WO2020098793A1

WO2020098793A1 - Crispr-cas12a enzyme and system

Info

Publication number: WO2020098793A1
Application number: PCT/CN2019/118871
Authority: WO
Inventors: 赖锦盛; 朱金洁; 易飞; 王莹莹; 张继红; 李英男; 吕梦璐; 周英思; 赵海铭; 宋伟彬
Original assignee: 中国农业大学
Priority date: 2018-11-15
Filing date: 2019-11-15
Publication date: 2020-05-22
Also published as: CN113015798A; CN113015798B

Abstract

Related are Cas effect proteins, a fusion protein comprising the proteins, and nucleic acid molecules encoding same. A complex and composition for use in nucleic acid editing, such as gene or genome editing, comprising the relevant proteins or the fusion protein or the nucleic acid molecules encoding the proteins. Also, a method for use in nucleic acid editing, such as gene or genome editing; the method using the relevant proteins or the fusion protein.

Description

CRISPR-Cas12a enzyme and system

Technical field

The invention relates to the field of nucleic acid editing, in particular to the field of regularly clustered short palindrome repeat (CRISPR) technology. In particular, the present invention relates to Cas effector proteins, fusion proteins containing such proteins, and nucleic acid molecules encoding them. The invention also relates to complexes and compositions for nucleic acid editing (eg, gene or genome editing), which comprise the protein or fusion protein of the invention, or nucleic acid molecules encoding them. The present invention also relates to a method for nucleic acid editing (eg, gene or genome editing), which uses a protein or fusion protein comprising the present invention.

Background technique

CRISPR / Cas technology is a widely used gene editing technology. It uses RNA guidance to specifically bind the target sequence on the genome and cut the DNA to produce double-strand breaks. It uses biological non-homologous end connection or homologous recombination to target. Gene editing.

The CRISPR / Cas9 system is the most commonly used type II CRISPR system. It recognizes the PAM motif of 3’-NGG and blunt ends the target sequence. The CRISPR / Cas Type V system is a new type of CRISPR system discovered in the past two years. It has a 5’-TTN motif and performs cohesive end cleavage of target sequences, such as Cpf1, C2c1, CasX, CasY. However, the different CRISPR / Cas currently exist have different advantages and disadvantages. For example, Cas9, C2c1 and CasX all need two RNAs to guide RNA, while Cpf1 only needs one guide RNA and can be used for multiple gene editing. CasX has a size of 980 amino acids, while common Cas9, C2c1, CasY and Cpf1 are usually around 1300 amino acids. In addition, the PAM sequences of Cas9, Cpf1, CasX, and CasY are more complex and diverse, and C2c1 recognizes the rigorous 5'-TTN, so its target site is easier to predict than other systems and reduces the potential off-target effect.

In short, in view of the fact that currently available CRISPR / Cas systems are limited by some shortcomings, the development of a more robust new CRISPR / Cas system with many aspects of good performance is of great significance to the development of biotechnology.

Summary of the invention

After a lot of experiments and repeated explorations, the inventor of the present application discovered a Cpf1 enzyme, also known as Cas12a or SmCpf1, in microorganisms. Based on this discovery, the inventors developed a new CRISPR / SmCpf1 system and a gene editing method based on the system.

Cas effector protein

Therefore, in the first aspect, the present invention provides a protein having the amino acid sequence shown in SEQ ID NO: 1 or an ortholog, homolog, variant or functional fragment thereof; wherein, the ortholog Homologues, homologues, variants or functional fragments substantially retain the biological function of the sequence from which they originate.

In the present invention, the biological functions of the above sequence include, but are not limited to, the activity of binding to the guide RNA, endonuclease activity, and the activity of binding to and cleaving at a specific site of the target sequence under the guidance of the guide RNA.

In certain embodiments, the orthologs, homologs, and variants have at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the sequence from which they were derived Sequence identity.

In certain embodiments, the ortholog, homolog, or variant has at least 95%, at least 96%, at least 97%, at least 98%, or at least 95%, compared to the sequence shown in SEQ ID NO: 1, or At least 99% sequence identity, and basically retains the biological function of the sequence from which it originates (for example, the activity of binding to the guide RNA, endonuclease activity, binding to a specific site of the target sequence under the guidance of the guide RNA And cutting activity).

In certain embodiments, the protein is an effector protein in the CRISPR / Cas system. In certain embodiments, the protein is derived from Smithella sp. M82. In certain embodiments, the protein is a Cas protein derived from Smithella sp. M82.

In certain embodiments, the protein of the invention comprises or consists of a sequence selected from the following:

(i) The sequence shown in SEQ ID NO: 1;

(ii) One or more amino acid substitutions, deletions, or additions compared to the sequence shown in SEQ ID NO: 1 (

eg

1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, deletions, or additions); or

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

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

Derived protein

The protein of the present invention can be derivatized, for example, linked to another molecule (eg, another polypeptide or protein). Generally, derivatization (eg, labeling) of a protein does not adversely affect the desired activity of the protein (eg, activity of binding to the guide RNA, endonuclease activity, binding and cleavage at specific sites of the target sequence under the guidance of the guide RNA Activity). Therefore, the protein of the present invention is also intended to include such derivatized forms. For example, the protein of the present invention can be functionally linked (by chemical coupling, gene fusion, non-covalent linking or other means) to one or more other molecular groups, such as another protein or polypeptide, detection reagents, pharmaceutical reagents Wait.

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 increase the ability of the protein of the 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 label to facilitate detection of the protein of the invention. For example, it can be linked to an epitope tag to facilitate expression, detection, tracing and / or purification of the protein of the invention.

Conjugate

Therefore, in a second aspect, the present invention provides a conjugate comprising a protein and a modified moiety as described above.

In certain embodiments, the modified moiety is selected from additional proteins or polypeptides, detectable labels, or any combination thereof.

In certain embodiments, the additional protein or polypeptide is selected from epitope tags, reporter gene sequences, nuclear localization signal (NLS) sequences, targeting moieties, transcription activation domains (eg, VP64), transcription repression domains (For example, KRAB domain or SID domain), nuclease domain (for example, Fok1), a domain with an activity selected from the group consisting of nucleotide deaminase, methylase activity, demethylase , Transcription activation activity, transcription inhibitory activity, transcription 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.

In certain embodiments, the conjugates of the invention comprise one or more NLS sequences, such as the NLS of the SV40 viral large T antigen. In certain exemplary embodiments, the NLS sequence is shown in SEQ ID NO: 7. In certain embodiments, the NLS sequence is located at, near, or near the terminus (eg, N-terminus or C-terminus) of the protein of the present invention. In certain exemplary embodiments, the NLS sequence is located at, near, or near the C-terminus of the protein of the present invention.

In certain embodiments, the conjugates of the present invention comprise epitope tags. 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 use the desired purpose (for example, Purification, detection or tracing) select the appropriate epitope tag.

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, and the like.

In certain embodiments, the conjugates of the present invention comprise domains capable of binding to DNA molecules or intracellular molecules, such as maltose binding protein (MBP), Lex A's DNA binding domain (DBD), GAL4's DBD, etc. .

In certain embodiments, the conjugates of the invention contain a detectable label, such as a fluorescent dye, such as FITC or DAPI.

In certain embodiments, the protein of the invention is optionally coupled, conjugated, or fused to the modified moiety via a linker.

In certain embodiments, the modified portion is directly connected to the N-terminus or C-terminus of the protein of the present invention.

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

Fusion protein

In a third aspect, the invention provides a fusion protein comprising the protein of the invention and additional protein or polypeptide.

In certain embodiments, the fusion protein of the invention comprises one or more NLS sequences, such as the NLS of the SV40 viral large T antigen. In certain embodiments, the NLS sequence is located at, near, or near the terminus (eg, N-terminus or C-terminus) of the protein of the present invention. In certain exemplary embodiments, the NLS sequence is located at, near, or near the C-terminus of the protein of the present invention.

In certain embodiments, the fusion protein of the present invention contains an epitope tag.

In certain embodiments, the fusion protein of the invention comprises a reporter gene sequence.

In certain embodiments, the fusion protein of the present invention comprises a domain capable of binding to DNA molecules or intracellular molecules.

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

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

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

In certain exemplary embodiments, the fusion protein of the present invention has an amino acid sequence selected from the group consisting of SEQ ID NO: 8.

The protein of the present invention, the conjugate of the present invention, or the fusion protein of the present invention are not limited by the manner of production. For example, they can be produced by genetic engineering methods (recombinant technology) or chemical synthesis methods.

