybridize to the condition on the non-target sequence substantially. Stringent conditions are usually sequence-dependent, and vary depending on many factors. Generally speaking, the longer the sequence, the higher the temperature at which the sequence-specific hybridizes to its target sequence. Non-limiting examples of stringent conditions are described in Tijssen (1993) " Laboratory Techniques In Biochemistry And Molecular Biology - Hybridization With Nucleic Acid Probes ", Part I, Chapter II, " Overview of Principles of Hybridization and the Strategy of Nucleic Acid Probe Assay ", Elsevier, New York.

[0211] 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 may include two chains forming a duplex, three or more chains forming a multi-chain complex, a single self-hybridizing chain, or any combination of these. Hybridization reactions can constitute a step in a more extensive process (such as the beginning of PCR or via the cutting of a polynucleotide of an enzyme). A sequence that can hybridize with a given sequence is referred to as the "complement" of the given sequence.

[0212] 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. Transcripts and encoded polypeptides may be collectively referred to as "gene products." If the polynucleotide is derived from genomic DNA, expression may include splicing of mRNA in eukaryotic cells.

[0213] As used herein, the term "joint" refers to a linear polypeptide formed by connecting a plurality of amino acid residues via peptide bonds. The joint 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 joint 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).

[0214] 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.

[0215] As used herein, the term "subject" includes, but is not limited to, various animals, such as mammals, such as bovines, equines, ovines, swine, canines, felines, lagomorphs, rodents (e.g., mice or rats), non-human primates (e.g., macaques or cynomolgus monkeys) or humans. In certain embodiments, the subject (e.g., human) suffers from a disorder (e.g., a disorder caused by a disease-related gene defect).

[0216] Advantageous Effects of the Invention

[0217] Compared with the prior art, the Cas protein and system of the present invention have significant advantages. For example, the molecular size of the Cas effector protein of the present invention is smaller than that of Cas9, C2c1, CasY and Cpf1 proteins, so the transfection efficiency is better than that of Cas9, C2c1, CasY and Cpf1 proteins. For example, the PAM domain of the Cas effector protein of the present invention is a 5'-RYR structure (R is A or G, Y is T or C), and there are 8 recognizable PAM combinations, namely ATG, ACG, ATA, ACA, GTG, GTA, GCG and GCA, which have a wider recognition site than the currently reported NGG PAM recognized by SpCas9 and the TTN PAM recognized by LbCas12a.

[0218] Embodiments of the present invention will be described in detail below in conjunction with the accompanying drawings and examples, but it will be appreciated by those skilled in the art that the following drawings and examples are only used to illustrate the present invention, rather than to limit the scope of the present invention. Various objects and advantages of the present invention will become apparent to those skilled in the art based on the following detailed description of the accompanying drawings and preferred embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0219] Figure 1A to Figure 1B This is the PAM structure analysis and verification results of Casδ-1 in Example 3.

[0220] Figure 2 This is the in vivo verification result of the PAM domain in Escherichia coli in Example 3.

[0221] Figure 3 Casδ-1 protein in Example 3 Analysis of target sequence cleavage characteristics .

[0222] FIG. 4A to FIG. 4B This is the result of the trans-cleavage activity analysis of Casδ-1 in Example 4.

[0223] Figure 5 This is an analysis of the editing activity of Casδ-1 at 14 target sites in HELA cells in Example 5.

[0224] FIG. 6A to FIG. 6B These are the results of analyzing the editing activity of Casδ-1 on AAVS1 target sequences at different PAM sites in HELA cells in Example 5.

[0225] Figure 7 The editing activity of Casδ-1 in Example 5 on crRNA composed of different lengths of direct repeat sequences Repeat and guide sequences Target in HELA cells was analyzed. In the figure, R represents Repeat, T represents Target, and the numbers represent the number of bases in the corresponding sequence.

[0226] Fig. 8A A fragment of the expression vector containing the Casδ-1 protein constructed in Example 6; Figure 8B This is the sequence result of the Casδ-1 protein editing the target site in corn in Example 6.

[0227] Fig. 9 This is the result of cleavage of double-stranded DNA by Casδ-1D31 in Example 9.

[0228] Sequence information

[0229] The information of the partial sequences involved in the present invention is provided in Table 1 below.

[0230] Table 1: Description of sequences

[0231]

[0232]

[0233]

[0234]

[0235]

[0236]

[0237]

[0238]

[0239]

[0240]

[0241]

[0242]

[0243] DETAILED DESCRIPTION

[0244] The invention will now be described with reference to the following examples which are intended to illustrate the invention rather than to limit the invention.

[0245] Unless otherwise specified, the experiments and methods described in the embodiments are carried out basically according to the conventional methods well known in the art and described in various references. For example, the conventional techniques such as immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics and recombinant DNA used in the present invention can be found in Sambrook, Fritsch and Maniatis, MOLECULAR CLONING: A LABORATORY MANUAL, 2nd edition (1989); CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (FM Ausubel et al., ed., (1987)); METHODS IN ENZYMOLOGY series (Academic Publishing Company): PCR 2: A PRACTICAL METHOD. APPROACH) (MJ MacPherson, BD Hames, and GR Taylor, eds. (1995)), ANTIBODIES, A LABORATORY MANUAL, eds. Harlow and Lane, eds. (1988), and ANIMAL CELL CULTURE (RI Freshney, ed. (1987)).

