WO2019201331A1 - Protéine effectrice crispr/cas et système - Google Patents

Protéine effectrice crispr/cas et système Download PDF

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WO2019201331A1
WO2019201331A1 PCT/CN2019/083418 CN2019083418W WO2019201331A1 WO 2019201331 A1 WO2019201331 A1 WO 2019201331A1 CN 2019083418 W CN2019083418 W CN 2019083418W WO 2019201331 A1 WO2019201331 A1 WO 2019201331A1
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sequence
protein
nucleic acid
acid molecule
cell
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PCT/CN2019/083418
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English (en)
Chinese (zh)
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赖锦盛
周英思
朱金洁
张湘博
赵海铭
宋伟彬
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中国农业大学
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Priority to CN201980027152.1A priority Critical patent/CN112004932B/zh
Publication of WO2019201331A1 publication Critical patent/WO2019201331A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/90—Stable introduction of foreign DNA into chromosome

Definitions

  • the invention relates to the field of nucleic acid editing, in particular to the field of regularly clustered short palindrome repetition (CRISPR) technology.
  • CRISPR regularly clustered short palindrome repetition
  • the invention relates to Cas effector proteins, fusion proteins comprising such proteins, and nucleic acid molecules encoding the same.
  • the invention also relates to complexes and compositions for nucleic acid editing (e.g., gene or genome editing) comprising a protein or fusion protein of the invention, or a nucleic acid molecule encoding the same.
  • the invention also relates to methods for nucleic acid editing (eg, gene or genome editing) using a protein or fusion protein comprising the invention.
  • CRISPR/Cas technology is a widely used gene editing technology that specifically binds to target sequences on the genome by RNA guidance and cleaves DNA to generate double-strand breaks, using biological non-homologous end joining or homologous recombination. Gene editing.
  • the CRISPR/Cas9 system is the most commonly used type II CRISPR system, which recognizes the PAM motif of 3'-NGG and blunt-ends the target sequence.
  • the CRISPR/Cas Type V system is a newly discovered CRISPR system in the past two years. It has a 5'-TTN motif for viscous end cleavage of target sequences, such as Cpf1, C2c1, CasX, CasY.
  • the different CRISPR/Cas currently exist have different advantages and disadvantages.
  • Cas9, C2c1 and CasX require two RNAs for guide RNA, while Cpf1 requires only one guide RNA and can be used for multiple gene editing.
  • CasX is 980 amino acids in size, while common Cas9, C2c1, CasY and Cpf1 are usually around 1300 amino acids.
  • PAM sequences of Cas9, Cpf1, CasX, and CasY are more complex and diverse, while C2c1 recognizes the rigorous 5'-TTN, so its target site is easier to predict than other systems and thus reduces potential off-target effects.
  • the inventors of the present application have unexpectedly discovered a novel RNA-directed endonuclease after extensive experimentation and repeated exploration. Based on this finding, the inventors developed a new CRISPR/Cas system and a gene editing method based on the system.
  • the invention provides a protein having the amino acid sequence set forth in any one of SEQ ID NOs: 1, 2, 3 or an ortholog, homologue, variant or functionality thereof a fragment; wherein the ortholog, homologue, variant or functional fragment substantially retains the biological function of the sequence from which it is derived.
  • the biological functions of the above sequences include, but are not limited to, activity binding to a targeting RNA, endonuclease activity, binding to a specific site of a target sequence under the guidance of a targeting RNA, and cleavage.
  • the ortholog, homolog, variant has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least compared to the sequence from which it is derived. 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
  • the ortholog, homolog, variant has at least 80%, at least 85%, at least 90 compared to the sequence set forth in any one of SEQ ID NOs: 1, 2, 3. %, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity, and substantially retains its source
  • the biological function of the sequence eg, activity bound to the targeting RNA, endonuclease activity, activity that binds to a specific site of the target sequence and cleaves under the guidance of a targeting RNA).
  • the protein is an effector protein in the CRISPR/Cas system.
  • the protein of the invention comprises a sequence selected from the group consisting of: or consists of a sequence selected from the group consisting of:
  • the protein of the invention comprises a sequence selected from the group consisting of: or consists of a sequence selected from the group consisting of:
  • the protein of the invention has the amino acid sequence set forth in SEQ ID NO:1.
  • the protein of the invention comprises a sequence selected from the group consisting of: or consists of a sequence selected from the group consisting of:
  • the protein of the invention has the amino acid sequence set forth in SEQ ID NO:2.
  • the protein of the invention comprises a sequence selected from the group consisting of: or consists of a sequence selected from the group consisting of:
  • the protein of the invention has the amino acid sequence set forth in SEQ ID NO:3.
  • a protein of the invention can be derivatized, for example, linked to another molecule (e.g., another polypeptide or protein).
  • derivatization eg, labeling
  • the proteins of the invention are also intended to include such derivatized forms.
  • a protein of the invention can be functionally linked (by chemical coupling, gene fusion, non-covalent attachment or otherwise) to one or more other molecular groups, such as another protein or polypeptide, a detection reagent, a pharmaceutical reagent Wait.
  • the proteins of the invention may be linked to other functional units.
  • it can be ligated to a nuclear localization signal (NLS) sequence to increase the ability of the proteins of the invention to enter the nucleus.
  • NLS nuclear localization signal
  • it can be linked to a targeting moiety to render the protein of the invention targeted.
  • it can be linked to a detectable label to facilitate detection of the proteins of the invention.
  • it can be linked to an epitope tag to facilitate expression, detection, tracing, and/or purification of the proteins of the invention.
  • the invention provides a conjugate comprising a protein and a modified moiety as described above.
  • the modified moiety is selected from another protein or polypeptide, a detectable label, or any combination thereof.
  • a nuclease domain eg, Fok1
  • a nuclease domain having a domain selected from the group consisting of methylase activity, demethylase, transcriptional activation activity, transcriptional repression Activity, transcription release factor activity, histone modification activity, nuclease activity, single-strand RNA cleavage activity, double-stranded RNA cleavage activity, single-strand DNA cleavage activity, double-strand DNA cleavage activity and nucleic acid binding activity; and any combination thereof.
  • a conjugate of the invention comprises one or more NLS sequences, such as the NLS of the SV40 viral large T antigen.
  • the NLS sequence is set forth in SEQ ID NO:19.
  • the NLS sequence is at, near, or near the end of the protein of the invention (eg, the N-terminus or the C-terminus).
  • the NLS sequence is located at, near, or near the C-terminus of the protein of the invention.
  • the conjugates of the invention comprise an epitope tag.
  • epitope tags are well known to those skilled in the art, examples of which include, but are not limited to, His, V5, FLAG, HA, Myc, VSV-G, Trx, etc., and those skilled in the art know how to achieve the desired purpose (eg, Purify, test or trace) Select the appropriate epitope tag.
  • a conjugate of the invention comprises a reporter gene sequence.
  • reporter genes are well known to those skilled in the art, and examples include, but are not limited to, GST, HRP, CAT, GFP, HcRed, DsRed, CFP, YFP, BFP, and the like.
  • the conjugates of the invention comprise a domain capable of binding to a DNA molecule or an intracellular molecule, such as a maltose binding protein (MBP), a DNA binding domain of Lex A (DBD), a DBD of GAL4, and the like.
  • MBP maltose binding protein
  • DBD DNA binding domain of Lex A
  • GAL4 GAL4
  • the conjugates of the invention comprise a detectable label, such as a fluorescent dye, such as FITC or DAPI.
  • a protein of the invention is optionally coupled, conjugated or fused to the modified moiety by a linker.
  • the modified moiety is directly linked to the N-terminus or C-terminus of the protein of the invention.
  • the modified moiety is linked to the N-terminus or C-terminus of the protein of the invention by a linker.
  • linkers are well known in the art, examples of which include, but are not limited to, one or more (eg, 1, 2, 3, 4 or 5) amino acids (eg, Glu or Ser) or amino acid derivatives.
  • a linker eg, Ahx, ⁇ -Ala, GABA, or Ava), or PEG, and the like.