Direct repeat sequence

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

(i) SEQ ID NO: the sequence shown in 3 or 5;

(ii) One or more base substitutions, deletions, or additions compared to the sequence shown in SEQ ID NO: 3 or 5 (

eg

1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 base substitutions, deletions or additions) sequences;

(iii) a sequence having at least 20%, at least 30%, and at least 95% sequence identity with the sequence shown in SEQ ID NO: 3 or 5;

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

(v) the complement of the sequence described in any one of (i)-(iii);

In addition, the sequence described in any one of (ii)-(v) basically retains the biological function of the sequence from which it originates. The biological function of the sequence refers to, as the same direction in the CRISPR-Cas system Repeated sequence activity.

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

In certain embodiments, the nucleic acid molecule comprises or consists of a sequence selected from:

(a) SEQ ID NO: nucleotide sequence shown in 3 or 5;

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

(c) The complement of the sequence described in (a).

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

CRISPR / Cas complex

In a fifth aspect, the invention provides a composite comprising:

(i) a protein component selected from: the protein, conjugate or fusion protein of the present invention, and any combination thereof; and

(ii) a nucleic acid component comprising, from the 5 'to 3' direction, an isolated nucleic acid molecule as described above and a targeting sequence capable of hybridizing with a target sequence,

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

In certain embodiments, the targeting sequence is attached to the 3 'end of the nucleic acid molecule.

In certain embodiments, the targeting sequence comprises the complement of the target sequence.

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

In certain embodiments, the nucleic acid molecule is RNA.

In certain embodiments, the complex does not contain trans-acting crRNA (tracrRNA).

In certain embodiments, the targeting sequence is at least 5, at least 10 in length, in certain embodiments, the targeting sequence is 10-30, or 15-25 in length, or 15-22, or 19-25 or 19-22 nucleotides.

In certain embodiments, the isolated nucleic acid molecule is 55-70 nucleotides in length, such as 55-65 nucleotides, such as 60-65 nucleotides, such as 62-65 nucleosides Acid, for example 63-64 nucleotides. In certain embodiments, the isolated nucleic acid molecule is 15-30 nucleotides in length, such as 15-25 nucleotides, such as 20-25 nucleotides, such as 22-24 nucleosides Acid, for example 23 nucleotides.

Encoding nucleic acid, vector and host cell

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

(i) The nucleotide sequence encoding the protein or fusion protein of the present invention;

(ii) encodes the isolated nucleic acid molecule according to the fourth aspect; or

(iii) The nucleotide sequence comprising (i) and (ii).

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

In a seventh aspect, the invention also provides a vector comprising the isolated nucleic acid molecule according to the sixth 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, cosmid, bacteriophage, cosmid, and the like. In certain alternative embodiments, the vector is capable of expressing the protein of the invention, the fusion protein, the isolated nucleic acid molecule according to the fourth aspect or the fifth aspect in a subject (eg mammal, eg human) The compound.

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

Composition and carrier composition

In a ninth aspect, the present invention also provides a composition comprising:

(i) a first component selected from the group consisting of: the protein, conjugate, fusion protein, nucleotide sequence encoding the protein or fusion protein of the invention, 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;

Wherein, the guide RNA includes a repeat sequence and a guide sequence from the 5 'to 3' direction, and the guide sequence can hybridize with the target sequence;

The guide RNA can form a complex with the protein, conjugate or fusion protein described in (i).

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

In certain embodiments, the targeting sequence is linked to the 3 'end of the direct repeat sequence. In certain embodiments, the targeting sequence comprises the complement of the target sequence.

In certain embodiments, the composition does not include tracrRNA.

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.

In certain embodiments, when the target sequence is DNA, the target sequence is located at the 3 ′ end of the original spacer sequence adjacent to the motif (PAM), and the PAM has the sequence shown by 5′-TTN, wherein , N is selected from A, G, T, C. Preferably, N is selected from A, G, C.

In certain embodiments, when the target sequence is RNA, the target sequence does not have PAM domain restrictions.

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

In certain embodiments, the target sequence is present in the cell. In certain embodiments, the target sequence is present in the nucleus or cytoplasm (eg, organelle). In certain embodiments, the cell is a eukaryotic cell. In certain embodiments, the cell is a prokaryotic cell.

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

In a tenth aspect, the invention also provides a composition comprising one or more carriers, the one or more carriers comprising:

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

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

among them:

The first nucleic acid and the second nucleic acid exist on the same or different carriers;

The guide RNA contains a repeat sequence and a guide sequence from the 5 'to 3' direction, and the guide sequence can hybridize with the target sequence;

The guide RNA can form a complex with the effector protein or fusion protein described in (i).

In certain embodiments, the composition does not include tracrRNA.

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 regulatory element is a promoter, such as an inducible promoter.

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

In certain embodiments, when the target sequence is DNA, the target sequence is located at the 3 ′ end of the original spacer sequence adjacent to the motif (PAM), and the PAM has the sequence shown by 5′-TTN, wherein , N is selected from A, G, T, C.

In certain embodiments, one type of vector is a plasmid, which refers to a circular double-stranded DNA loop into which additional DNA fragments can be inserted, for example, by standard molecular cloning techniques. Another type of vector is a viral vector in which virus-derived DNA or RNA sequences are present for packaging viruses (eg, retroviruses, replication-deficient retroviruses, adenoviruses, replication-deficient adenoviruses, and adeno-associated Virus). Viral vectors also contain polynucleotides carried by viruses used to transfect into a host cell. Certain vectors (eg, bacterial vectors with an origin of bacterial replication and episomal mammalian vectors) are capable of autonomous replication in the host cell into which they are introduced. Other vectors (eg, non-episomal mammalian vectors) integrate into the host cell's genome after introduction into the host cell, and thereby replicate with the host genome. Moreover, certain vectors can direct the expression of genes to which they are operably linked. Such vectors are referred to herein as "expression vectors". Common expression vectors used in recombinant DNA technology are usually in the form of plasmids.

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

Delivery and delivery composition

The protein, conjugate, fusion protein of the present invention, the isolated nucleic acid molecule according to the fourth aspect, the complex of the present invention, the isolated nucleic acid molecule according to the sixth aspect, the carrier according to the seventh aspect The composition according to the ninth aspect and the tenth 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, magnetic transfection, lipofection, puncture transfection, optical transfection, reagent-enhanced nucleic acid uptake, and via liposomes, immunoliposomes, viral particles, artificial viruses Delivery.

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

In certain embodiments, the delivery vehicle is a particle.

In certain embodiments, the delivery vehicle is selected from lipid particles, sugar particles, metal particles, protein particles, liposomes, exosomes, microvesicles, gene guns, or viral vectors (eg, replication-deficient reverse transcription) Virus, lentivirus, adenovirus or adeno-associated virus).

Reagent test kit

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

In certain embodiments, the kit of the invention comprises the composition as described in the ninth aspect. In some embodiments, the kit also includes instructions for using the composition.

In certain embodiments, the kit of the invention comprises the composition of the tenth aspect. In some embodiments, the kit also includes instructions for using the composition.

In some embodiments, the components included in the kit of the present invention may be provided in any suitable container.

In certain embodiments, the kit also contains one or more buffers. The buffer may be any buffer, including but not limited to sodium carbonate buffer, sodium bicarbonate buffer, borate buffer, Tris buffer, MOPS buffer, HEPES buffer, and combinations thereof. In certain embodiments, the buffer is alkaline. In certain embodiments, the buffer has a pH of from about 7 to about 10.

In certain embodiments, the kit further includes one or more oligonucleotides, the one or more oligonucleotides corresponding to a guide sequence for insertion into the vector, so as to be operably linked to the guide Sequence and regulatory elements. In certain embodiments, the kit includes homologous recombination template polynucleotides.

Method and use

In another aspect, the present invention provides a method for modifying a target gene, comprising: combining the complex according to the fifth aspect, the composition according to the ninth aspect, or the composition according to the tenth aspect Contact with the target gene, or deliver to the cell containing the target gene; the target sequence is present in the target gene.

In certain embodiments, the target gene is present in the cell. In certain embodiments, the cell is a prokaryotic cell. In certain embodiments, the cell is a eukaryotic cell. In certain embodiments, the cell is a mammalian cell. In certain embodiments, the cell is a human cell. In certain embodiments, the cell is selected from non-human primate, bovine, porcine, 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 possessed by cultivated plants (such as cassava, corn, sorghum, wheat, or rice), algae, trees, or vegetables.

In certain embodiments, the target gene is present in a nucleic acid molecule (eg, plasmid) in vitro. In certain embodiments, the target gene is present in a plasmid.

In certain embodiments, 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.