[0246] In addition, if the specific conditions are not specified in the examples, they are carried out according to the conventional conditions or the conditions recommended by the manufacturer. If the manufacturer is not specified in the reagents or instruments used, they are all conventional products that can be obtained commercially. It is known to those skilled in the art that the embodiments describe the present invention by way of example and are not intended to limit the scope of the present invention. All public cases and other references mentioned herein are incorporated herein by reference in their entirety.

[0247] The sources of some of the reagents involved in the following examples are as follows:

[0248] LB liquid medium: 10g Tryptone, 5g Yeast Extract, 10g NaCl, dilute to 1L, sterilize. If antibiotics are needed, add them after the medium cools down, with a final concentration of 50μg / mL.

[0249] Chloroform / isoamyl alcohol: Add 10 mL of isoamyl alcohol to 240 mL of chloroform and mix well.

[0250] RNP buffer: 100 mM NaCl, 50 mM Tris-HCl, 10 mM MgCl 2 , 100 μg / mL BSA, pH 7.9.

[0251] Prokaryotic expression vectors pET-30a, pUC19 and pACYCDuet-1 were purchased from Beijing Quanshijin Biotechnology Co., Ltd.

[0252] Escherichia coli competent TSC-E03 was purchased from Beijing Qingke Biotechnology Co., Ltd.

[0253] Example 1. Acquisition of Casδ gene and Casδ guide RNA

[0254] 1. Annotation of CRISPR and genes: Prodigal was used to annotate the microbial genome and metagenomic data from the NCBI and JGI databases to obtain all proteins, and Piler-CR was used to annotate the CRISPR loci. All parameters were default.

[0255] 2. Protein filtering: remove redundancy of annotated proteins through sequence consistency and remove proteins with completely identical sequences.

[0256] 3. Acquisition of CRISPR-associated proteins: Each CRISPR locus will be extended 10Kb upstream and downstream to identify non-redundant proteins in the CRISPR adjacent region.

[0257] 4. Clustering of CRISPR-related proteins: Use BLASTP to perform internal pairwise alignment of non-redundant CRISPR-related proteins and output alignment results with Evalue < 1E-10. Use MCL to perform cluster analysis on the output results of BLASTP to identify CRISPR-related protein families.

[0258] 5. Identification of CRISPR-enriched protein families: Use BLASTP to align the proteins of the CRISPR-related protein family to the non-redundant protein database without CRISPR-related proteins, and output the alignment results with Evalue < 1E-10. If the homologous protein found in a non-CRISPR-related protein database is less than 100%, it means that the protein of this family is enriched in the CRISPR region. Through this method, we identify the CRISPR-enriched protein family.

[0259] 6. Annotation of protein functions and domains: CRISPR-enriched protein families were annotated using the Pfam database, NR database, and Cas proteins collected from NCBI to obtain new CRISPR / Cas protein families. Mafft was used to perform multiple sequence alignments on each CRISPR / Cas family protein, and then JPred and HHpred were used to perform conserved domain analysis to identify protein families containing the RuvC domain.

[0260] On this basis, the inventors obtained some new Cas effector proteins, which were named Casδ-1 and Casδ-2, respectively. The protein sequences are shown in SEQ ID NO: 1 and SEQ ID NO: 2, and the nucleotide sequences encoding the proteins are shown in SEQ ID NO: 4 and SEQ ID NO: 5. The prototype direct repeat sequences (repeat sequences contained in pre-crRNA) corresponding to Casδ-1 and Casδ-2 are shown in SEQ ID NO: 7 and SEQ ID NO: 8.

[0261] Example 2. Description of the sequence structure of the Casδ gene

[0262] 1. The CRISPR / Casδ sequence fragment was synthesized by Beijing Qingke Biotechnology Co., Ltd. and constructed into the protein expression vector pET-30a(+), and confirmed by first-generation sequencing. According to the sequencing results, the recombinant plasmid pET-30a+CRISPR / Casδ is described as follows:

[0263] (1) The recombinant plasmid pET-30a+CRISPR / Casδ-1 contains an expression cassette, and the expression cassette sequence is shown in SEQ ID NO: 31. In the sequence shown in SEQ ID NO: 31, positions 1 to 27 from the 5' end are the nucleotide sequence of SV40-NLS, positions 28 to 96 are the nucleotide sequence of 3×FLAG, positions 97 to 2904 are the nucleotide sequence of Casδ-1, and positions 2905 to 2964 are the nucleoplasmin NLS signal peptide.

[0264] (2) The recombinant plasmid pET-30a+CRISPR / Casδ-2 contains an expression cassette, and the expression cassette sequence is shown in SEQ ID NO: 32. In the sequence shown in SEQ ID NO: 32, from the 5' end, positions 1 to 27 are the nucleotide sequence of SV40-NLS, positions 28 to 96 are the nucleotide sequence of 3×FLAG, positions 97 to 2697 are the nucleotide sequence of Casδ-2, and positions 2698 to 2757 are the nucleoplasmin NLS signal peptide.

[0265] Example 3. Identification of PAM and DNA cleavage patterns in the CRISPR / Casδ system

[0266] 1. In vitro expression and purification of Casδ protein

[0267] The steps for in vitro expression and purification of Casδ protein are as follows:

[0268] 1. Artificially synthesize the nucleotide sequence shown in SEQ ID NO:31-32.