  • the invention provides a fusion protein comprising a protein of the invention and an additional protein or polypeptide.
  • a nuclease domain eg, Fok1
  • a nuclease domain having a domain selected from the group consisting of methylase activity, demethylase, transcriptional activation activity, transcriptional repression Activity, transcription release factor activity, histone modification activity, nuclease activity, single-strand RNA cleavage activity, double-stranded RNA cleavage activity, single-strand DNA cleavage activity, double-strand DNA cleavage activity and nucleic acid binding activity; and any combination thereof.
  • a fusion protein of the invention comprises one or more NLS sequences, such as the NLS of the SV40 viral large T antigen.
  • the NLS sequence is at, near, or near the end of the protein of the invention (eg, the N-terminus or the C-terminus). In certain exemplary embodiments, the NLS sequence is located at, near, or near the C-terminus of the protein of the invention.
  • a fusion protein of the invention comprises an epitope tag.
  • a fusion protein of the invention comprises a reporter gene sequence.
  • a fusion protein of the invention comprises a domain capable of binding to a DNA molecule or an intracellular molecule.
  • a protein of the invention is optionally fused to the additional protein or polypeptide via a linker.
  • the additional protein or polypeptide is directly linked to the N-terminus or C-terminus of the protein of the invention.
  • the additional protein or polypeptide is linked to the N-terminus or C-terminus of the protein of the invention by a linker.
  • the fusion proteins of the invention have an amino acid sequence selected from the group consisting of SEQ ID NOs: 20-22.
  • the protein of the present invention, the conjugate of the present invention or the fusion protein of the present invention is not limited by the manner in which it is produced, for example, it can be produced by a genetic engineering method (recombination technique) or can be produced by a chemical synthesis method.
  • the invention provides an isolated nucleic acid molecule comprising a sequence selected from the group consisting of: or consisting of:
  • sequence of any one of (ii)-(v) substantially retains the biological function of the sequence from which it is derived, the biological function of the sequence being referred to as the same direction in the CRISPR-Cas system Repeat the activity of the sequence.
  • the isolated nucleic acid molecule is a direct repeat in a CRISPR-Cas system.
  • the nucleic acid molecule comprises a sequence selected from the group consisting of: or consists of a sequence selected from the group consisting of:
  • the isolated nucleic acid molecule is RNA.
  • the isolated nucleic acid molecule comprises a sequence selected from the group consisting of: or consists of:
  • the isolated nucleic acid molecule comprises a sequence selected from the group consisting of: or consists of:
  • the isolated nucleic acid molecule comprises a sequence selected from the group consisting of: or consists of:
  • the isolated nucleic acid molecule comprises a sequence selected from the group consisting of: or consists of:
  • the isolated nucleic acid molecule comprises a sequence selected from the group consisting of: or consists of:
  • the isolated nucleic acid molecule comprises a sequence selected from the group consisting of: or consists of:
  • the invention provides a composite comprising:
  • a protein component selected from the group consisting of a protein, conjugate or fusion protein of the invention, and any combination thereof;
  • 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 to the target sequence
  • the targeting sequence is linked to the 3' end of the nucleic acid molecule.
  • the targeting sequence comprises the complement of the target sequence.
  • the nucleic acid component is a targeting RNA in a CRISPR-Cas system.
  • the nucleic acid molecule is RNA.
  • the complex does not comprise a trans-acting crRNA (tracrRNA).
  • the targeting sequence is at least 5, at least 10 in length, and in certain embodiments, the targeting sequence is 10-30, or 15-25 in length, or 15-22, or 19-25 or 19-22 nucleotides.
  • 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.
  • the invention provides an isolated nucleic acid molecule comprising:
  • nucleotide sequence set forth in any of (i)-(iii) is codon optimized for expression in a prokaryotic cell. In certain embodiments, the nucleotide sequence set forth in any of (i)-(iii) is codon optimized for expression in eukaryotic cells.
  • the invention provides a vector comprising the isolated nucleic acid molecule of the sixth aspect.
  • the vector of the present invention may be a cloning vector or an expression vector.
  • vectors of the invention are, for example, plasmids, cosmids, phage, cosmid, and the like.
  • the vector is capable of expressing a protein of the invention, a fusion protein, an isolated nucleic acid molecule of the fourth aspect, or a fifth aspect, in a subject (eg, a mammal, eg, a human) Said complex.
  • the invention also provides a host cell comprising an isolated nucleic acid molecule or vector as described above.
  • 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 (eg, mammalian cells, such as mouse cells, human cells, etc.).
  • the cells of the invention may also be cell lines, such as 293T cells.
  • compositions and carrier composition are Composition and carrier composition
  • the present invention also provides a composition comprising:
  • a first component selected from the group consisting of a protein, a conjugate, a fusion protein, a nucleotide sequence encoding the protein or fusion protein, and any combination thereof;
  • a second component which is a nucleotide sequence comprising a targeting RNA or a nucleotide sequence encoding the nucleotide sequence comprising the targeting RNA;
  • the targeting RNA comprises a homologous repeat sequence and a targeting sequence from the 5' to 3' direction, the targeting sequence being capable of hybridizing to the target sequence;
  • the targeting RNA is capable of forming a complex with the protein, conjugate or fusion protein described in (i).
  • the isotropic repeat is an isolated nucleic acid molecule as defined in the fourth aspect.
  • the targeting sequence is ligated to the 3' end of the isotropic repeat. In certain embodiments, the targeting sequence comprises the complement of the target sequence.
  • the composition does not comprise tracrRNA.
  • the composition is non-naturally occurring or modified. In certain embodiments, at least one component of the composition is non-naturally occurring or modified. In certain embodiments, the first component is non-naturally occurring or modified; and/or the second component is non-naturally occurring or modified.
  • the target sequence when the target sequence is DNA, the target sequence is located at the 3' end of the proximate spacer adjacent motif (PAM), and the PAM has a sequence indicated by 5'-TTN, wherein , N is selected from A, G, T, and C.
  • PAM proximate spacer adjacent motif
  • the target sequence when the target sequence is RNA, the target sequence does not have a PAM domain restriction.
  • the target sequence is a DNA or RNA sequence derived from a prokaryotic or eukaryotic cell. In certain embodiments, the target sequence is a non-naturally occurring DNA or RNA sequence.
  • the target sequence is present in a cell. In certain embodiments, the target sequence is present within the nucleus or within the cytoplasm (eg, an organelle). In certain embodiments, the cell is a prokaryotic cell. In certain embodiments, the cell is a eukaryotic cell.
  • the protein is linked to one or more NLS sequences.
  • the conjugate or fusion protein comprises one or more NLS sequences.
  • the NLS sequence is linked to the N-terminus or C-terminus of the protein.
  • the NLS sequence is fused to the N-terminus or C-terminus of the protein.
  • the present invention also provides a composition comprising one or more carriers, the one or more carriers comprising:
  • a first nucleic acid which is a nucleotide sequence encoding a protein or fusion protein of the invention; optionally the first nucleic acid is operably linked to a first regulatory element;
  • a second nucleic acid encoding a nucleotide sequence comprising a targeting RNA; optionally the second nucleic acid is operably linked to a second regulatory element;
  • the first nucleic acid and the second nucleic acid are present on the same or different carrier;
  • the targeting RNA comprises a homologous repeat sequence and a targeting sequence from the 5' to 3' direction, the targeting sequence being capable of hybridizing to the target sequence;
  • the targeting RNA is capable of forming a complex with the effector protein or fusion protein described in (i).
  • the isotropic repeat is an isolated nucleic acid molecule as defined in the fourth aspect.
  • the targeting sequence is ligated to the 3' end of the isotropic repeat. In certain embodiments, the targeting sequence comprises the complement of the target sequence.
  • the composition does not comprise tracrRNA.
  • the composition is non-naturally occurring or modified. In certain embodiments, at least one component of the composition is non-naturally occurring or modified.
  • the first regulatory element is a promoter, such as an inducible promoter.
  • the second regulatory element is a promoter, such as an inducible promoter.