In certain embodiments, the break results in reduced transcription of the target gene.

In certain embodiments, the method further comprises: contacting the editing template with the target gene, or delivering to the cell containing the target gene. In such embodiments, the method repairs the broken target gene by homologous recombination with an exogenous template polynucleotide, wherein the repair results in a mutation, including one or more nucleosides of the target gene Acid insertion, deletion, or substitution. In certain embodiments, the mutation results in one or more amino acid changes in the protein expressed from the gene containing the target sequence.

Therefore, in certain embodiments, the modification further includes inserting an editing template (eg, exogenous nucleic acid) into the break.

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

In certain embodiments, the delivery vehicle is selected from lipid particles, sugar particles, metal particles, protein particles, liposomes, exosomes, viral vectors (eg, replication-defective retroviruses, lentiviruses, adenoviruses) Or adeno-associated virus).

In certain embodiments, the method is used to modify one or more target sequences in a target gene or nucleic acid molecule encoding a target gene product to modify a cell, cell line, or organism.

In another aspect, the present invention provides a method of altering the expression of a gene product, comprising: combining the complex according to the fifth aspect, the composition according to the ninth aspect, or the composition according to the tenth aspect The composition is contacted with a nucleic acid molecule encoding the gene product or delivered to a cell containing the nucleic acid molecule, and the target sequence is present in the nucleic acid molecule.

In certain embodiments, the nucleic acid molecule is present in the cell. In certain embodiments, the cell is a prokaryotic cell. In certain embodiments, the cell is a eukaryotic cell. In certain embodiments, the cell is a mammalian cell. In certain embodiments, the cell is a human cell. In certain embodiments, the cell is selected from non-human primate, bovine, porcine, 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 possessed by cultivated plants (such as cassava, corn, sorghum, wheat, or rice), algae, trees, or vegetables.

In certain embodiments, the nucleic acid molecule is present in a nucleic acid molecule (eg, plasmid) in vitro. In certain embodiments, the nucleic acid molecule is present in a plasmid.

In certain embodiments, the expression of the gene product is altered (eg, increased or decreased). In certain embodiments, the expression of the gene product is enhanced. In certain embodiments, the expression of the gene product is reduced.

In certain embodiments, the gene product is a protein.

In another aspect, the invention relates to the protein according to the first aspect, the conjugate according to the second aspect, the fusion protein according to the third aspect, the isolated nucleic acid molecule according to the fourth aspect, The complex according to the fifth aspect, the isolated nucleic acid molecule according to the sixth aspect, the vector according to the seventh aspect, the composition according to the ninth aspect, the composition according to the tenth aspect 3. The kit or delivery composition of the present invention is used for nucleic acid editing.

In certain embodiments, the nucleic acid editing includes gene or genome editing, such as modifying genes, knocking out genes, changing the expression of gene products, repairing mutations, and / or inserting polynucleotides.

In another aspect, the invention relates to the protein according to the first aspect, the conjugate according to the second aspect, the fusion protein according to the third aspect, the isolated nucleic acid molecule according to the fourth aspect, The complex according to the fifth aspect, the isolated nucleic acid molecule according to the sixth aspect, the vector according to the seventh aspect, the composition according to the ninth aspect, the composition according to the tenth aspect 2. The use of the kit or delivery composition of the present invention in the preparation of a formulation, the formulation being used for:

(i) In vitro gene or genome editing;

(ii) Detection of isolated single-stranded DNA;

(iii) Edit target sequences in target loci to modify biological or non-human organisms;

(iv) Treatment of disorders caused by defects in target sequences in target loci.

Cells and cell progeny

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

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

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

The invention also relates to an in vitro, ex vivo or in vivo cell or cell line or their progeny, said cell or cell line or their progeny comprising: the protein according to the first aspect, such as the second The conjugate according to aspect, the fusion protein according to third aspect, the isolated nucleic acid molecule according to fourth aspect, the complex according to fifth aspect, the isolated nucleic acid according to sixth aspect Molecules, the carrier according to the seventh aspect, the composition according to the ninth aspect, the composition according to the tenth aspect, the kit or the delivery composition of the present invention.

In certain embodiments, the cell is a prokaryotic cell.

In certain embodiments, the cell is a eukaryotic cell. In certain embodiments, the cell is a mammalian cell. In certain embodiments, the cell is a human cell. In certain embodiments, the cell is a non-human mammalian cell, such as a non-human primate, bovine, ovine, porcine, canine, monkey, rabbit, rodent (e.g., rat or mouse) cell. In certain embodiments, the cell is a non-mammalian eukaryotic cell, such as a poultry bird (eg, chicken), fish, or crustacean (eg, clam, shrimp) cell. In certain embodiments, the cells are plant cells, such as those possessed by monocotyledonous or dicotyledonous plants or cultivated plants or food crops such as cassava, corn, sorghum, soybean, wheat, oats or rice, for example Algae, trees or production plants, fruits or vegetables (for example, trees such as citrus trees, nut trees; nightshade plants, cotton, tobacco, tomatoes, grapes, coffee, cocoa, etc.).

In certain embodiments, the cells are stem cells or stem cell lines.

Definition of Terms

In the present invention, unless otherwise stated, the scientific and technical terms used herein have the meaning 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 in this article are all routine procedures widely used in the corresponding fields. . Meanwhile, in order to better understand the present invention, definitions and explanations of related terms are provided below.

In the present invention, the expression "SmCpf1" refers to a Cpf1 effector protein discovered and identified by the inventors for the first time, which has an amino acid sequence selected from the following:

(i) The sequence shown in SEQ ID NO: 1;

eg

The SmCpf1 of the present invention is an endonuclease that binds to and cuts a specific site of a target sequence under the guidance of a guide RNA, and has both DNA and RNA endonuclease activity.

As used herein, the term "regularly clustered short palindrome repeat (CRISPR) -CRISPR-related (Cas) (CRISPR-Cas) system" or "CRISPR system" is used interchangeably and has the skill in the art A person generally understands the meaning that it usually contains a transcript or other element related to the expression of a CRISPR-related ("Cas") gene, or a transcript or other element that can direct the activity of the Cas gene. Such transcripts or other elements may contain sequences encoding Cas effector proteins and guide RNAs containing CRISPR RNA (crRNA), and trans-acting crRNA (tracrRNA) sequences contained in the CRISPR-Cas9 system, or from CRISPR loci Other sequences or transcripts. In the CRISPR system based on SmCpf1 according to the present invention, no tracrRNA sequence is required.

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

As used herein, the terms "guide RNA" and "mature crRNA" are used interchangeably and have the meaning commonly understood by those skilled in the art. In general, the guide RNA can contain direct repeats and guide sequences, or consist essentially of or consist of direct repeats and guide sequences (also known as spacer sequences in the context of endogenous CRISPR systems) (spacer)) composition. In some cases, the targeting sequence is any polynucleotide sequence that is sufficiently complementary to the target sequence to hybridize to the target sequence and direct specific binding of the CRISPR / Cas complex to the target sequence. In certain embodiments, when optimally aligned, the degree of complementarity between the targeting 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 ability of those of ordinary skill in the art to determine the best alignment. For example, there are publicly and commercially available alignment algorithms and programs, such as but not limited to ClustalW, Smith-Waterman in Matlab, Bowtie, Geneious, Biopython, and SeqMan.

In some cases, the targeting 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 some cases, the length of the guide sequence is not more than 50, 45, 40, 35, 30, 25, 24, 23, 22, 21, 20, 15 , 10 or fewer nucleotides. In certain embodiments, the targeting sequence is 10-30, or 15-25, or 15-22, or 19-25 or 19-22 nucleotides in length.

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 in length 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 some cases, the length of the direct repeat sequence is not more than 70, 65, 64, 63, 62, 61, 60, 59, 58, 57, 57 or 56 , 55, 50, 45, 40, 35, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 15 , 10 or fewer nucleotides. In certain embodiments, the direct repeat sequence is 55-70 nucleotides in length, such as 55-65 nucleotides, such as 60-65 nucleotides, such as 62-65 nucleosides Acid, for example 63-64 nucleotides. In certain embodiments, the direct repeat sequence is 15-30 nucleotides in length, such as 15-25 nucleotides, such as 20-25 nucleotides, such as 22-24 nucleosides Acid, for example 23 nucleotides.

As used herein, the term "CRISPR / Cas complex" refers to a ribonucleoprotein complex formed by the combination of guide RNA or mature crRNA with Cas protein, which includes hybridization to the target sequence and with the Cas Targeting sequence for protein binding. The ribonucleoprotein complex can recognize and cleave polynucleotides that can hybridize to the guide RNA or mature crRNA.