[0269] 2. The recombinant plasmids pET-30a-CRISPR / Casδ-1 and 2 were introduced into E. coli TSC-E03 to obtain recombinant bacteria, which were named TSC-E03-CRISPR / Casδ-1 and 2. Single clones of TSC-E03-CRISPR / Casδ-1 and 2 were picked and inoculated into 100 mL LB liquid medium (containing 50 μg / mL kanamycin), and cultured at 37°C and 200 rpm for 12 h to obtain a culture solution.

[0270] 3. Take the culture solution and inoculate it into 50mL LB liquid medium (containing 50μg / mL kanamycin) 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 18°C ​​and 220 rpm for 14 h, and the cells were centrifuged at 4°C and 7000 rpm for 10 min to collect the bacterial precipitate.

[0271] 5. Take the bacterial precipitate, 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, stop 6 s, total 20 min), then centrifuge at 4°C, 10000 rpm for 10 min, and collect supernatant A.

[0272] 6. Take supernatant A, centrifuge at 4°C, 12000 rpm for 10 min, and collect supernatant B.

[0273] 7. The supernatant B was purified using a nickel column produced by GE (refer to the instructions of the nickel column for the specific steps of purification), and then the Casδ-1 to Casδ-3 proteins were quantified using a protein quantification kit produced by Thermo Fisher Scientific.

[0274] 2. Transcription and purification of Casδ protein guide RNA:

[0275] 1. Design the templates for guide RNA transcription respectively. The structure of the transcription template is: (1) T7 promoter + prototype direct repeat sequences of Casδ-1 and Casδ-2 (SEQ ID NO: 7-8) + guide sequence (SEQ ID NO: 36). Primer 5.0 software was used to design the primers, ensuring that the forward primer and reward primer had at least 18 bp of overlapping sequence.

[0276] 2. Prepare the following reaction system, gently blow and mix, centrifuge briefly, and place in a PCR instrument for slow annealing. The PCR system is as follows:

[0277]

[0278] 3. Use MinElute PCR Purification Kit to purify the template. The steps are as follows:

[0279] 1) Add 5 volumes of PB to the PCR product, place a MinElute column on a 2 mL collection tube, let stand at room temperature for 2 min, and centrifuge at 12000g for 1 min;

[0280] 2) Discard the waste liquid, add 750 μL Buffer PE (remember to add ethanol before use), and centrifuge at 12000g for 1 min;

[0281] 3) Discard the waste liquid, add 350 μL Buffer PE, centrifuge at 12000 g for 1 min, discard the waste liquid, and centrifuge at 12000 g for 2 min;

[0282] 4) Transfer the MinElute column to a new 1.5 mL centrifuge tube, open the tube lid, and incubate at 65°C for 2 min.

[0283] 5) Add 20 μL of preheated EB solution, let stand for 2 minutes, and centrifuge at 12000g for 2 minutes. To improve the recovery rate, the contents of the centrifuge tube can be passed through the MinElute centrifuge column 2-3 times;

[0284] 6) Determine the concentration using Nanodrop and store at -20℃ for later use.

[0285] 4. Purification of guide RNA: Phenol: chloroform: isoamyl alcohol (25:24:1) extraction to remove DNaseI in the system;

[0286] 1) Add 80 μL RNA free H 2 O, adjust the volume to 100 μL;

[0287] 2) Take out 2 mL of Phase Lock Gel (PLG) Heavy, centrifuge at 15000g for 2 min, add 100 μL of phenol:chloroform:isoamyl alcohol (25:24:1) and 100 μL of RNA digested with DNAseI, flick the Phase-Lock tube 5-10 times by hand to mix evenly, and then centrifuge at 15°C, 16000g for 12 min;

[0288] 3) Take a new RNA-free 1.5 mL centrifuge tube, aspirate the supernatant from the previous step into the centrifuge tube, be careful not to aspirate the gel, add isopropanol with the same volume as the supernatant and one-tenth volume of sodium acetate solution, mix with a pipette tip, and place in a -20°C refrigerator for 1 hour or overnight;

[0289] 4) Centrifuge at 4°C, 16000g for 30min, discard the supernatant, add 75% pre-cooled ethanol, mix the precipitate by pipetting, centrifuge at 4°C, 16000g for 12min, discard the supernatant, let stand in a fume hood for 2-3min, dry the ethanol on the RNA surface, add 100μL RNAfree H 2 O, pipette and mix.

[0290] 5. Use Nanodrop to measure the concentration of purified crRNA, and uniformly dilute to 250 ng / μL, divide into 200 μL PCR centrifuge tubes, and freeze at -80°C for later use.

[0291] 3. In vitro cleavage and PAM consumption of Casδ protein:

[0292] 1. Establishment of double-stranded DNA enzyme digestion system:

[0293] (1) Prepare the following reaction system, gently pipette to mix, and then briefly centrifuge. Incubate at 37°C for 15 min. The DNA cleavage reaction system is as follows:

[0294]

[0295] (2) Add 300 ng of substrate DNA (100 ng / μL), 3 μL, gently pipette to mix, and centrifuge briefly. Incubate at 37°C for 8 h;

[0296] (3) Add RNAse and incubate at 37°C for 15 min to fully digest RNA impurities in the system;

[0297] (4) adding proteinase K and incubating at 58°C for 15 min to digest Casδ-1 to 3 proteins;

[0298] (5) Agarose gel running test.

[0299] The gel running results showed that Casδ-1 can effectively cut double-stranded DNA.