  • the target sequence when the target sequence is DNA, the target sequence is located at the 3' end of the proximate spacer adjacent motif (PAM), and the PAM has a sequence indicated by 5'-TTN, wherein , N is selected from A, G, T, and C.
  • PAM proximate spacer adjacent motif
  • the target sequence when the target sequence is RNA, the target sequence does not have a PAM domain restriction.
  • the target sequence is a DNA or RNA sequence derived from a prokaryotic or eukaryotic cell. In certain embodiments, the target sequence is a non-naturally occurring DNA or RNA sequence.
  • the target sequence is present in a cell. In certain embodiments, the target sequence is present within the nucleus or within the cytoplasm (eg, an organelle). In certain embodiments, the cell is a prokaryotic cell. In certain embodiments, the cell is a eukaryotic cell.
  • the protein is linked to one or more NLS sequences.
  • the conjugate or fusion protein comprises one or more NLS sequences.
  • the NLS sequence is ligated to the N-terminus or C-terminus of the protein.
  • the NLS sequence is fused to the N-terminus or C-terminus of the protein.
  • one type of vector is a plasmid, which refers to a circular double stranded DNA loop in which additional DNA fragments can be inserted, for example, by standard molecular cloning techniques.
  • a viral vector in which a virus-derived DNA or RNA sequence is present for packaging a virus (eg, retrovirus, replication-defective retrovirus, adenovirus, replication-defective adenovirus, and adeno-associated In the vector of the virus).
  • the viral vector also comprises a polynucleotide carried by a virus for transfection into a host cell.
  • vectors e.g., bacterial vectors having bacterial origins of replication and episomal mammalian vectors
  • Other vectors e.g., non-episomal mammalian vectors
  • certain vectors are capable of directing the expression of genes to which they are operably linked.
  • Such vectors are referred to herein as "expression vectors.”
  • Common expression vectors used in recombinant DNA techniques are typically in the form of plasmids.
  • the recombinant expression vector can comprise a nucleic acid molecule of the invention in a form suitable for expression of the nucleic acid in a host cell, which means that the recombinant expression vector comprises 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.
  • compositions of the ninth and tenth aspects can be delivered by any method known in the art.
  • Such methods include, but are not limited to, electroporation, lipofection, nuclear transfection, microinjection, sonoporation, gene gun, calcium phosphate mediated transfection, cation transfection, lipofection, 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 of body, etc.
  • the present invention provides a delivery composition
  • a delivery composition comprising a delivery vehicle, and one or more selected from the group consisting of a protein, a conjugate, a fusion protein of the invention, as in the fourth aspect
  • the delivery vehicle is a particle.
  • X. Virus lentivirus, adenovirus or adeno-associated virus.
  • the invention provides a kit comprising one or more of the components described above.
  • the kit comprises one or more components selected from the group consisting of a protein, a conjugate, a fusion protein of the invention, an isolated nucleic acid molecule of the fourth aspect, the invention The complex, the isolated nucleic acid molecule of the sixth aspect, the carrier of the seventh aspect, the composition of the ninth and tenth aspects.
  • the kit of the invention comprises the composition of the ninth aspect. In certain embodiments, the kit further comprises instructions for using the composition.
  • the kit of the invention comprises the composition of the tenth aspect. In certain embodiments, the kit further comprises instructions for using the composition.
  • kits of the invention can be provided in any suitable container.
  • the kit further comprises one or more buffers.
  • the buffer can be any buffer including, but not limited to, sodium carbonate buffer, sodium bicarbonate buffer, borate buffer, Tris buffer, MOPS buffer, HEPES buffer, and combinations thereof.
  • the buffer is basic.
  • the buffer has a pH of from about 7 to about 10.
  • the kit further comprises one or more oligonucleotides corresponding to a targeting sequence for insertion into a vector for operably linking the guide Sequence and adjustment elements.
  • the kit comprises a homologous recombination template polynucleotide.
  • the invention provides a method of modifying a target gene, comprising: the complex of the fifth aspect, the composition of the ninth aspect, or the composition of the tenth aspect Contacting the target gene or delivering to a cell comprising the target gene; the target sequence is present in the target gene.
  • the methods are used to modify a target gene in vitro or ex vivo.
  • the method is not a method of treating a human or animal by therapy.
  • the method does not include the step of modifying the genetic characteristics of the human germline.
  • the target gene is present in a cell.
  • the cell is a prokaryotic cell.
  • the cell is a eukaryotic cell.
  • the cell is a mammalian cell.
  • the cell is a human cell.
  • the cell is selected from a non-human primate, bovine, porcine or rodent cell.
  • the cell is a non-mammalian eukaryotic cell, such as a poultry or fish.
  • the cell is a plant cell, such as a cell of a cultivated plant (such as cassava, corn, sorghum, wheat, or rice), algae, tree, or vegetable.
  • the target gene is present in a nucleic acid molecule (eg, a plasmid) in vitro. In certain embodiments, the target gene is present in a plasmid.
  • a nucleic acid molecule eg, a plasmid
  • the modification refers to cleavage of the target sequence, such as double-strand breaks in DNA or single-strand breaks in RNA.
  • the cleavage results in a decrease in transcription of the target gene.
  • the method further comprises contacting the editing template with the target gene or delivering to a cell comprising the target gene.
  • the method repairs the disrupted target gene by homologous recombination with an exogenous template polynucleotide, wherein the repair results in a mutation comprising one or more nucleosides of the target gene Insertion, deletion, or substitution of an acid.
  • the mutation results in a change in one or more amino acids in a protein expressed from a gene comprising the target sequence.
  • the modification further comprises inserting an editing template (eg, an exogenous nucleic acid) into the fragmentation.
  • an editing template eg, an exogenous nucleic acid
  • the protein, conjugate, fusion protein, isolated nucleic acid molecule, complex, vector or composition is included in a delivery vehicle.
  • the delivery vehicle is selected from the group consisting of a lipid particle, a sugar particle, a metal particle, a protein particle, a liposome, an exosome, a viral vector (eg, a replication defective retrovirus, a lentivirus, an adenovirus) Or adeno-associated virus).
  • a viral vector eg, a replication defective retrovirus, a lentivirus, an adenovirus
  • the methods are used to modify a target gene or one or more target sequences in a nucleic acid molecule encoding a target gene product to modify a cell, cell line or organism.
  • the invention provides a method of altering the expression of a gene product, comprising: the complex of the fifth aspect, the composition of the ninth aspect, or the The composition is contacted with a nucleic acid molecule encoding the gene product or delivered to a cell comprising the nucleic acid molecule, the target sequence being present in the nucleic acid molecule.
  • the methods are used to alter expression of a gene product in vitro or ex vivo.
  • the method is not a method of treating a human or animal by therapy.
  • the method does not include the step of modifying the genetic characteristics of the human germline.
  • the nucleic acid molecule is present in a cell.
  • the cell is a prokaryotic cell.
  • the cell is a eukaryotic cell.
  • the cell is a mammalian cell.
  • the cell is a human cell.
  • the cell is selected from a non-human primate, bovine, porcine or rodent cell.
  • the cell is a non-mammalian eukaryotic cell, such as a poultry or fish.
  • the cell is a plant cell, such as a cell of a cultivated plant (such as cassava, corn, sorghum, wheat, or rice), algae, tree, or vegetable.
  • the nucleic acid molecule is present in a nucleic acid molecule (eg, a plasmid) in vitro. In certain embodiments, the nucleic acid molecule is present in a plasmid.
  • 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.
  • the gene product is a protein.
  • the protein, conjugate, fusion protein, isolated nucleic acid molecule, complex, vector or composition is included in a delivery vehicle.
  • the delivery vehicle is selected from the group consisting of a lipid particle, a sugar particle, a metal particle, a protein particle, a liposome, an exosome, a viral vector (eg, a replication defective retrovirus, a lentivirus, an adenovirus) Or adeno-associated virus).
  • a viral vector eg, a replication defective retrovirus, a lentivirus, an adenovirus
  • the methods are used to modify a target gene or one or more target sequences in a nucleic acid molecule encoding a target gene product to modify a cell, cell line or organism.