Therefore, in the case of forming a CRISPR / Cas complex, "target sequence" refers to a polynucleotide that is designed to be targeted by a targeting sequence, such as a sequence complementary to the targeting sequence, wherein the target The hybridization between the sequence and the targeting sequence will promote the formation of CRISPR / Cas complex. Complete complementarity is not necessary, as long as there is sufficient complementarity to cause hybridization and promote the formation of a CRISPR / Cas complex. The target sequence may comprise any polynucleotide, such as DNA or RNA. In some cases, the target sequence is located in the nucleus or cytoplasm of the cell. In some cases, the target sequence may be located in an organelle of eukaryotic cells, such as mitochondria or chloroplasts. A sequence or template that can be used to recombine into a target locus containing the target sequence is called an "edit template" or "edit polynucleotide" or "edit sequence". In certain embodiments, the editing template is an exogenous nucleic acid. In certain embodiments, the recombination is homologous recombination.

In the present invention, the expression "target sequence" or "target polynucleotide" may be any endogenous or exogenous polynucleotide for a cell (eg, eukaryotic cell). For example, the target polynucleotide may be a polynucleotide present in the nucleus of eukaryotic cells. The target polynucleotide may be a sequence encoding a gene product (eg, protein) or a non-coding sequence (eg, regulatory polynucleotide or useless DNA). In some cases, it is believed that the target sequence should be related to the proximate motif (PAM) of the spacer sequence. The exact sequence and length requirements for PAM vary depending on the Cas effect enzyme used, but PAM is typically a 2-5 base pair sequence adjacent to the original spacer sequence (ie, the target sequence). One skilled in the art can identify the PAM sequence used with a given Cas effector protein.

In some cases, the target sequence or target polynucleotide may include multiple disease-related genes and polynucleotides and signaling biochemical pathway-related genes and polynucleotides. Non-limiting examples of such target sequences or target polynucleotides include US provisional patent applications 61 / 736,527 and 61 / 748,427 filed on December 12, 2012 and January 2, 2013, filed on December 12, 2013 Those listed in the international application PCT / US2013 / 074667 dated December 12, all of which are incorporated herein by reference.

In some cases, examples of target sequences or target polynucleotides include sequences related to signaling biochemical pathways, such as signaling biochemical pathway related genes or polynucleotides. Examples of target polynucleotides include disease-related genes or polynucleotides. "Disease-related" gene or polynucleotide refers to any gene or polynucleoside that produces transcription or translation products at abnormal levels or in abnormal forms in cells derived from disease-affected tissues compared to non-disease-controlled tissues or cells acid. In the case where the altered expression is related to the appearance and / or progression of the disease, it may be a gene expressed at an abnormally high level; or, it may be a gene expressed at an abnormally low level. Disease-related genes also refer to genes that have one or more mutations or genetic mutations that are directly responsible for or are unbalanced with one or more genes responsible for the etiology of the disease. The transcribed or translated product may be known or unknown, and may be at normal or abnormal levels.

As used herein, the term "wild type" has a meaning commonly understood by those skilled in the art, which represents a typical form of an organism, a strain, a gene or a characteristic that distinguishes it from a mutant or variant form when it exists in nature It can be isolated from sources in nature and has not been intentionally modified by man.

As used herein, the terms "non-naturally occurring" or "engineered" are used interchangeably and refer to manual participation. 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 another component to which they are bound in nature or as found in nature.

As used herein, the term "ortholog (ortholog)" has the meaning commonly understood by those skilled in the art. As a further guide, "orthologs" of proteins as described herein refer to proteins belonging to different species that perform the same or similar functions as the proteins that are their orthologs.

As used herein, the term "identity" is used to refer to the sequence matching between two polypeptides or between two nucleic acids. When a position in two compared sequences is occupied by the same base or amino acid monomer subunit (for example, a position in each of two DNA molecules is occupied by adenine, or two A certain position in each of the polypeptides is occupied by lysine), then each molecule is the same at this 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 for comparison x 100. For example, if 6 of the 10 positions of the two sequences match, the two sequences have 60% identity. For example, the DNA sequences CTGACT and CAGGTT share 50% identity (3 out of 6 positions match). Generally, comparisons are made when 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 is conveniently performed by a computer program such as the Align program (DNAstar, Inc.). You can also use the algorithms of E. Meyers and W. Miller (Comput. Appl. Biosci., 4: 11-17 (1988)) that have been integrated into the ALIGN program (version 2.0), and use the PAM120 weight residue table (weight residue table) , A gap length penalty of 12, and a gap penalty of 4 to determine the percent identity between two amino acid sequences. In addition, the Needleman and Wunsch (JMoI Biol. 48: 444-453 (1970)) algorithms in the GAP program integrated into the GCG software package (available at www.gcg.com) can be used, and the Blossum 62 matrix or PAM250 matrix and gap weights of 16, 14, 12, 10, 8, 6, or 4 and length weights of 1, 2, 3, 4, 5, or 6 to determine the percent identity between two amino acid sequences .

As used herein, the term "vector" refers to a nucleic acid vehicle into which a polynucleotide can be inserted. When the vector enables expression of the protein encoded by the inserted polynucleotide, the vector is called an expression vector. The vector can be introduced into the host cell by transformation, transduction or transfection, so that the genetic material elements carried by it can be expressed in the host cell. Vectors are well known to those skilled in the art and include, but are not limited to: plasmids; phagemids; Kos plasmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or P1 derived artificial chromosomes (PAC) ; Phages such as lambda phage or M13 phage and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillae Polyoma vacuolar virus (such as SV40). A vector may contain multiple 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 contain an origin of replication.

As used herein, the term "host cell" refers to a cell that can be used to introduce a vector, including, but not limited to, prokaryotic cells such as E. 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, HEK293 cells or human cells.

Those skilled in the art will understand that the design of the expression vector may depend on factors such as the choice of host cell to be transformed, the desired level of expression, and the like. A vector can be introduced into a host cell to thereby produce transcripts, proteins, or peptides, including proteins, fusion proteins, isolated nucleic acid molecules, etc. as described herein (eg, CRISPR transcripts, such as nucleic acid transcripts , Protein, or enzyme).

As used herein, the term "regulatory element" is intended to include promoters, enhancers, internal ribosome entry sites (IRES), and other expression control elements (eg, transcription termination signals, such as polyadenylation signals and Multi-U sequence), for a detailed description, please refer to Goeddel, "Gene Expression Technology: Enzymatic Methods" (GENE EXPRESSION TECHNOLOGY: METHODS IN INZYMOLOGY) 185, Academic Press, San Diego , California (1990). In some cases, regulatory elements include those that direct the constitutive expression of a nucleotide sequence in many types of host cells and those that direct the nucleotide sequence to be expressed only in certain host cells (eg, Organization-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 specific cell types (e.g. Lymphocytes). In some cases, regulatory elements can also direct expression in a time-dependent manner (eg, 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., Section 8 ( 1) Volume, pages 466-472, 1988); the SV40 enhancer; and the intron sequence between

exons

2 and 3 of rabbit β-globin (Proc. Natl. Acad. Sci. USA., Volume 78 (3), pages 1527-31, 1981).

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 upstream of a gene that can initiate expression of a downstream gene. A constitutive promoter is a nucleotide sequence that, when it is operably linked to a polynucleotide encoding or defining a gene product, under most or all physiological conditions of the cell, it results in the gene product in the cell Of generation. An inducible promoter is a nucleotide sequence that, when operably linked to a polynucleotide encoding or defining a gene product, is basically only caused when the inducer corresponding to the promoter is present in the cell The gene product is produced inside the cell. A tissue-specific promoter is a nucleotide sequence that, when operably linked to a polynucleotide encoding or defining a gene product, basically only results when the cell is a tissue-type cell corresponding to the promoter Gene products are produced in cells.

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 expression of the nucleotide sequence (e.g. , In an in vitro transcription / translation system or when the vector is introduced into a host cell, in the host cell).

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 conventional Watson-Crick or other non-traditional types. Percent complementarity indicates the percentage of residues in a nucleic acid molecule that can form hydrogen bonds with a second nucleic acid sequence (eg, Watson-Crick base pairing) (eg, 5, 6, 7, 8 out of 10) , 9, 10 are 50%, 60%, 70%, 80%, 90%, and 100% complementary). "Completely complementary" 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 having 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, At least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98 on the region of 30, 35, 40, 45, 50 or more nucleotides %, 99%, or 100% degree of complementarity, or refers to two nucleic acids that hybridize under stringent conditions.