[0300] 2. PAM site identification:

[0301] (1) The reaction system was configured as in step 6 above, and the substrate DNA was replaced with a plasmid library with 8 random bases before the target site. The reaction was placed at 37° C. for 8 h. The secondary control sample was a sample with Casδ added but no crRNA added. Each protein was repeated three times.

[0302] (2) After the reaction is completed, the reaction sample is purified by column, and the purified product is used as a template for the construction of the second-generation library. The system and method of library construction are the same as the library construction method in step 2 of the in vivo PAM library consumption of Escherichia coli. The specific operation process is as follows:

[0303] (Each sample corresponds to one R primer and multiple F primers), prepare the following reagents:

[0304]

[0305] Place the prepared reaction system in the PCR instrument, and the procedure is as follows:

[0306]

[0307]

[0308] 1G sequencing per sample;

[0309] (3) The number of occurrences of the combined PAM sequences in the experimental group and the control group were counted respectively, and the results were normalized by the number of all PAM sequences in each group. For any PAM sequence, when log2 (normalized value of the control group / normalized value of the experimental group) was greater than 3.5, we considered that this PAM was significantly consumed. We obtained the significantly consumed PAM sequences from all PAM sequences. In addition, we used Weblogo to predict the significantly consumed PAM sequences, and finally obtained the PAM domain of Casδ ( Figure 1A and Figure 1B ).

[0310] (4) Verification of the PAM library domain: Through the PAM library depletion experiment, we obtained the PAM domain of Casδ-1. In order to verify the rigor of this domain, we set up ATG PAM for in vivo experiments to test the editing activity of Casδ-1 on this PAM. First, we integrated the T7 promoter with the 26nt target of the corresponding PAM site and the sequence of the T7 terminator into the pET30a-Casδ-1 vector, and then co-transformed it with the pACYCDuet-1 plasmid and coated it on kanamycin and chloramphenicol resistance plates for screening. Select the double-resistant monoclonal plaques for shaking the bacteria, and IPTG induction for 12 hours with an OD value of 1.0. Then, the induced bacteria before and after induction were gradiently diluted and spotted for observation. If the chloramphenicol gene is edited, the growth on the chloramphenicol resistance plate will be poor. Through experimental results ( Figure 2 and Figure 3 ), we can see that CRISPR / Casδ-1 can only effectively edit target sequences with specific PAM domains (e.g., ATG), but has no editing activity on other target sequences (e.g., CCC), thus verifying the accuracy of Casδ-1's PAM domain recognition. The above experimental results confirm that Casδ-1 has a rigorous PAM recognition method, so Casδ-1 can significantly reduce off-target effects.

[0311] (5) Analysis of dsDNA cleavage mode of Casδ-1 protein:

[0312] In order to further determine the cleavage site and substrate cleavage mode of Casδ-1 protein when targeting dsDNA, the cleavage bands after Casδ-1 protein targeted substrate dsDNA cleavage were respectively excised and recovered. Since the distance between the 26nt target site and the 5' end is greater than the 3' end when the dsDNA fragment is designed, the longer fragments were sequenced with the F-direction primers for amplifying linear dsDNA fragments, and the recovered shorter fragments were sequenced with the R-direction primers for amplification. By aligning the results of the first-generation sequencing with the reference dsDNA sequence, the cleavage site of Casδ-1 protein for the substrate is located at 18nt and 19nt far from the PAM, forming a sticky end with a 1nt protrusion ( Figure 3 ).

[0313] Example 4. Analysis of trans-cleavage activity of the CRISPR / Casδ system

[0314] A single-stranded or double-stranded DNA that does not contain SEQ ID NO: 36 is designed as an enzyme substrate for trans-cleavage activity analysis; a single-stranded or double-stranded DNA containing SEQ ID NO: 36 is used as a targeted substrate, and the purified protein and the corresponding crRNA are configured with the following reaction system as shown in the following table.

[0315] Components volume Cas proteins 1μL(60nM) crRNA 1μL(120nM) ssDNA 1μL(30nM) 10X ssDNA Cleavage Buffer 2μL

[0316] Place the above reaction system in a PCR instrument and incubate at 25°C for 10 minutes; then add 2μL of single-stranded DNA without target sequence and incubate at 37°C for 1h; add 1μL RNase A and incubate at 37°C for 30min; add 1μL Proteinase K and incubate at 55°C for 30min; add 4μL 6X DNA loading buffer and run 1.5% agarose gel for detection. If the targeted substrate is dsDNA, it is necessary to synthesize a primer of about 90nt without the target sequence, dilute it to 1μM, and store it at -20°C as a random substrate in the trans-cleavage experiment. The dsDNA fragment in the in vitro dsDNA enzyme digestion experiment is used as the targeted substrate, and the purified protein and the corresponding crRNA are prepared into a reaction system as shown in the following table.

[0317] Components volume Cas proteins 1μL(200nM) crRNA 1μL(800nM) dsDNA 1μL(30nM) 10X ssDNA Cleavage Buffer 2μL Nuclease-free water Make up to 18 μL

[0318] The above reaction system was placed in a PCR instrument and incubated at 37°C for 15 min; (1) 2 μL (100 nM) of diluted random substrate ssDNA was added and incubated at 37°C for 2 h; (2) 1 μL RNase A was added and incubated at 37°C for 30 min; (3) 1 μL Proteinase K was added and incubated at 55°C for 30 min; (4) 2X RNA Loading Buffer was added and the nucleic acid was run on 10% nucleic acid denaturing acrylamide gel for detection.