  • the invention relates to the protein of the first aspect, the conjugate of the second aspect, the fusion protein of the third aspect, the isolated nucleic acid molecule of the fourth aspect, The complex of claim 5, the isolated nucleic acid molecule of the sixth aspect, the carrier of the seventh aspect, the composition of the ninth aspect, the composition of the tenth aspect
  • a kit or delivery composition of the invention for use in nucleic acid editing eg, in vitro or ex vivo nucleic acid editing
  • nucleic acid editing eg, in vitro or ex vivo nucleic acid editing
  • the nucleic acid to be edited is present within the cell.
  • the cell is a prokaryotic cell or a eukaryotic cell.
  • the nucleic acid to be edited is present in a nucleic acid molecule (eg, a plasmid) in vitro.
  • the nucleic acid editing comprises genetic or genomic editing, such as modifying a gene, knocking out a gene, altering the expression of a gene product, repairing a mutation, and/or inserting a polynucleotide.
  • the gene or genome editing does not include the step of modifying the genetic characteristics of the human germline.
  • the use is not a method of treating a human or animal by therapy.
  • the use further comprises repairing the edited target sequence by homologous recombination with an exogenous template polynucleotide, wherein the repair can produce a mutation in the target sequence, including one or more nuclei Insertion, deletion or substitution of a nucleotide.
  • the invention relates to the protein of the first aspect, the conjugate of the second aspect, the fusion protein of the third aspect, the isolated nucleic acid molecule of the fourth aspect, The complex of claim 5, the isolated nucleic acid molecule of the sixth aspect, the carrier of the seventh aspect, the composition of the ninth aspect, the composition of the tenth aspect
  • a kit or delivery composition of the invention in the preparation of a formulation for: (i) in vitro or ex vivo DNA detection; (ii) editing of a target sequence in a target locus to modify a biological or non- Human organisms (eg, prokaryotes).
  • the formulation is for the detection of single-stranded DNA or double-stranded DNA (eg, detection of single-stranded or double-stranded DNA in prokaryotic cells).
  • the DNA assay is for detecting a tumor, virus or bacterium.
  • target DNA eg, tumor-specific label, virus or bacterial-specific label
  • the detection of viruses or bacteria can be achieved by detecting single-stranded DNA and detecting that the single-stranded DNA is non-specifically cleaved.
  • the invention also relates to a method of detecting target DNA in a sample comprising the steps of:
  • composition according to the fifth aspect the composition according to the ninth aspect or the composition according to the tenth aspect, and the single chain having the label DNA; among them,
  • the targeting sequence contained in the complex or composition is capable of hybridizing to a target DNA
  • the single-stranded DNA does not hybridize to the targeting sequence
  • the target DNA is viral DNA or bacterial DNA.
  • the target DNA is tumor cell DNA.
  • the target DNA is single stranded or double stranded.
  • the detectable signal is determined by one or more methods selected from the group consisting of: imaging based detection, sensor based detection, color detection, gold nanoparticle based detection, fluorescence polarization, colloidal phase Variable/dispersion, electrochemical detection and semiconductor-based sensing.
  • the method further comprises the step of amplifying the target DNA in the sample.
  • modifications introduced to cells by the methods of the invention can cause the cells and their progeny to be altered to improve the production of their biological products, such as antibodies, starch, ethanol, or other desired cellular output.
  • modifications introduced into the cell by the methods of the invention can cause the cell and its progeny to include changes that result in a change in the produced biological product.
  • the invention relates to a cell or a progeny thereof obtained by the method as described above, wherein the cell contains a modification that is not found in its wild type.
  • the invention also relates to a cell product of a cell or a progeny thereof as described above.
  • the invention also relates to an in vitro, ex vivo or in vivo cell or cell line or a progeny thereof, the cell or cell line or a progeny thereof comprising: the protein of the first aspect, such as the second The conjugate of the aspect, the fusion protein of the third aspect, the isolated nucleic acid molecule of the fourth aspect, the complex of the fifth aspect, the isolated nucleic acid of the sixth aspect A molecule, a carrier according to the seventh aspect, a composition according to the ninth aspect, a composition according to the tenth aspect, a kit of the invention or a delivery composition.
  • the cell is a prokaryotic cell.
  • the cell is a eukaryotic cell. In certain embodiments, the cell is a mammalian cell. In certain embodiments, the cell is a human cell. In certain embodiments, the cell is a non-human mammalian cell, such as a cell of a non-human primate, cow, sheep, pig, dog, monkey, rabbit, rodent (eg, rat or mouse). In certain embodiments, the cell is a non-mammalian eukaryotic cell, such as a poultry bird (eg, chicken), a fish or a crustacean (eg, scorpion, shrimp) cells.
  • a poultry bird eg, chicken
  • fish or a crustacean eg, scorpion, shrimp
  • the cell is a plant cell, such as a cell possessed by a monocot or a dicot or a cultivated plant or a cell of a food crop such as cassava, corn, sorghum, soybean, wheat, oat or rice, for example Algae, tree or production of plants, fruits or vegetables (for example, trees such as citrus, nut trees; nightshade, cotton, tobacco, tomatoes, grapes, coffee, cocoa, etc.).
  • a plant cell such as a cell possessed by a monocot or a dicot or a cultivated plant or a cell of a food crop such as cassava, corn, sorghum, soybean, wheat, oat or rice, for example Algae, tree or production of plants, fruits or vegetables (for example, trees such as citrus, nut trees; nightshade, cotton, tobacco, tomatoes, grapes, coffee, cocoa, etc.).
  • the cell is a stem cell or stem cell line.
  • Cas12i refers to a Cas effector protein first discovered and identified by the present inventors having an amino acid sequence selected from the group consisting of:
  • the Cas12i of the present invention is an endonuclease which binds to a specific site of a target sequence and cleaves under the guidance of a guide RNA, and has both DNA and RNA endonuclease activity.
  • CRISPR complex regional short palindrome repeat
  • Cas CRISPR-CRISPR-related
  • CRISPR system CRISPR system
  • Such transcription products or other elements may comprise a sequence encoding a Cas effector protein and a targeting RNA comprising CRISPR RNA (crRNA), and a trans-acting crRNA (tracrRNA) sequence contained in the CRISPR-Cas9 system, or from a CRISPR locus Other sequences or transcripts.
  • crRNA CRISPR RNA
  • tracrRNA trans-acting crRNA
  • Cas effector protein As used herein, the terms “Cas effector protein”, “Cas effector enzyme” are used interchangeably and refer to any of the proteins presented in the CRISPR-Cas system that are greater than 900 amino acids in length. In some cases, such proteins refer to proteins identified from the Cas locus.
  • the terms “guide RNA”, “mature crRNA” are used interchangeably and have the meaning as commonly understood by one of ordinary skill in the art.
  • the targeting RNA may comprise a direct repeat sequence and a guide sequence, or consist essentially of or consist of a homologous repeat sequence and a guide sequence (also referred to as a spacer sequence in the context of an endogenous CRISPR system). (spacer)) composition.
  • the targeting sequence is any polynucleotide sequence that is sufficiently complementary to the target sequence to hybridize to the target sequence and direct the specific binding of the CRISPR/Cas complex to the target sequence.
  • 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%. Determining the optimal alignment is within the abilities of one of ordinary skill in the art. For example, there are publicly available and commercially available alignment algorithms and programs such as, but not limited to, ClustalW, Smith-Waterman in Matlab, Bowtie, Geneious, Biopython, and SeqMan.
  • 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 in length, 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.
  • the guide sequence is no more than 50, 45, 40, 35, 30, 25, 24, 23, 22, 21, 20, 15 in length. , 10 or fewer nucleotides.
  • the targeting sequence is 10-30, or 15-25, or 15-22, or 19-25 or 19-22 nucleotides in length.
  • the isotropic repeats are 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 .
  • the same direction repeat sequence is no more than 70, 65, 64, 63, 62, 61, 60, 59, 58, 57, 56 in length.
  • the isotropic repeat 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 isotropic repeat is 15-30 nucleotides in length, such as 15-25 nucleotides, such as 20-25 nucleotides, such as 22-24 nucleosides. Acid, for example 2 3 nucleotides.