As used herein, "stringent conditions" for hybridization refer to conditions under which a nucleic acid that is complementary to a target sequence mainly hybridizes to the target sequence and does not substantially hybridize to non-target sequences. Stringent conditions are usually sequence-dependent and vary depending on many factors. In general, the longer the sequence, the higher the temperature at which the sequence specifically hybridizes to its target sequence. Non-limiting examples of stringent conditions are described in Tijssen (1993) "Laboratory Techniques in Biochemistry and Molecular Biology-Nucleic Acid Probe Hybridization" (Laboratory Techniques In Biochemistry And Molecular Biology-Hybridization With Nucleic Acid Probes) , Part I, Chapter 2, "Overview of Hybridization Principles and Strategies for Analysis of Nucleic Acid Probes" ("Overview of principles" of hybridization and the strategy of acrylic probe "), Elsevier, New York.

As used herein, the term "hybridization" refers to a reaction in which one or more polynucleotides react to form a complex that is bonded via hydrogen bonding of the bases between the nucleotide residues And stabilize. Hydrogen bonding can occur by means of Watson-Crick base pairing, Hoogstein binding, or in any other sequence-specific manner. The complex may comprise two strands forming a duplex, three or more strands forming a multi-strand complex, a single self-hybridizing strand, or any combination of these. The hybridization reaction can constitute a step in a broader process (such as the initiation of PCR, or the cleavage of a polynucleotide via an enzyme). The sequence that can hybridize with a given sequence is called the "complement" of the given sequence.

As used herein, the term "expression" refers to the process by which a polynucleotide (eg, transcribed into mRNA or other RNA transcript) is transcribed from a DNA template and / or the transcribed mRNA is subsequently translated into a peptide, The process of peptides or proteins. 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.

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

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

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, Rabbits, rodents (eg, mice or rats), non-human primates (eg, macaques or cynomolgus monkeys), or humans. In certain embodiments, the subject (e.g., human) has a disorder (e.g., a disorder caused by a disease-related genetic defect).

Beneficial effects of invention

Compared with the prior art, the SmCpf1 protein and system of the present invention have significant advantages. For example, the PAM domain of the SmCpf1 effector protein of the present invention has a 5'-TTN structure. For example, the Cas effect protein of the present invention can efficiently perform DNA cleavage in eukaryotic organisms, and is superior to FnCpf1 whose reported PAM domain is 5'-TTN.

The embodiments of the present invention will be described in detail below in conjunction with the drawings and examples, but those skilled in the art will understand that the following drawings and examples are only used to illustrate the present invention, not to limit the scope of the present invention. The various objects and advantageous aspects of the invention will become apparent to those skilled in the art from the following detailed description of the drawings and preferred embodiments.

BRIEF DESCRIPTION

Figure 1 shows the results of amino acid sequence alignment between SmCpf1 and other Cpf1 proteins.

Figure 2a shows the results of SmCpf1 processing of pre-crRNA in vivo.

Figure 2b is the result of crm structure analysis of SmCpf1, showing the secondary structure of the same repeat sequence.

Figure 3a shows the results of PAM domain analysis.

Figure 3b is a plasmid wear-out analysis of SmCpf1 for plasmids containing different PAM sequences.

Figure 4aSmCpf1 cleavage of VEGFA in human cell lines.

Figure 4b is a schematic diagram of SmCpf1 cleaving DNMT1 in a human cell line.

Sequence information

Information on the partial sequence involved in the present invention is provided in Table 1 below.

Table 1: Description of the sequence

SEQ ID NO:SEQ ID: NO:	描述 description
11	SmCpf1的氨基酸序列Amino acid sequence of SmCpf1
22	SmCpf1的编码核苷酸序列SmCpf1 encoding nucleotide sequence
33	SmCpf1的原型同向重复序列SmCpf1 Prototype Direct Repeat Sequence
44	SmCpf1的原型同向重复序列的编码核酸序列SmCpf1 prototype direct repeat sequence encoding nucleic acid sequence
55	SmCpf1成熟同向重复序列SmCpf1 mature direct repeat sequence
66	SmCpf1成熟同向重复序列的编码核酸序列SmCpf1 mature direct repeat sequence encoding nucleic acid sequence
77	NLS序列NLS sequence

88	SmCpf1-NLS融合蛋白的氨基酸序列Amino acid sequence of SmCpf1-NLS fusion protein
99	表达SmCpf1系统的质粒Plasmid expressing SmCpf1 system
1010	PAM文库序列PAM library sequence
1111	PAM消耗导向RNAPAM depletion guide RNA
1212	真核编辑导向RNAEukaryotic editing guide RNA

detailed description

The invention will now be described with reference to the following examples which are intended to illustrate the invention (but not to limit it).

Unless otherwise specified, the experiments and methods described in the examples were basically performed according to conventional methods well known in the art and described in various references. For example, for conventional techniques such as immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics, and recombinant DNA used in the present invention, please refer to Sambrook, Fridge ( Fritsch and Maniatis, “MOLECULAR CLONING: A LABORATORY MANUAL”, 2nd edition (1989); “CURRENT PROTOCOLS IN MOLECULAR” BIOLOGY) (edited by FM Ausubel (FMAusubel) and others, (1987)); "METHODS IN ENZYMOLOGY" series (academic publishing company): "PCR 2: Practical Methods" (PCR 2: A PRACTICAL APPROACH) (MJ MacPherson, BD Hames and GR Taylor (1995)), Harlow and Lane (1988) Antibodies : Laboratory Manual (ANTIBODIES, A LABORATORY MANUAL), and "ANIMAL CELL" (ANIMAL CELL) CULTURE (edited by RI Freshney (1987)).

In addition, if no specific conditions are indicated in the examples, the conventional conditions or the conditions recommended by the manufacturer shall be used. The reagents or instruments used do not indicate the manufacturer, are all conventional products that are commercially available. Those skilled in the art know that the embodiments describe the present invention by way of example, and are not intended to limit the scope of the claimed invention. All publications and other references mentioned herein are incorporated by reference in their entirety.

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

LB liquid medium: 10g Tryptone, 5g Yeast Extract, 10g NaCl, constant volume to 1L, sterilized. If antibiotics are needed, add 50μg / ml final concentration after the medium is cooled.

Chloroform / Isoamyl alcohol: 240ml of chloroform plus 10ml of isoamyl alcohol, mix well.

RNP buffer: 100 mM sodium chloride, 50 mM Tris-HCl, 10 mM MgCl ₂ , 100 μg / ml BSA, pH 7.9.

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

E. coli competent EC100 was purchased from Epicentre.

Example 1. Obtaining SmCpf1 gene and SmCpf1 guide RNA

1. Annotation of CRISPR and genes: Prodigal was used to annotate the genomic data of the Smithella sp.M82 microbe of NCBI to get all proteins, and Piler-CR was used to annotate the CRISPR seat. The parameters were all default parameters.

2. Obtaining the SmCpf1 protein: align the LbCpf1 sequence to the annotation protein of the Smithella sp. M82 genome, and retain the alignment of Evalue <1e-50 to obtain the SmCpf1 protein sequence. Look for the CRSPR locus within 10 kb of the upstream and downstream of the SmCpf1 locus, thereby obtaining the repetitive sequence and spacer sequence of SmCpf1.

On this basis, the inventors obtained a new Cpf1 effector protein, SmCpf1 (also known as Cas12a), from the genome data of bacteria Smithella sp. M82. The protein sequence is shown in SEQ ID NO: 1, and the coding DNA is As shown in SEQ ID NO: 2. The prototype direct repeat sequence corresponding to SmCpf1 (repeat sequence contained in pre-crRNA) is shown in SEQ ID NO: 3. The mature direct repeat sequence corresponding to SmCpf1 (repeat sequence contained in the mature crRNA) is shown in SEQ ID NO: 5. The sequence alignment between SmCpf1 and other Cpf1 is shown in Figure 1.

Example 2. SmCpf1 gene processing of mature crRNA

1. The double-stranded DNA molecule shown in SEQ ID NO: 2 is artificially synthesized, and the double-stranded DNA molecule shown in SEQ ID NO: 4 is artificially synthesized at the same time.

2. Connect the double-stranded DNA molecule synthesized in step 1 to the prokaryotic expression vector pACYC-Duet-1 to obtain the recombinant plasmid pACYC-Duet-1 + CRISPR / SmCpf1.