[0319] The experimental results are as follows Figure 4A and 4B As shown, the experimental results confirmed that after the trans-cleavage activity of Casδ-1 protein was activated, both single-stranded DNA and double-stranded DNA substrates could be cut.

[0320] Example 5. Detection of Casδ editing activity in human cells

[0321] In this embodiment, 14 target sites, i.e., sg1 to sg14, were selected from the genome sequence of human HELA cells, and 14 guide sequences were designed for these target sites (the sequences are shown in SEQ ID NO:38 to SEQ ID NO:51, respectively). Further, the eukaryotic expression vector containing the Casδ-1 gene and the expression vector containing the U6 promoter and the guide RNA (containing the prototype direct repeat sequence shown in SEQ ID NO:7 and the editing guide sequence in human cells containing SEQ ID NO:38 to SEQ ID NO:51) were transferred into human HELA cells by liposome transfection and cultured at 37 degrees Celsius and 5% carbon dioxide concentration for 72 hours. The DNA of all cells was extracted, and the sequence containing 200bp of the target site was amplified, and the PCR product was sent to Beijing Jiyinga Medical Testing Laboratory Co., Ltd. for second-generation library construction and sequencing.

[0322] The sequencing results were statistically analyzed using bioinformatics and the original target sequences. The results showed that Casδ-1 could effectively edit all 14 target sites, with the editing efficiency reaching up to about 60% ( Figure 5 ).

[0323] Example 6. Analysis of editing activity of different PAM recognition sites of Casδ-1 in HELA cells

[0324] The PAM site recognized by Casδ-1 was identified as RYR (wherein R is A or G, and Y is T or C) by the experimental method of Example 3. In order to further confirm whether all recognized PAM sites have editing efficiency in eukaryotes, the editing efficiency was verified in HELA cells. 8 guide sequences were designed for these PAM recognition sites (the sequences are shown in SEQ ID NO:52 to SEQ ID NO:59, respectively). The target sequences of different PAM recognition sites were selected within the 200bp range of the AAVS1 gene, and the expression vector containing the U6 promoter and the guide RNA (containing the prototype direct repeat sequence shown in SEQ ID NO:7 and the editing guide sequence in human cells containing SEQ ID NO:52 to SEQ ID NO:59) was transferred into human HELA cells by liposome transfection and cultured at 37 degrees Celsius and 5% carbon dioxide concentration for 72h.

[0325] By constructing the second-generation library, sequencing and bioinformatics analysis of the transformed samples, it was found that the RYR PAM recognition sites of Casδ-1 all had different degrees of editing efficiency, among which the PAM recognition sites ATG and ACG had higher editing efficiency ( Fig. 6A and Figure 6B ).

[0326] Example 7. Analysis of editing activity of Casδ-1 at different Repeat and Target lengths

[0327] In order to further verify the effect of different lengths of direct repeat sequences Repeat and guide sequences Target on editing efficiency, we selected different lengths of Repeat (SEQ ID NO: 7 or 9-16) and Target (SEQ ID NO: 38) sequences for combination, and different lengths of Target (SEQ ID NO: 60-67) and Repeat (SEQ ID NO: 7) sequences for combination, and confirmed the difference in editing efficiency in eukaryotic organisms by animal cell transfection. For specific experimental procedures and steps, please refer to Example 5.

[0328] Through bioinformatics analysis, it was found that Repeat still retained a certain editing activity when 18 bases or less were cut from the 5' end, and the Target had editing activity in the range of 16 to 26 bases ( Figure 7 ).

[0329] Example 8. Analysis of Casδ-1 editing activity in stable genetic transformation of maize

[0330] The target site of the maize ZmGL2 gene was designed (wherein the direct repeat sequence of the Casδ-1 protein is shown in SEQ ID NO: 7, and the guide sequence is shown in SEQ ID NO: 37), and the crRNA sequence was connected to the expression vector of the OsU3 promoter. At the same time, the Ubi promoter and the protein coding sequence of Casδ-1 were constructed into the same expression vector ( Fig. 8A ). Through genetic transformation screening of maize callus, positive plants were obtained, and the first-generation sequencing experimental method was used to analyze whether the ZmGL2 target site was edited, and a total of 6 mutants with different deletion types were obtained ( Figure 8B ).

[0331] The above experiments confirmed that the Casδ-1 protein successfully edited the target site in corn.

[0332] Example 9. Analysis of editing activity of Casδ-1 truncated protein

[0333] In order to further reduce the size of the protein to improve the delivery efficiency, we deleted the 31 amino acids at the N-terminus of Casδ-1 to obtain a truncated form of Casδ-1, and named the truncated form Casδ-1D31 (the amino acid sequence is shown in SEQ ID NO:3, and the nucleotide sequence is shown in SEQ ID NO:6). Further, based on its amino acid sequence, the amino acid sequence of the Casδ-1D31-NLS fusion protein shown in SEQ ID NO:30 was synthesized, and constructed into the pET30a vector by homologous recombination experimental methods. The steps of prokaryotic protein purification and in vitro enzyme digestion experiments were the same as those described in Example 3.

[0334] In vitro enzyme cleavage results showed that Casδ-1D31 still has double-stranded DNA cleavage activity ( Fig. 9 ).