  • CRISPR/Cas complex refers to a ribonucleoprotein complex formed by the binding of a guide RNA or a mature crRNA to a Cas protein, which comprises hybridization to a target sequence and with Cas Protein-directed targeting sequences.
  • the ribonucleoprotein complex is capable of recognizing and cleaving a polynucleotide that hybridizes to the targeting RNA or mature crRNA.
  • a target sequence refers to a polynucleotide that is designed to be targeted by a targeting sequence, such as a sequence that is complementary to the targeting sequence, wherein the target Hybridization between the sequence and the targeting sequence will promote the formation of the CRISPR/Cas complex. Complete complementarity is not required as long as sufficient complementarity exists to cause hybridization and promote the formation of a CRISPR/Cas complex.
  • the target sequence can comprise any polynucleotide, such as DNA or RNA. In some cases, the target sequence is located in the nucleus or cytoplasm of the cell.
  • the target sequence can be located in an organelle of a eukaryotic cell, such as a mitochondria or chloroplast. Sequences or templates that can be used for recombination into a target locus comprising the target sequence are referred to as "editing templates” or “editing polynucleotides” or “editing sequences.”
  • the editing template is an exogenous nucleic acid.
  • the recombination is homologous recombination.
  • the expression "target sequence” or “target polynucleotide” may be any endogenous or exogenous polynucleotide to a cell (eg, a eukaryotic cell).
  • the target polynucleotide can be a polynucleotide present in the nucleus of a eukaryotic cell.
  • the target polynucleotide can be a sequence encoding a gene product (eg, a protein) or a non-coding sequence (eg, a regulatory polynucleotide or unwanted DNA). In some cases, it is believed that the target sequence should be associated with the original spacer sequence adjacent motif (PAM).
  • PAM spacer sequence adjacent motif
  • PAM protein acetylase
  • sequence and length requirements for PAM will vary depending on the Cas effector enzyme used, but PAM is typically a 2-5 base pair sequence adjacent to the original spacer sequence (ie, the target sequence).
  • PAM is typically a 2-5 base pair sequence adjacent to the original spacer sequence (ie, the target sequence).
  • One skilled in the art will be able to identify PAM sequences for use with a given Cas effector protein.
  • the target sequence or target polynucleotide can include a plurality of disease associated genes and polynucleotides as well as signaling biochemical pathway related genes and polynucleotides.
  • Non-limiting examples of such target sequences or target polynucleotides include U.S. Provisional Patent Application Nos. 61/736,527 and 61/748,427, filed on Dec. 12, 2012, and January 2, 2013, filed on Dec. Those listed in International Application No. PCT/US2013/074667, the entire contents of which is incorporated herein by reference.
  • target sequences or target polynucleotides include sequences associated with a signaling biochemical pathway, such as a signaling biochemical pathway-related gene or polynucleotide.
  • target polynucleotides include disease-related genes or polynucleotides.
  • a "disease-associated" gene or polynucleotide refers to any gene or polynucleoside that produces a transcriptional or translational product at an abnormal level or in an abnormal form in a cell derived from a disease-affected tissue as compared to a non-disease-controlled tissue or cell. acid.
  • the altered expression is associated with the appearance and/or progression of the disease, it may be a gene that is expressed at an abnormally high level; alternatively, it may be a gene that is expressed at an abnormally low level.
  • a disease-associated gene also refers to a gene having one or more mutations or a genetic variation that is directly responsible for or incompatible 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.
  • wild type has the meaning commonly understood by those skilled in the art to mean a typical form of a organism, a strain, a gene, or a feature that distinguishes it from a mutant or variant when it exists in nature. It can be isolated from sources in nature and not intentionally modified.
  • nucleic acid molecule or polypeptide As used herein, the terms “non-naturally occurring” or “engineered” are used interchangeably and refer to artificial participation. When these terms are used to describe a nucleic acid molecule or polypeptide, it is meant that the nucleic acid molecule or polypeptide is at least substantially freed from at least one other component of its association in nature or as found in nature.
  • an "ortholog" of a protein as referred to herein refers to a protein belonging to a different species that performs the same or similar function as a protein that is an ortholog thereof.
  • identity is used to mean the matching of sequences between two polypeptides or between two nucleic acids.
  • a position in the two sequences being compared is occupied by the same base or amino acid monomer subunit (for example, a position in each of the two DNA molecules is occupied by adenine, or two
  • Each position in each of the polypeptides is occupied by lysine, and then each molecule is identical at that position.
  • the "percent identity" between the two sequences is a function of the number of matching positions shared by the two sequences divided by the number of positions to be compared x 100. For example, if 6 of the 10 positions of the two sequences match, then the two sequences have 60% identity.
  • the DNA sequences CTGACT and CAGGTT share 50% identity (3 out of a total of 6 positions match).
  • the comparison is made when the two sequences are aligned to produce maximum identity.
  • Such alignment can be achieved by, for example, the method of Needleman et al. (1970) J. Mol. Biol. 48: 443-453, which can be conveniently performed by a computer program such as the Align program (DNAstar, Inc.). It is also possible to use the algorithm of E. Meyers and W. Miller (Comput. Appl Biosci., 4: 11-17 (1988)) integrated into the ALIGN program (version 2.0), using the PAM 120 weight residue table.
  • the gap length penalty of 12 and the gap penalty of 4 were used to determine the percent identity between the two amino acid sequences.
  • the Needleman and Wunsch (J MoI Biol. 48: 444-453 (1970)) algorithms in the GAP program integrated into the GCG software package can be used, using the Blossum 62 matrix or The PAM250 matrix and the gap weight of 16, 14, 12, 10, 8, 6 or 4 and the length weight of 1, 2, 3, 4, 5 or 6 to determine the percent identity between two amino acid sequences .
  • vector refers to a nucleic acid vehicle into which a polynucleotide can be inserted.
  • a vector is referred to as an expression vector when the vector enables expression of the protein encoded by the inserted polynucleotide.
  • the vector can be introduced into the host cell by transformation, transduction or transfection, and the genetic material element carried thereby 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; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or P1 derived artificial chromosomes (PAC).
  • Phage such as lambda phage or M13 phage and animal virus.
  • Animal viruses useful as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, nipples Multi-tumor vacuolar virus (such as SV40).
  • a vector may contain a variety of elements that control expression, including, but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. In addition, the vector may also contain an origin of replication.
  • the term "host cell” refers to a cell that can be used to introduce a vector, including, but not limited to, a prokaryotic cell such as Escherichia coli or Bacillus subtilis, such as a fungal cell such as a yeast cell or an Aspergillus.
  • a prokaryotic cell such as Escherichia coli or Bacillus subtilis
  • a fungal cell such as a yeast cell or an Aspergillus.
  • S2 Drosophila cells or insect cells such as Sf9
  • animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells or human cells.
  • a vector can be introduced into a host cell to thereby produce a transcript, protein, or peptide, including a protein, fusion protein, isolated nucleic acid molecule, etc. as described herein (eg, a CRISPR transcript, such as a nucleic acid transcript) , protein, or enzyme).
  • a CRISPR transcript such as a nucleic acid transcript
  • 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 Poly U sequence), a detailed description can be found in Goeddel, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego Diego), California (1990).
  • regulatory elements include those sequences that direct constitutive expression of a nucleotide sequence in a plurality of types of host cells, as well as those sequences that direct expression of the nucleotide sequence only in certain host cells (eg, Tissue-specific regulatory sequence).
  • Tissue-specific promoters can primarily direct expression in a desired tissue of interest, such as muscle, neurons, bone, skin, blood, specific organs (eg, liver, pancreas), or specific cell types (eg, Lymphocytes).
  • the regulatory elements may 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.
  • the term "regulatory element” encompasses enhancer elements such as WPRE; CMV enhancer; R-U5' fragment in LTR of HTLV-I ((Mol. Cell. Biol., 8th ( 1) Vol., pp. 466-472, 1988); SV40 enhancer; and intron sequence between exons 2 and 3 of rabbit ⁇ -globin (Proc. Natl. Acad. Sci. USA., Vol. 78(3), pp. 1527-31, 1981).