The recombinant plasmid pACYC-Duet-1 + CRISPR / SmCpf1 was sequenced. The sequencing results show that the recombinant plasmid pACYC-Duet-1 + CRISPR / SmCpf1 contains the sequences shown in SEQ ID NO: 2 and SEQ ID NO: 4, and expresses the SmCpf1 protein shown in SEQ ID NO: 1 and SEQ ID NO: The prototype of SmCpf1 shown in Figure 3 repeats in the same direction. The recombinant plasmid pACYC-Duet-1 + CRISPR / SmCpf1 was introduced into E. coli EC100 to obtain recombinant bacteria, which was named EC100 / pACYC-Duet-1 + CRISPR / SmCpf1.

3. Take the EC100 / pACYC-Duet-1 + CRISPR / SmCpf1 monoclonal cells, inoculate 100mL of LB liquid medium (containing 50μg / mL ampicillin), and incubate at 37 ° C and 200rpm for 12h with shaking to obtain culture solution.

4. Extraction of bacterial RNA: Transfer 1.5 mL of bacterial culture to a pre-chilled microcentrifuge tube, centrifuge at 6000 × g at 4 ° C for 5 minutes. After centrifugation, the supernatant was discarded, and the cell pellet was resuspended in 200 μL Max Bacterial Enhancement Reagent preheated to 95 ° C, and mixed by blowing and mixing. Incubate at 95 ° C for 4 minutes. Add 1 mL to the lysate

Reagent and mix by pipetting, and incubate at room temperature for 5 minutes. Add 0.2mL of cold chloroform, mix the tube by hand for 15 seconds, and incubate at room temperature for 2-3 minutes. Centrifuge at 12,000 × g for 15 minutes at 4 ° C. Take 600 μL of supernatant in a new tube, add 0.5 mL of cold isopropanol to precipitate RNA, mix by inverting, and incubate at room temperature for 10 minutes. Centrifuge at 15,000 × g for 10 minutes at 4 ° C, discard the supernatant, add 1 mL of 75% ethanol, and vortex to mix. Centrifuge at 7500 × g for 5 minutes at 4 ° C. Discard the supernatant and air dry. The RNA pellet was dissolved in 50 μL RNase-free water and incubated at 60 ° C for 10 minutes.

5. Digestion of DNA: 20ugRNA was dissolved in 39.5μL dH ₂ O, 65 ℃, 5min. On ice for 5min, add 0.5μL RNAI, 5μL buffer, 5μL DNaseI, 37 ℃ 45min (50μL system). Add 50 μL dH ₂ O and adjust the volume to 100 μL. After centrifugation at 26000 Phase-Lock tube 16000g for 30s, add 100μL of phenol: chloroform: isoamyl alcohol (25: 24: 1), 100μL of digested RNA, shake for 15s, 15 ℃, centrifuge at 16000g for 12min. Take the supernatant in a new 1.5mL centrifuge tube, add equal volume of isopropanol 1/10 NaoAC with the supernatant, and react for 1h or -20 ℃ overnight. Centrifuge at 16000g for 30min at 4 ° C and discard the supernatant. Add 350 μL of 75% ethanol to wash the precipitate, centrifuge at 16000g at 4 ° C for 10 min, and discard the supernatant. Air dry, add 20μL RNase-free water, 65 ℃, 5min to dissolve the precipitate. NanoDrop measures the concentration and runs the glue.

6. 3 'Dephosphorylation and 5' Phosphorylation: ~ 20ug of digested RNA, add water to each 42.5μL, 90 ℃ 2min. Cool on ice for 5 minutes. Add 5μL 10 × T4 PNK buffer, 0.5μL RNaI, 2μL T4 PNK (50μL), 37 ℃ for 6h. Add 1μL T4 PNK, 1.25μL (100mM) ATP, 37 ° C for 1h. Add 47.75 μL dH ₂ O and adjust the volume to 100 μL. After centrifugation at 26000 Phase-Lock tube 16000g for 30s, add 100μL of phenol: chloroform: isoamyl alcohol (25: 24: 1), 100μL of digested RNA, shake for 15s, 15 ℃, centrifuge at 16000g for 12min. Take the supernatant in a new 1.5mL centrifuge tube, add equal volume of isopropanol to the supernatant, the total volume is 1/10 NaoAC, react for 1h or -20 ℃ overnight. Centrifuge at 16000g for 30min at 4 ° C and discard the supernatant. Add 350 μL of 75% ethanol to wash the precipitate, centrifuge at 16000g at 4 ° C for 10 min, and discard the supernatant. Air dry, add 21μL RNase-free water, 65 ℃, 5min to dissolve the precipitate, NanoDrop to measure the concentration.

7. RNA monophosphorylation: 20 μL RNA, 90 ° C for 1 min, and cooling on ice for 5 min. Add 2μL RNA 5'Polphosphatase 10 × Reaction buffer, 0.5μL Inhibitor, 1μL RNA 5'Polphosphatase (20Units), add RNase-free water to 20μL, 37 ℃ for 60min. Add 80 μL dH ₂ O and adjust the volume to 100 μL. After centrifuging at 16000g for 2mL Phase-Lock tube for 30s, add 100μL of phenol: chloroform: isoamyl alcohol (25: 24: 1), 100μL of digested RNA, shake for 15s, 15 ℃, centrifuge at 16000g for 12min. Take the supernatant in a new 1.5mL centrifuge tube, add equal volume of isopropanol to the supernatant, the total volume is 1/10 NaoAC, react for 1h or -20 ℃ overnight. Centrifuge at 16000g for 30min at 4 ° C, discard the supernatant, wash the precipitate with 350μL of 75% ethanol, centrifuge at 16000g at 4 ° C for 10min, discard the supernatant. Air dry, add 21μL RNase-free water, 65 ℃, 5min to dissolve the precipitate, NanoDrop to measure the concentration.

8. Preparation of cDNA library: 16.5 μL RNase-free water. 5μL Poly (A) Polymerase 10 × Reaction buffer. 5 μL of 10 mM ATP. 1.5μL RiboGuard RNase Inhibitor. 20 μL RNA Substrate. 2μL Poly (A) Polymerase (4Units). 50 μL total volume. 37 ° C for 20min. Add 50 μL dH ₂ O and adjust the volume to 100 μL. After centrifuging at 16000g for 2mL Phase-Lock tube for 30s, add 100μL of phenol: chloroform: isoamyl alcohol (25: 24: 1), 100μL of digested RNA, shake for 15s, 15 ℃, centrifuge at 16000g for 12min. Take the supernatant in a new 1.5mL centrifuge tube, add equal volume of isopropanol to the supernatant, the total volume is 1/10 NaoAC, react for 1h or -20 ℃ overnight. Centrifuge at 16000g for 30min at 4 ℃, discard the supernatant, dry it, add 11μL of RNase-free water, dissolve the precipitate at 65 ℃ for 5min, measure the concentration by NanoDrop.

9. Send the cDNA library with sequencing adapter to Beijing Berry Hekang for sequencing.

10. Perform quality filtering on the original data to remove sequences with an average base quality value below 30. After removing the linker from the sequence, reserve the RNA sequence from 25 to 50 nt and use bowtie to align it to the reference sequence of the CRISPR array.

11. By comparison, we found that the pre-crRNA of SmCpf1 can be successfully processed into 39-42nt mature crRNA in E. coli, which consists of 19nt Repeat sequence and 20-23nt targeting sequence (Figure 2a).

12. Using ViennaRNA and VARNA for structural prediction and visual analysis of mature crRNA, we found that a 3 base end of the repeat sequence of crRNA can form a 6-base neck ring (Figure 2b).

Example 3. Identification of the PAM domain of the SmCpf1 gene

1. Construct the recombinant plasmid pACYC-Duet-1 + CRISPR / SmCpf1 and sequence. According to the sequencing results, the recombinant plasmid pACYC-Duet-1 + CRISPR / SmCpf1 is structurally described as follows: the small fragment between the recognition sequence of the restriction enzyme PmlI and KpnI of the vector pACYC-Duet-1 is replaced with SEQ ID NO The double-stranded DNA molecule shown in positions 1 to 3753 from the 5 'end in the sequence shown in: 2. The recombinant plasmid pACYC-Duet-1 + CRISPR / SmCpf1 expresses the prototype direct repeat sequence of the SmCpf1 protein shown in SEQ ID NO: 1 and the SmCpf1 shown in SEQ ID NO: 3.

2. The recombinant plasmid pACYC-Duet-1 + CRISPR / SmCpf1 contains the expression cassette, and the nucleotide sequence of the expression cassette is shown in SEQ ID NO: 9. In the sequence shown in SEQ ID NO: 9, positions 1 to 44 from the 5 'end are the nucleotide sequence of the pLacZ promoter, positions 45 to 3797 are the nucleotide sequence of the SmCpf1 gene, positions 3798 to 3862 The nucleotide sequence of the terminator (used to terminate transcription). From the 5 'end, positions 3863 to 3919 are the nucleotide sequence of the J23119 promoter, positions 3920 to 4081 are the nucleotide sequence of the CRISPR array, and positions 4082 to 4108 are the nucleotide sequence of the rrnB-T1 terminator (Used to terminate transcription).