[0335] Although the specific embodiments of the present invention have been described in detail, it will be understood by those skilled in the art that various modifications and changes may be made to the details according to all the teachings that have been published, and these changes are within the scope of protection of the present invention. The entire invention is given by the attached claims and any equivalents thereof.

Claims

1. A protein having an amino acid sequence as shown in any one of SEQ ID NOs: 1, 2, 3 or an ortholog, homolog, variant or functional fragment thereof; wherein: The ortholog, homolog, variant or functional fragment substantially retains the biological function of the sequence from which it is derived; For example, the ortholog, homolog, variant has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity compared to the sequence from which it is derived; For example, the orthologs, homologs, variants have at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity compared to the sequence shown in any one of SEQ ID NOs: 1, 2, and 3, and substantially retain the biological function of the sequence from which it is derived; For example, the protein is an effector protein in a CRISPR / Cas system.

2. The protein of claim 1, comprising or consisting of a sequence selected from the following: (i) a sequence shown in any one of SEQ ID NOs: 1, 2, and 3; (ii) a sequence having one or more amino acid substitutions, deletions or additions compared to the sequence shown in any one of SEQ ID NOs: 1, 2, or 3 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, and 40 amino acid substitutions, deletions, or additions); or (iii) a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the sequence shown in any one of SEQ ID NOs: 1, 2, and 3; For example, the protein has an amino acid sequence shown in any one of SEQ ID NOs: 1, 2, and 3.

3. A protein truncation, wherein the protein truncation is truncated by one or more amino acids (e.g., 1-10, 11-20, 21-30, 31-40, 41-50, 51-60 or more amino acids) at the N-terminus and / or C-terminus compared to the protein of claim 1 or 2; Preferably, the protein truncation is truncated by 31 amino acids at the N-terminus compared to the protein according to claim 1 or 2; Preferably, the protein truncation is truncated by 31 amino acids at the N-terminus compared to the sequence shown in any one of SEQ ID NO: 1 or 2; Preferably, the protein truncation comprises a sequence selected from the following, or consists of a sequence selected from the following: (i) the sequence shown in SEQ ID NO: 3; (ii) a sequence having one or more amino acid substitutions, deletions or additions compared to the sequence shown in SEQ ID NO: 3 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 and 40 amino acid substitutions, deletions or additions); or (iii) a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the sequence set forth in SEQ ID NO:3; For example, the protein truncation has the amino acid sequence shown in SEQ ID NO:

3.

4. A conjugate comprising the protein of claim 1 or 2 or the protein truncation of claim 3 and a modified portion; For example, the modifying moiety is selected from another protein or polypeptide, a detectable label, and any combination thereof; For example, the modification portion is optionally linked to the N-terminus or C-terminus of the protein or protein truncation via a linker; For example, the modified portion is fused to the N-terminus or C-terminus of the protein or protein truncation; For example, 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 following: 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; For example, the conjugate comprises an epitope tag; For example, the conjugate comprises an NLS sequence; For example, the NLS sequence is shown in SEQ ID NO: 27; For example, the NLS sequence is located at, near or proximal to a terminus (eg, the N-terminus or the C-terminus) of the protein or protein truncation.

5. A fusion protein comprising the protein according to claim 1 or 2 or the protein truncation according to claim 3 and another protein or polypeptide; For example, the additional protein or polypeptide is optionally linked to the N-terminus or C-terminus of the protein or protein truncation via a linker; For example, 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 following: 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; For example, the fusion protein comprises an epitope tag; For example, the fusion protein comprises an NLS sequence; For example, the NLS sequence is shown in SEQ ID NO: 27; For example, the NLS sequence is located at, near or proximal to a terminus (e.g., N-terminus or C-terminus) of the protein or protein truncation; For example, the fusion protein has an amino acid sequence as shown in any one of SEQ ID NOs: 28-30.

6. An isolated nucleic acid molecule comprising or consisting of a sequence selected from the group consisting of: (i) a sequence shown in any one of SEQ ID NO: 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16; (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 any one of SEQ ID NOs: 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16; (iii) a sequence having at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% sequence identity to the sequence shown in any one of SEQ ID NOs: 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16; (iv) a sequence that hybridizes to the sequence described in any one of (i) to (iii) under stringent conditions; or (v) a complementary sequence of the sequence described in any one of (i) to (iii); Furthermore, the sequence described in any one of (ii) to (v) substantially retains the biological function of the sequence from which it is derived; For example, the nucleic acid molecule comprises one or more stem-loops or optimized secondary structures; For example, the sequence of any of (ii)-(v) retains the secondary structure of the sequence from which it is derived; For example, the nucleic acid molecule comprises a sequence selected from the following, or consists of a sequence selected from the following composition: (a) the nucleotide sequence shown in any one of SEQ ID NO: 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16; (b) a sequence that hybridizes under stringent conditions to the sequence described in (a); or (c) the complementary sequence of the sequence described in (a); For example, the isolated nucleic acid molecule is RNA; For example, the isolated nucleic acid molecule is a direct repeat sequence in a CRISPR / Cas system.

7. A composite comprising: (i) a protein component selected from the group consisting of: the protein of claim 1 or 2, the protein truncation of claim 3, the conjugate of claim 4, the fusion protein of claim 5, and any combination thereof; and (ii) a nucleic acid component comprising, from 5' to 3' direction, the isolated nucleic acid molecule of claim 6 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.