  • promoter has the meaning well-known to those skilled in the art and refers to a non-coding nucleotide sequence located upstream of the gene that initiates expression of the downstream gene.
  • a constitutive promoter is a nucleotide sequence that, when operably linked to a polynucleotide encoding or defining a gene product, results in a gene product in the cell under most or all physiological conditions of the cell. The production.
  • An inducible promoter is a nucleotide sequence that, when operably linked to a polynucleotide encoding or defining a gene product, results in substantially only when an inducer corresponding to the promoter is present in the cell The gene product is produced intracellularly.
  • a tissue-specific promoter is a nucleotide sequence that, when operably linked to a polynucleotide encoding or defining a gene product, is substantially only caused when the cell is a cell of the tissue type corresponding to the promoter Gene products are produced in the cells.
  • operably linked is intended to mean that a nucleotide sequence of interest is linked to the one or more regulatory elements in a manner that allows expression of the nucleotide sequence (eg, In an in vitro transcription/translation system or in the host cell when the vector is introduced into a host cell).
  • 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 complement indicates the percentage of residues in a nucleic acid molecule that can form a hydrogen bond (eg, Watson-Crick base pairing) with a second nucleic acid sequence (eg, 5, 6, 7, 8 out of 10) 9, 10, that is 50%, 60%, 70%, 80%, 90%, and 100% complementary). "Completely complementary” means that all contiguous residues of one nucleic acid sequence form a hydrogen bond with the same number of contiguous residues in a second nucleic acid sequence.
  • substantially complementary means 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 in the region of 30, 35, 40, 45, 50 or more nucleotides %, 99%, or 100% complementarity, or two nucleic acids that hybridize under stringent conditions.
  • stringent conditions for hybridization refers to conditions under which a nucleic acid that is complementary to a target sequence primarily hybridizes to the target sequence and does not substantially hybridize to a non-target sequence. 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 “Technology Techniques In Biochemi stry And Molecular Biology-Hybridization With Nucleic Acid Probes" by Tijssen (1993). ), Part I, Chapter 2, “Overview of principles of hybridization and t he strategy of nucleic acid probe assay", Elsevier, New York.
  • hybridization refers to a reaction in which one or more polynucleotides react to form a complex that hydrogen bonds through the bases between these nucleotide residues. And stabilized. 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 chains forming one duplex, three or more chains 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. A sequence that is capable of hybridizing to a given sequence is referred to as the "complement" of the given sequence.
  • the term "expression” refers to a process by which a DNA template is transcribed into a polynucleotide (eg, transcribed into mRNA or other RNA transcript) and/or transcribed mRNA, which is subsequently translated into a peptide, The process of a polypeptide or protein.
  • the transcripts and encoded polypeptides may be collectively referred to as "gene products.” If the polynucleotide is derived from genomic DNA, expression can include splicing of mRNA in eukaryotic cells.
  • linker refers to a linear polypeptide formed by the joining of multiple amino acid residues by peptide bonds.
  • the linker of the invention may be a synthetic amino acid sequence, or a naturally occurring polypeptide sequence, such as a polypeptide having the function of a hinge region.
  • 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 ).
  • treating refers to treating or curing a condition, delaying the onset of symptoms of the condition, and/or delaying the progression of the condition.
  • the term "subject” includes, but is not limited to, various animals, such as mammals, such as bovine, equine, ovine, porcine, canine, feline, A rabbit, a rodent (eg, a mouse or rat), a non-human primate (eg, a macaque or a cynomolgus monkey) or a human.
  • the subject eg, a human
  • has a condition eg, a condition caused by a disease-related gene defect.
  • the PAM domain of the Cas effector of the present invention is a strict 5'-TTN structure, and nearly 100% of the second and third bases in front of the target sequence are T, and other positions may be arbitrary sequences.
  • C2c1 which is the most rigorous PAM recognition that has been reported so far, has a more rigorous PAM recognition method, which significantly reduces the off-target effect.
  • the Cas effector of the present invention is capable of DNA cleavage in eukaryotes and is about 200-300 amino acids smaller in size than Cpf1 and Cas9 proteins, and thus is significantly more efficient than Cpf1 and Cas9 in transfection efficiency.
  • 1A-1B are the results of in vivo processing and structural analysis of crRNA of Cas12i.1 in Example 2.
  • Figure 7 is a graph showing the results of PAM domain analysis of Cas12i.2 in Example 4 in Example 4.
  • Figure 8 is a graph showing the results of PAM domain analysis of Cas12i.3 in Example 5 of Example 5.
  • 9A-9B are the results of in vitro cleavage mode identification of CRISPR/Cas12i.1 in Example 6.
  • 10A-10B are in vitro digestion results of different truncated crRNAs in Example 7.
  • 11A-11B are results of in vitro digestion experiments of the crRNA containing point mutation in Example 7.
  • 12A to 12B are results of in vitro digestion experiments of CrRNAs containing point mutations at different positions in Example 7.
  • 13A to 13B are results of in vitro digestion experiments of the crRNA comprising the 3'-terminal mutation in Example 7.
  • Figure 14A shows the results of non-specific cleavage of single-stranded DNA by cas12i.1 under target DNA activation in Example 7 (lane 1: Cas12i.1 protein + M13 + dsDNA + crRNA (targeting DNA); lane 2: Cas12i. 1 protein + M13 + dsDNA).
  • Figure 14B shows the results of non-specific cleavage of cas12i.3 against single-stranded DNA under target DNA activation in Example 7 (lane 1: Cas12i.3 protein + M13 + dsDNA + crRNA (targeting DNA); lane 2: Cas12i. 3 protein + M13 + dsDNA).
  • LB liquid medium 10 g tryptone, 5 g yeast extract (Yeast Extract), 10 g NaCl, made up to 1 L, sterilized. If antibiotics are required, add the final concentration of 50 ⁇ g/ml after the medium is cooled.
  • Chloroform / isoamyl alcohol 240 ml of chloroform plus 10 ml of isoamyl alcohol, and mix.
  • RNP buffer 100 mM sodium chloride, 50 mM Tris-HCl, 10 mM MgCl 2 , 100 ⁇ g/ml BSA, pH 7.9.
  • the prokaryotic expression vectors pACYC-Duet-1 and pUC19 were purchased from Beijing Quanjin Biotechnology Co., Ltd.
  • E. coli competent EC100 was purchased from Epicentre.
  • Phage M13mp18 Single-stranded DNA was purchased from NEB.
  • RNAaseA, Dnase and Protease-free and Proteinase K were purchased from Thermo Scientific.
  • CRISPR and gene annotation Prodigal was used to genetically annotate the microbial genome and metagenomic data of the NCBI and JGI databases to obtain all proteins, and the Pyrer-CR was used to annotate the CRISPR block. The parameters are default parameters.
  • Protein filtration The sequence protein is de-redundant by sequence identity, and the proteins with completely identical sequences are removed, and proteins with a length of more than 800 amino acids are classified into macromolecular proteins. Since all second-generation CRISPR/Cas systems have effector proteins longer than 900 amino acids, in order to reduce computational complexity, only macromolecular proteins are considered when mining CRISPR effector proteins.
  • BLASTP was used to perform internal pairwise alignment of non-redundant macromolecular CRISPR-related proteins, and the results of the alignment of Evalue ⁇ 1E-10 were output.
  • the MCL was used to cluster the output of BLASTP, a family of CRISPR-related proteins.
  • CRISPR-enriched macromolecular protein families BLASTP was used to compare the proteins of the CRISPR-related protein family to a database of non-redundant macromolecular proteins from which the CRISPR-related proteins were removed, and the results of the alignment of Evalue ⁇ 1E-10 were output. If a non-CRISPR-related protein database finds less than 100% homologous protein, then this family of proteins is enriched in the CRISPR region, and the CRISPR-enriched macromolecular protein family is identified by this method.