3. Obtaining recombinant E. coli: The recombinant plasmid pACYC-Duet-1 + CRISPR / SmCpf1 was introduced into E. coli EC100 to obtain recombinant E. coli, named EC100 / pACYC-Duet-1 + CRISPR / SmCpf1. The recombinant plasmid pACYC-Duet-1 was introduced into E. coli EC100 to obtain recombinant Agrobacterium, which was named EC100 / pACYC-Duet-1.

4. Construction of PAM library: artificially synthesize the sequence shown in SEQ ID NO: 10 and connect it to pUC19 vector, in which the sequence shown in SEQ ID NO: 10 includes 8 random bases at 5 'end and target sequence. Eight random bases were designed in front of the 5 'end of the target sequence of the PAM library to construct a plasmid library. The plasmids were transferred into E. coli containing the CRISPR / SmCpf1 locus and E. coli not containing the CRISPR / SmCpf1 locus. After processing at 37 ° C for 1 hour, we extracted the plasmid and PCR amplified and sequenced the sequence of the PAM region.

5. Acquisition of PAM library domains: The number of occurrences of 65,536 combinations of PAM sequences in the experimental group and the control group were counted and standardized with the number of all PAM sequences in the respective groups. For any PAM sequence, when log2 (normalized value of control group / normalized value of experimental group) is greater than 3.5, we believe that this PAM is significantly consumed, and we have a total of 4,431 significantly consumed PAM sequences, all of which account for 6.76% . We used Weblogo to predict the significantly consumed PAM sequence and found that the PAM domain of SmCpf1 is a 5'-TTN structure (Figure 3a).

6. In order to verify that the PAM sequence specifically recognized by SmCpf1 is 5'TTN, the SmCpf1 expression vector (SEQ ID NO: 9, expressing SmCpf1 and guide RNA (SEQ ID NO: 11)) and PACYC-Duet1 blank vector are transformed by electric shock Transfer into commercial electrocompetence, select positive clones to prepare electrocompetence containing SmCpf1 expression vector and PACYC-Duet1 blank vector, and then use PAM sequences 5'TTA, 5'TTC, 5'TTT, 5 Plasmids of 'TTA and 5'TAT were subjected to plasmid depletion analysis experiments. In this experiment, PACYC-Duet1 blank vector served as a control. By diluting the spotting plate and counting the number of monoclonals, it can be seen that SmCpf1 shows a significant consumption effect on the plasmid with the PAM sequence of 5'TTN, but not on the 5'TAT plasmid with the PAM sequence of 5'TTN. Out of the plasmid. However, compared with 5'TTA, 5'TTC and 5'TTG, SmCpf1 has a relatively weak effect on the consumption of plasmids with PAM sequence of 5'TTT. This experiment confirmed once again that SmCpf1 specifically recognizes the PAM sequence of 5'TTN (Figure 3b).

Example 4. SmCpf1 cleavage in human cell lines

The eukaryotic expression vector containing the SmCpf1 gene and the PCR product containing the U6 promoter and crRNA (SEQ ID NO: 12) were introduced into human HEK293T cells by liposome transfection, and cultured at 37 ° C under 5% carbon dioxide 72h. Extract the DNA of all cells, and amplify the sequence containing 700bp of the target site, connect the PCR product to the B-simple vector for first-generation sequencing, the sequencing is completed by Thermo Fisher Scientific, and the sequencing results are compared to the VEGFA gene of the human genome On the previous page, it was identified that the editing efficiency of SmCpf1 for VEGFA reached 3.2% (Figure 4a). At the same time, the PCR product was constructed by Tn5 for the second-generation sequencing library. The editing efficiency reached 8.13%. SmCpf1 also identified the DNMT1 gene cleavage (Figure 4b).

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

Claims

A protein having the amino acid sequence shown in SEQ ID NO: 1 or an ortholog, homolog, variant or functional fragment thereof; wherein the ortholog, homolog, The variant or functional fragment basically retains the biological function of the sequence from which it originates;

For example, the ortholog, homolog, or variant has 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 originated;

For example, the orthologues, homologues, and variants have at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence compared to the sequence shown in SEQ ID NO: 1 Identity, and basically retains the biological function of the sequence from which it originates;

For example, the protein is an effector protein in the CRISPR / Cas system;

For example, the protein is derived from Smithella sp. M82.
The protein of claim 1, which comprises or consists of a sequence selected from:

(i) The sequence shown in SEQ ID NO: 1;

(ii) One or more amino acid substitutions, deletions, or additions compared to the sequence shown in SEQ ID NO: 1 (eg 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, deletions, or additions); or

(iii) a sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence shown in SEQ ID NO: 1;

For example, the protein has the amino acid sequence shown in SEQ ID NO: 1.
A conjugate comprising the protein of claim 1 or 2 and a modified portion;

For example, the modified portion is selected from additional proteins or polypeptides, detectable labels, and any combination thereof;

For example, the modified portion is optionally connected to the N-terminus or C-terminus of the protein via a linker;

For example, the modified portion is fused to the N-terminus or C-terminus of the protein;

For example, the additional protein or polypeptide is selected from epitope tags, reporter gene sequences, nuclear localization signal (NLS) sequences, targeting moieties, transcription activation domains (e.g., VP64), transcription repression domains (e.g., KRAB structure Domain or SID domain), nuclease domain (for example, Fok1), a domain with an activity selected from the following: nucleotide deaminase, methylase activity, demethylase, transcription activation activity, Transcription inhibitory activity, transcription 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 contains an epitope tag;

For example, the conjugate contains an NLS sequence;

For example, the NLS sequence is shown in SEQ ID NO: 7;

For example, the NLS sequence is located at, near, or near the terminus of the protein (eg, N-terminus or C-terminus).
A fusion protein comprising the protein of claim 1 or 2 and another protein or polypeptide;

For example, the additional protein or polypeptide is optionally connected to the N-terminus or C-terminus of the protein via a linker;

For example, the additional protein or polypeptide is selected from epitope tags, reporter gene sequences, nuclear localization signal (NLS) sequences, targeting moieties, transcription activation domains (e.g., VP64), transcription repression domains (e.g., KRAB structure Domain or SID domain), nuclease domain (for example, Fok1), a domain with an activity selected from the group consisting of nucleotide deaminase, methylase activity, demethylase, transcription activation activity, Transcription inhibitory activity, transcription 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 contains an epitope tag;

For example, the fusion protein contains NLS sequence;

For example, the NLS sequence is shown in SEQ ID NO: 7;

For example, the NLS sequence is located at, near, or near the end of the protein (eg, N-terminal or C-terminal);

For example, the fusion protein has an amino acid sequence selected from the group consisting of SEQ ID NO: 8.
An isolated nucleic acid molecule comprising or consisting of a sequence selected from:

(i) The sequence shown in SEQ ID NO: 3 or SEQ ID NO: 5;

(ii) One or more base substitutions, deletions, or additions compared to the sequence shown in SEQ ID NO: 3 or SEQ ID NO: 5 (eg 1, 2, 3, 4, 5 , 6, 7, 8, 9 or 10 base substitutions, deletions or additions);

(iv) a sequence having at least 95% sequence identity with the sequence shown in SEQ ID NO: 3 or SEQ ID NO: 5;

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

(vi) the complement of the sequence described in any one of (i)-(iii);

Moreover, the sequence described in any one of (ii)-(v) basically retains the biological function of the sequence from which it originates;

For example, the nucleic acid molecule contains one or more stem loops or optimized secondary structure;

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

For example, the nucleic acid molecule comprises or consists of a sequence selected from the following:

(a) The nucleotide sequence shown in SEQ ID NO: 3 or SEQ ID NO: 5;

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

(c) the complement of the sequence described in (a);

For example, the isolated nucleic acid molecule is RNA;

For example, the isolated nucleic acid molecule is a repeat sequence in the CRISPR / Cas system.
A complex comprising:

(i) a protein component selected from: the protein of any one of claims 1-2, the conjugate of claim 3, the fusion protein of claim 4, and any combination thereof; and

(ii) a nucleic acid component comprising the isolated nucleic acid molecule of claim 5 and a targeting sequence capable of hybridizing to a target sequence from 5 'to 3' direction,

Wherein, the protein component and the nucleic acid component combine to form a complex;