8. The complex according to claim 7, wherein The guide sequence is connected to the 3' end of the nucleic acid molecule; For example, the guide sequence comprises a complementary sequence to the target sequence; For example, the nucleic acid component is a guide RNA in a CRISPR / Cas system; For example, the nucleic acid molecule is RNA; For example, the complex does not include a trans-acting crRNA (tracrRNA); Preferably, the complex targets a third component, which is a double-stranded polynucleotide containing a target sequence, and the target sequence is adjacent to the motif sequence recognized by the protein component; Preferably, the target sequence is located at the 3' end of the motif sequence.

9. An isolated nucleic acid molecule comprising: (i) a nucleotide sequence encoding the protein according to claim 1 or 2, or the protein truncation according to claim 3, or the fusion protein according to claim 5; (ii) a nucleotide sequence encoding the isolated nucleic acid molecule of claim 6; and / or, (iii) a nucleotide sequence comprising (i) and (ii); For example, the nucleotide sequence described in any one of (i)-(iii) is codon-optimized for expression in prokaryotes or eukaryotic cells.

10. A vector comprising the isolated nucleic acid molecule of claim 9.

11. A host cell comprising the isolated nucleic acid molecule of claim 9 or the vector of claim 10.

12. A composition comprising: (i) a first component selected from the group consisting of: a protein according to claim 1 or 2, a protein truncation according to claim 3, a conjugate according to claim 4, a fusion protein according to claim 5, a nucleotide sequence encoding the protein, protein truncation or fusion protein, and any combination thereof; and (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; 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, protein truncation, conjugate or fusion protein described in (i); For example, the direct repeat sequence is an isolated nucleic acid molecule as defined in claim 6; For example, the guide sequence is linked to the 3' end of the direct repeat sequence; for example, the guide sequence comprises a complementary sequence to the target sequence; For example, the composition does not include trans-acting crRNA (tracrRNA); For example, the composition is non-naturally occurring or modified; for example, at least one component of the composition is non-naturally occurring or modified; For example, the first component is non-naturally occurring or modified; and / or, the second component is non-naturally occurring or modified.

13. A composition comprising one or more carriers, wherein the one or more carriers comprise: (i) a first nucleic acid comprising a nucleotide sequence encoding the protein of claim 1 or 2, the protein truncation of claim 3, or the fusion protein of claim 5; optionally, the first nucleic acid is operably linked to a first regulatory element; and (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; 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, protein truncation or fusion protein described in (i); For example, the direct repeat sequence is an isolated nucleic acid molecule as defined in claim 6; For example, the guide sequence is linked to the 3' end of the direct repeat sequence; for example, the guide sequence comprises a complementary sequence to the target sequence; For example, the composition does not include trans-acting crRNA (tracrRNA); For example, the composition is non-naturally occurring or modified; for example, at least one component of the composition is non-naturally occurring or modified; For example, the first regulatory element is a promoter, such as an inducible promoter; for example, the second regulatory element is a promoter, such as an inducible promoter.

14. The composition according to claim 12 or 13, wherein When the target sequence is DNA, the target sequence is located at the 3' end of the protospacer adjacent motif (PAM), and the PAM has a sequence represented by 5'-RYR, wherein R is A or G, and Y is T or C; Preferably, the sequence of the PAM is selected from ATG, ACG, GTG, ATA, ACA, GCA, GTA and / or GCG.

15. The composition according to any one of claims 12 to 14, wherein The target sequence is a DNA or RNA sequence from a prokaryotic cell or a eukaryotic cell; alternatively, the target sequence is a non-naturally occurring DNA or RNA sequence.

16. The composition according to any one of claims 12 to 15, wherein The target sequence is present in a cell; alternatively, the target sequence is present in a nucleic acid molecule (e.g., a plasmid) in vitro; For example, the target sequence is present in the nucleus or in the cytoplasm (eg, an organelle) of a cell; for example, the cell is a prokaryotic cell; for example, the cell is a eukaryotic cell.

17. The composition according to any one of claims 12 to 16, wherein The protein or protein truncation is linked to one or more NLS sequences, or the conjugate or fusion protein comprises one or more NLS sequences; For example, the NLS sequence is linked to the N-terminus or C-terminus of the protein or protein truncation; for example, the NLS sequence is fused to the N-terminus or C-terminus of the protein or protein truncation.

18. A kit comprising one or more components selected from the following: the protein of claim 1 or 2, the protein truncation of claim 3, the conjugate of claim 4, the fusion protein of claim 5, the isolated nucleic acid molecule of claim 6, the complex of claim 7 or 8, the isolated nucleic acid molecule of claim 9, the vector of claim 10, the composition of any one of claims 12 to 17; For example, the kit comprises the composition of any one of claims 12-17, and instructions for using the composition.

19. A delivery composition comprising a delivery vector and one or more selected from the following: the protein of claim 1 or 2, the protein truncation of claim 3, the conjugate of claim 4, the fusion protein of claim 5, the isolated nucleic acid molecule of claim 6, the complex of claim 7 or 8, the isolated nucleic acid molecule of claim 9, the vector of claim 10, and the composition of any one of claims 12 to 17; For example, the delivery vehicle is a particle; For example, the delivery vehicle is selected from lipid particles, sugar particles, metal particles, protein particles, liposomes, exosomes, microvesicles, gene guns or viral vectors (e.g., replication-defective retroviruses, lentiviruses, adenoviruses or adeno-associated viruses).