  • the CRISPR/Cas protein family was obtained by annotating the CRISPR-enriched macromolecular protein family using the Pfam database, the NR database, and the Cas protein collected from NCBI. Multiple sequence alignments of each CRISPR/Cas family protein were performed using Mafft, followed by conserved domain analysis with JPred and HHpred to identify a family of proteins containing the RuvC domain.
  • Cas12i Cas12i
  • Cas12i.1 SEQ ID NO: 1
  • Cas12i.2 SEQ. ID NO: 2
  • Cas12i.3 SEQ ID NO: 3
  • the prototype homologous repeat sequences corresponding to Cas12i.1, Cas12i.2, and Cas12i.3 are shown in SEQ ID NOs: 7, 8, and 9, respectively.
  • the mature homologous repeat sequences (repeat sequences contained in the mature crRNA) corresponding to Cas12i.1, Cas12i.2, and Cas12i.3 are shown in SEQ ID NOs: 13, 14, and 15, respectively.
  • a double-stranded DNA molecule of SEQ ID NO: 4 is artificially synthesized, and a double-stranded DNA molecule of SEQ ID NO: 10 is artificially synthesized.
  • the double-stranded DNA molecule synthesized in the step 1 was ligated to the prokaryotic expression vector pACYC-Duet-1 to obtain a recombinant plasmid pACYC-Duet-1-CRISPR/Cas12i.1.
  • the recombinant plasmid pACYC-Duet-1-CRISPR/Cas12.i was sequenced. The sequencing results indicated that the recombinant plasmid pACYC-Duet-1-CRISPR/Cas12i.1 contained the sequence of SEQ ID NO: 4 and SEQ ID NO: 10, and expressed the Cas12i.1 protein represented by SEQ ID NO: 1. Cas12i.1 targeting RNA set forth in SEQ ID NO:7.
  • the recombinant plasmid pCYC-Duet-1-CRISPR/Cas12i.1 was introduced into Escherichia coli EC100 to obtain a recombinant strain, and the recombinant strain was named EC100-CRISPR/Cas12i.1.
  • Extraction of bacterial RNA Transfer 1.5 mL of the bacterial culture to a pre-chilled microcentrifuge tube, centrifuge at 6,000 x g for 5 minutes at 4 °C. After centrifugation, the supernatant was discarded, and the cell pellet was resuspended in 200 ⁇ L Max Bacterial Enhancement Reagent preheated to 95 ° C, and mixed by pipetting. Incubate at 95 ° C for 4 minutes. Add 1 mL to the lysate Reagent and mix by pipetting and incubate for 5 minutes at room temperature. Add 0.2 mL of cold chloroform, mix by shaking the tube for 15 seconds, and incubate for 2-3 minutes at room temperature.
  • RNA pellet was dissolved in 50 ⁇ L of RNase-free water and incubated at 60 ° C for 10 minutes.
  • RNAI Digestion of DNA: 20 ⁇ g of RNA was dissolved in 39.5 ⁇ L dH O at 65 ° C for 5 min. On ice for 5 min, 0.5 ⁇ L of RNAI, 5 ⁇ L of buffer, 5 ⁇ L of DNase I, and 37 ° C for 45 min (50 ⁇ L system) were added. Add 50 ⁇ L dH O and adjust the volume to 100 ⁇ L.
  • the precipitate was washed by adding 350 ⁇ L of 75% ethanol, centrifuged at 16000 g for 10 min at 4 ° C, and the supernatant was discarded. Dry, add 20 ⁇ L of RNase-free water, 65 ° C, and dissolve the precipitate for 5 min. NanoDrop measures the concentration and runs the glue.
  • the precipitate was washed by adding 350 ⁇ L of 75% ethanol, centrifuged at 16000 g for 10 min at 4 ° C, and the supernatant was discarded. Dry, add 21 ⁇ L of RNase-free water, 65 ° C, dissolve the precipitate for 5 min, and measure the concentration by NanoDrop.
  • RNA monophosphorylation 20 ⁇ L of RNA, 1 min at 90 ° C, and cooled on ice for 5 min. 2 ⁇ L of RNA 5'Polphosphatase 10 ⁇ Reaction buffer, 0.5 ⁇ L of Inhibitor, 1 ⁇ L of RNA 5'Polphosphatase (20 Units), RNase-free water to 20 ⁇ L, and 37 ° C for 60 min were added. Add 80 ⁇ L dH O and adjust the volume to 100 ⁇ L.
  • cDNA library 16.5 ⁇ L RNase-free water. 5 ⁇ L Poly(A) Polymerase 10 ⁇ Reaction buffer. 5 ⁇ L of 10 mM ATP. 1.5 ⁇ L RiboGuard RNase Inhibitor. 20 ⁇ L RNA Substrate. 2 ⁇ L Poly(A) Polymerase (4 Units). 50 ⁇ L total volume. 37 ° C for 20 min. Add 50 ⁇ L dH 2 O and adjust the volume to 100 ⁇ L.
  • the cDNA library was added to the sequencing linker and sent to Beijing Berry Hekang for sequencing.
  • RNA sequence of 25 nt to 50 nt was retained and aligned to the reference sequence of the CRISPR array using bowtie.
  • the results are shown in Fig. 1A.
  • the peak shape is the structure of the second-generation sequencing sequence alignment of the CRISPR block.
  • the vertical line shows the enzyme cleavage site
  • the gray rectangle is the schematic structure of the Repeat structure
  • the light gray diamond shape is the schematic structure of the spacer sequence.
  • the cleavage site information obtained by the Cas12i.1 alignment showed that the pre-crRNA of Cas12i.1 was successfully processed into 45 nt mature crRNA by E. coli in Cas12i.1, which consisted of a 23 nt Repeat sequence and 19
  • the -22nt guide sequence consists of.
  • the recombinant plasmid pACYC-Duet-1+CRISPR/Cas12i.1 expresses the Cas12i.1 protein shown in SEQ ID NO: 1 and the Cas12i.1 targeting RNA shown in SEQ ID NO: 27.
  • the recombinant plasmid pACYC-Duet-1+CRISPR/Cas12i.1 contains an expression cassette, and the nucleotide sequence of the expression cassette is shown in SEQ ID NO:23.
  • SEQ ID NO:23 the nucleotide sequence of the pLacZ promoter from positions 1 to 44 from the 5' end, the nucleotide sequence of the Cas12i.1 gene at positions 45 to 3, 326, and the third, 327 to The 3,412 position is the nucleotide sequence of the terminator (used to terminate transcription).
  • the 3,413 to 3,452 positions are the nucleotide sequence of the J23119 promoter
  • the 3,453 to 3,628 are the nucleotide sequence of the CRISPR array
  • the 3,627 to 3,713 are the nucleotide sequence of the rrnB-T1 terminator. (used to terminate transcription).
  • SEQ ID NO: 26 The sequence shown in SEQ ID NO: 26 was artificially synthesized and ligated into the pUC19 vector, wherein the sequence shown by SEQ ID NO: 26 includes eight random bases and a target sequence at the 5' end. Eight random base construction plasmid libraries were designed in front of the 5' end of the target sequence of the PAM library. The plasmids were separately transferred into E. coli containing the Cas12i.1 locus and E. coli containing no Cas.12i.1 locus. After treatment at 37 ° C for 1 hour, the plasmid was extracted, and the PAM region sequence was subjected to PCR amplification and sequencing.
  • the PAM domain of Cas12i.1 was obtained by the above PAM library consumption experiment. In order to verify the stringency of this domain, 10 sets of PAM (TTA, TTT, TTC, TTG, TAT) were set up. , TCT, TGT, ATT, CTT, GTT) were tested in vivo to test the editing activity of Cas12i on these PAMs.
  • TTA, TTT, TTC, TTG, TAT 10 sets of PAM (TTA, TTT, TTC, TTG, TAT) were set up. , TCT, TGT, ATT, CTT, GTT) were tested in vivo to test the editing activity of Cas12i on these PAMs.
  • SEQ ID NO: 30 the 30 nt target sequence
  • the PAM sequence into the non-conserved position of the kucana gene of the pUC19 plasmid, and then proceeded with a complex formed by CRSPR/Cas12i.1 and guide RNA.