For example, the targeting sequence is linked to the 3 'end of the nucleic acid molecule;

For example, the targeting sequence includes the complementary sequence of the target sequence;

For example, the nucleic acid component is a guide RNA in the CRISPR / Cas system;

For example, the nucleic acid molecule is RNA;

For example, the complex does not contain trans-acting crRNA (tracrRNA).
The composite of claim 6, comprising:

(i) a protein component selected from: the protein of claims 1-2, a conjugate or fusion protein comprising the protein; and

(ii) A nucleic acid component comprising the isolated nucleic acid molecule of claim 5 and the targeting sequence.
An isolated nucleic acid molecule, comprising:

(i) the nucleotide sequence encoding the protein of claims 1-2, or the fusion protein of claim 4;

(ii) a nucleotide sequence encoding the isolated nucleic acid molecule of claim 5; and / or,

(iii) the nucleotide sequence comprising (i) and (ii);

For example, the nucleotide sequence described in any one of (i)-(iii) is codon-optimized for expression in prokaryotic cells or eukaryotic cells.
A vector comprising the isolated nucleic acid molecule of claim 8.
A host cell comprising the isolated nucleic acid molecule of claim 8 or the vector of claim 9.
A composition comprising:

(i) a first component selected from: the protein of claims 1-2, the conjugate of claim 3, the fusion protein of claim 4, the core encoding the protein or the fusion protein Nucleotide sequence, 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;

Wherein, the guide RNA includes a repeat sequence and a guide sequence from the 5 'to 3' direction, and the guide sequence can hybridize with the target sequence;

The guide RNA can form a complex with the protein, conjugate or fusion protein described in (i);

For example, the direct repeat sequence is an isolated nucleic acid molecule as defined in claim 5;

For example, the guide sequence is connected to the 3 'end of the direct repeat sequence;

For example, the targeting sequence includes the complementary sequence of the target sequence;

For example, the composition does not contain trans-acting crRNA (tracrRNA);

For example, the composition is non-naturally occurring or modified;

For example, at least one component in 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.
A composition comprising one or more carriers, the one or more carriers comprising:

(i) a first nucleic acid which is a nucleotide sequence encoding the protein of any one of claims 1-4 or the fusion protein of claim 5; optionally the first nucleic acid is operably linked to The first regulating element; and

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

among them:

The first nucleic acid and the second nucleic acid exist on the same or different carriers;

The guide RNA contains a repeat sequence and a guide sequence from the 5 'to 3' direction, and the guide sequence can hybridize with the target sequence;

The guide RNA can form a complex with the effector protein or fusion protein described in (i);

For example, the direct repeat sequence is an isolated nucleic acid molecule as defined in claim 5;

For example, the guide sequence is connected to the 3 'end of the direct repeat sequence;

For example, the targeting sequence includes the complementary sequence of the target sequence;

For example, the composition does not contain trans-acting crRNA (tracrRNA);

For example, the composition is non-naturally occurring or modified;

For example, at least one component in 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.
The composition of any one of claims 11-12, wherein, when the target sequence is DNA, the target sequence is located at the 3 ′ end of the pro-spacer sequence adjacent to the motif (PAM), and the PAM has 5 The sequence represented by '-TTN, wherein N is selected from A, G, T, and C; when the target sequence is RNA, the target RNA sequence does not have PAM domain restrictions.
The composition of any one of claims 11-13, wherein the target sequence is a DNA or RNA sequence from a prokaryotic or eukaryotic cell; or, the target sequence is a non-naturally occurring DNA or RNA sequence.
The composition of any one of claims 11-14, wherein the target sequence is present in a cell;

For example, the target sequence is present in the nucleus or cytoplasm (eg, organelle);

For example, the cell is a eukaryotic cell;

For example, the cell is a prokaryotic cell.
The composition of any one of claims 11-15, wherein the protein 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 connected to the N-terminus or C-terminus of the protein;

For example, the NLS sequence is fused to the N-terminus or C-terminus of the protein.
A kit comprising one or more components selected from the group consisting of the protein according to any one of claims 1-2, the conjugate according to claim 3, and the fusion protein according to claim 4 2. The isolated nucleic acid molecule according to any one of claims 5, the complex according to any one of claims 6 to 7, the isolated nucleic acid molecule according to claim 8, the vector according to claim 9, the claims The composition according to any one of 11-16;

For example, the kit contains the composition of claim 11 and instructions for using the composition;

For example, the kit contains the composition of claim 12, and instructions for using the composition.
A delivery composition comprising a delivery vehicle and one or more selected from the group consisting of the protein of any one of claims 1-2, the conjugate of claim 3, and the claim 4 Fusion protein, the isolated nucleic acid molecule of any of claims 5, the complex of any of claims 6-7, the isolated nucleic acid molecule of claim 8, the vector of claim 9 2. The composition of any one of claims 11-16;

For example, the delivery vehicle is a particle;

For example, the delivery vector is selected from lipid particles, sugar particles, metal particles, protein particles, liposomes, exosomes, microvesicles, gene guns, or viral vectors (eg, replication-defective retroviruses, lentiviruses, Adenovirus or adeno-associated virus).
A method of modifying a target gene, comprising: contacting the complex of any one of claims 6-7 or the composition of any one of claims 11-16 with the target gene, or delivering to In the cell of the target gene; the target sequence is present in the target gene;

For example, the target gene is present in the cell;

For example, the cell is a prokaryotic cell;

For example, the cell is a eukaryotic cell;

For example, the cells are selected from (eg, mammalian cells, such as human cells), plant cells;

For example, the target gene is present in a nucleic acid molecule (eg, plasmid) in vitro;

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 also includes inserting an exogenous nucleic acid into the break.
A method for changing the expression of a gene product, comprising: combining the complex according to any one of claims 6-7 or the composition according to any one of claims 11-16 with a nucleic acid molecule encoding the gene product Contacting, or delivering to a cell containing the nucleic acid molecule, the target sequence is present in the nucleic acid molecule;

For example, the nucleic acid molecule is present in the cell;

For example, the cell is a prokaryotic cell;

For example, the cell is a eukaryotic cell;

For example, the cell is selected from animal cells (eg, mammalian cells, such as human cells), plant cells;

For example, the nucleic acid molecule is present in a nucleic acid molecule (eg, plasmid) in vitro;

For example, the expression of the gene product is altered (eg, increased or decreased);

For example, the gene product is a protein.
The method of any one of claims 18-20, wherein the protein, conjugate, fusion protein, isolated nucleic acid molecule, complex, carrier, 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) .
The method of any one of claims 18-21, which is used to modify one or more target sequences in a target gene or a nucleic acid molecule encoding a target gene product to modify a cell, cell line, or organism.
A cell or progeny thereof obtained by the method of any one of claims 18-22, wherein the cell contains a modification that is not present in its wild type.
The cell product of the cell of claim 23 or a progeny thereof.
An in vitro, ex vivo or in vivo cell or cell line or progeny thereof, said cell or cell line or progeny thereof comprising: the protein of any one of claims 1-2, claim 3 The conjugate, the fusion protein of claim 4, the isolated nucleic acid molecule of claim 5, the complex of any one of claims 6-7, the isolated nucleic acid of claim 8 Molecule, carrier according to claim 9, composition according to any one of claims 11-16;

For example, the cell is a eukaryotic cell;

For example, the cell is an animal cell (eg, a mammalian cell, such as a human cell) or a plant cell;

For example, the cells are stem cells or stem cell lines.
The protein of any one of claims 1-2, the conjugate of claim 3, the fusion protein of claim 4, the isolated nucleic acid molecule of any one of claims 5, claim 6 The complex of any one of 7, the isolated nucleic acid molecule of claim 8, the vector of claim 9, the composition of any of claims 11-16, or the reagent of claim 18 Cassettes for nucleic acid editing (eg, gene or genome editing);

For example, the gene or genome editing includes modifying genes, knocking out genes, changing the expression of gene products, repairing mutations, and / or inserting polynucleotides.
The protein of any one of claims 1-2, the conjugate of claim 3, the fusion protein of claim 4, the isolated nucleic acid molecule of any one of claims 5, claim 6 The complex of any of 7, the isolated nucleic acid molecule of claim 8, the vector of claim 9, the composition of any of claims 11-16, or the reagent of claim 32 Box, use in the preparation of a formulation, the formulation is used for:

(i) In vitro gene or genome editing;

(ii) Detection of isolated single-stranded DNA;

(iii) Edit target sequences in target loci to modify biological or non-human organisms;

(iv) Treatment of disorders caused by defects in target sequences in target loci.