20. A method for modifying a target gene, comprising: Contacting the complex of claim 7 or 8 or the composition of any one of claims 12 to 17 with the target gene, or delivering it to a cell comprising the target gene; wherein the target sequence is present in the target gene; For example, the target gene is present in a cell, or the target gene is present in a nucleic acid molecule (e.g., a plasmid) in vitro; For example, the cell is a prokaryotic cell; for example, the cell is a eukaryotic cell; for example, the cell is selected from an animal cell (e.g., a mammalian cell, such as a human cell), a plant cell; For example, the modification refers to a break in the target sequence, such as a double-strand break in DNA or a single-strand break in RNA; For example, the modification further comprises inserting an exogenous nucleic acid into the break.

21. A method of altering the expression of a gene product, comprising: Contacting the complex of claim 7 or 8 or the composition of any one of claims 12 to 17 with a nucleic acid molecule encoding the gene product, or delivering it to a cell comprising the nucleic acid molecule; wherein the target sequence is present in the nucleic acid molecule; For example, the nucleic acid molecule is present in a cell, or the nucleic acid molecule is present in a nucleic acid molecule (e.g., a plasmid) in vitro; For example, the cell is a prokaryotic cell; for example, the cell is a eukaryotic cell; for example, the cell is selected from an animal cell (e.g., a mammalian cell, such as a human cell), a plant cell; For example, expression of the gene product is altered (eg, increased or decreased); for example, the gene product is a protein.

22. The method of claim 20 or 21, wherein the protein, protein truncation, conjugate, fusion protein, isolated nucleic acid molecule, complex, vector or composition is contained in a delivery vehicle; For example, 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).

23. The method of any one of claims 20 to 22, 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.

24. A cell or progeny thereof obtained by the method of any one of claims 20-23, wherein the cell comprises a modification not present in its wild type.

25. A cell product of the cell of claim 24 or its progeny.

26. An in vitro, ex vivo or in vivo cell or cell line or their progeny, the cell or cell line or their progeny comprising: the protein of claim 1 or 2, the protein truncation of claim 3, the conjugate of claim 4, the fusion protein of claim 5, the isolated nucleic acid molecule of claim 6, the complex of claim 7 or 8, the isolated nucleic acid molecule of claim 9, the vector of claim 10, the composition of any one of claims 12-17; For example, the cell is a prokaryotic cell or a eukaryotic cell.

27. Use of the protein of claim 1 or 2, the protein truncation of claim 3, the conjugate of claim 4, the fusion protein of claim 5, the isolated nucleic acid molecule of claim 6, the complex of claim 7 or 8, the isolated nucleic acid molecule of claim 9, the vector of claim 10, the composition of any one of claims 12 to 17, or the kit of claim 18 in the preparation of a formulation for nucleic acid editing (e.g., in vitro or ex vivo nucleic acid editing); For example, the nucleic acid editing includes gene or genome editing; For example, the gene or genome editing includes modifying a gene, knocking out a gene, altering the expression of a gene product, repairing a mutation, and / or inserting a polynucleotide.

28. Use of the protein of claim 1 or 2, the protein truncation of claim 3, the conjugate of claim 4, the fusion protein of claim 5, the isolated nucleic acid molecule of claim 6, the complex of claim 7 or 8, the isolated nucleic acid molecule of claim 9, the vector of claim 10, the composition of any one of claims 12-17 or the kit of claim 18 in the preparation of a preparation for: (i) in vitro or ex vivo DNA detection; and / or, (ii) editing a target sequence in a target locus to modify an organism or a non-human organism.

29. A method for detecting the presence of a target nucleic acid in a sample, comprising the following steps: (1) contacting the sample with a labeled DNA probe and any of the following components: The complex according to claim 7 or 8, the composition according to any one of claims 12 to 17, or the kit according to claim 18; Wherein, the guide sequence contained in the complex, composition or kit is capable of hybridizing with the target nucleic acid, and the DNA probe does not hybridize with the guide sequence; preferably, the DNA probe emits a detectable signal after being cleaved; (2) detecting a detectable signal generated by cleavage of the DNA probe by a protein or protein truncation contained in the complex, composition or kit, thereby determining whether a target nucleic acid is present in the sample; Preferably, one end (eg, the 5' end) of the DNA probe is labeled with a fluorescent group, and the other end (eg, the 3' end) is labeled with a quencher group.

30. The method of claim 29, wherein: The sequence of the target nucleic acid is a sequence obtained from a pathogen; preferably, the pathogen is selected from a virus, a bacterium, a fungus, a protozoa, a parasite or any combination thereof; Preferably, the sequence of the target nucleic acid is obtained from the genome of a tumor cell; Optionally, the method further comprises the step of contacting the sample with a reagent for reverse transcription; preferably, the reagent for reverse transcription is selected from reverse transcriptase, oligonucleotide primers, dNTPs or any combination thereof; Preferably, the target nucleic acid is single-stranded or double-stranded; preferably, the sequence of the target nucleic acid is a DNA or RNA sequence from a prokaryotic cell or a eukaryotic cell; or, the sequence of the target nucleic acid is a non-naturally occurring DNA or RNA sequence; Preferably, the detectable signal is determined by one or more methods selected from the group consisting of: imaging-based detection, sensor-based detection, color detection, gold nanoparticle-based detection, fluorescence polarization, colloidal phase transition / dispersion, electrochemical detection, and semiconductor-based sensing; Preferably, the method further comprises the step of amplifying the target nucleic acid in the sample.

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