  • the mixture was cultured for 8 hours, and the medium was Lb liquid medium at a temperature of 37 °C.
  • the ability of Cas12i to deplete different PAM sequences can be determined by plating and counting the number of colonies.
  • the results are shown in Figure 5.
  • the CRISPR/Cas12i.1 system can only effectively edit target sequences with 5'-TTA, 5'-TTT, 5'-TTC and 5'-TTG PAM.
  • the target sequences of 5'-TAT, 5'-TCT, 5'-TCG, 5'-ATT, 5'-CTT and 5'-GTT PAM have no editing activity, thus verifying the recognition of PAM domain of Cas12i.1 Rigorous. Further, the total number of colonies in the well was counted.
  • the editing activity of the CRISPR/Cas12i.1 system for 5'-TTA, 5'-TTT and 5'-TTC was significantly higher than that of 5'-TTG.
  • the recombinant plasmid pACYC-Duet-1+CRISPR/Cas12i.2 expresses the Cas12i.2 protein shown in SEQ ID NO: 2 and the Cas12i.2 targeting RNA shown in SEQ ID NO: 28.
  • the recombinant plasmid pACYC-Duet-1+CRISPR/Cas12i.2 contains an expression cassette, and the nucleotide sequence of the expression cassette is shown in SEQ ID NO: 24.
  • SEQ ID NO: 24 the nucleotide sequence of the pLacZ promoter from positions 1 to 44 from the 5' end, the nucleotide sequence of the Cas12i.2 gene at positions 45 to 3, 185, and the third, 186 to The 3,271 position is the nucleotide sequence of the terminator (used to terminate transcription).
  • the 3,272- to 3,311 positions are the nucleotide sequence of the J23119 promoter
  • the 3rd, 312th to 3rd, 480th are the nucleotide sequence of the CRISPR array
  • the 3rd, 481th to 3rd, 567th are the nucleotides of the rrnB-T1 terminator. Sequence (used to terminate transcription).
  • SEQ ID NO: 26 The sequence shown in SEQ ID NO: 26 was artificially synthesized and ligated into the pUC19 vector, wherein the sequence shown by SEQ ID NO: 26 includes eight random bases and a target sequence at the 5' end. Eight random base construction plasmid libraries were designed in front of the 5' end of the target sequence of the PAM library. The plasmids were separately transferred into E. coli containing the Cas12i.2 locus and E. coli containing no Cas.12i.2 locus. After treatment at 37 ° C for 1 hour, the plasmid was extracted, and the PAM region sequence was subjected to PCR amplification and sequencing.
  • the recombinant plasmid pACYC-Duet-1+CRISPR/Cas12i.3 expresses the Cas12i.3 protein shown in SEQ ID NO: 3 and the Cas12i.3 targeting RNA shown in SEQ ID NO:29.
  • the recombinant plasmid pACYC-Duet-1+CRISPR/Cas12i.3 contains an expression cassette, and the nucleotide sequence of the expression cassette is shown in SEQ ID NO: 25.
  • SEQ ID NO: 25 the nucleotide sequence of the pLacZ promoter from positions 1 to 44 from the 5' end, and the nucleotide sequence of the Cas12i.3 gene at positions 45 to 3, 146, from 3, 147 to The 3,232 position is the nucleotide sequence of the terminator (used to terminate transcription).
  • the 3, 233 to 3, 272 positions are the nucleotide sequence of the J23119 promoter
  • the 3rd, 273th to 3rd, 444th are the nucleotide sequence of the CRISPR array
  • the 3rd, 445th to 3rd, 531th are the nucleotide sequence of the rrnB-T1 terminator. (used to terminate transcription).
  • the recombinant plasmid pACYC-Duet-1+CRISPR/Cas12i.3 was introduced into Escherichia coli EC100 to obtain recombinant Escherichia coli, named EC100/pACYC-Duet-1+CRISPR/Cas12i.3.
  • the recombinant plasmid pACYC-Duet-1 was introduced into Escherichia coli EC100 to obtain a recombinant strain designated as EC100/pACYC-Duet-1.
  • SEQ ID NO: 26 The sequence shown in SEQ ID NO: 26 was artificially synthesized and ligated into the pUC19 vector, wherein the sequence shown by SEQ ID NO: 26 includes eight random bases and a target sequence at the 5' end. Eight random base construction plasmid libraries were designed in front of the 5' end of the target sequence of the PAM library. The plasmids were separately transferred into E. coli containing the Cas12i.3 locus and E. coli containing no Cas.12i.3 locus. After treatment at 37 ° C for 1 hour, the plasmid was extracted, and the PAM region sequence was subjected to PCR amplification and sequencing.
  • the DNA sequence shown in SEQ ID NO: 23 is artificially synthesized.
  • the double-stranded DNA molecule synthesized in the step 1 was ligated to the prokaryotic expression vector pET-30a(+) to obtain a recombinant plasmid pET-30a-CRISPR/Cas12i.1.
  • the recombinant plasmid pET-30a-CRISPR/Cas12i.1 was sequenced. The sequencing results indicated that the recombinant plasmid pET-30a-CRISPR/Cas12i.1 contained the sequence of SEQ ID NO: 23 and expressed the Cas12i.1 protein (SEQ ID NO: 20) with a nuclear localization signal.
  • the recombinant plasmid pET-30a-CRISPR/Cas12i.1 was introduced into Escherichia coli EC100 to obtain a recombinant strain, and the recombinant strain was named EC100-CRISPR/Cas12i.1.
  • the monoclonal clone of EC100-CRISPR/Cas12i was inoculated into 100 mL of LB liquid medium (containing 50 ⁇ g/mL ampicillin), and cultured at 37 ° C, shaking at 200 rpm for 12 hours to obtain a culture broth.
  • the culture solution was taken and inoculated into 50 mL of LB liquid medium (containing 50 ⁇ g/mL ampicillin) at a volume ratio of 1:100, and cultured at 37 ° C, shaking at 200 rpm until the OD 600nm value was 0.6, then IPTG was added and The concentration was 1 mM, cultured at 28 ° C, shaking at 220 rpm for 4 h, centrifuged at 10,000 rpm for 10 min at 4 ° C, and the cell pellet was collected.
  • LB liquid medium containing 50 ⁇ g/mL ampicillin
  • the target sequence (SEQ ID NO: 30) was subjected to in vitro cleavage using a complex of Cas12i.1 protein and targeting RNA (SEQ ID NO: 27), and the digestion buffer was RNP buffer at 37 °C. After 4 hours of reaction, the digested products were collected, and the sense and antisense strands of DNA were sequenced by Sanger sequencing, respectively, and the sequencing results are shown in Fig. 9A.
  • the sequencing results showed that Cas12i.1 cleaves at the 18th base of the target strand away from the PAM end, and simultaneously cleaves the 24th base of the non-target strand, and finally forms a 6nt-length viscous end.
  • Figure 9B shows.
  • Example 7 Mismatching of the targeting RNA of Cas12i.1 with the target sequence The effect of the activity of the CRISPR/Cas12i.1 system
  • the 5' end sequence of the crRNA was truncated to obtain the truncated body shown in Fig. 10A, and the in vitro digestion activity of the target sequence was examined for these truncated bodies.
  • the in vitro cutting conditions were consistent with Example 6.2 above.
  • the digested product was subjected to agarose gel electrophoresis with a mass fraction of 1.5% to determine the enzymatic activity of the Cas12i.1 protein in vitro. Specifically, 0.6 g of agarose and 40 ml of 0.5 ⁇ TBE solution were first mixed, boiled to be transparent, and after cooling to 60 ° C, 2 ⁇ l of YeaRed nucleic acid dye was added and shaken. Pour the glue into the installed glue machine.
  • the electrophoresis tank is spotted with DNA and electrophoresed for 40 minutes.
  • the electrophoresis parameters were set to a voltage of 80V and a current of 200A.
  • the electrophoresis results are shown in Figure 10B.
  • the Repeat sequence is truncated by 2 bases, the cleavage activity of Cas12i.1 is significantly decreased.
  • the Ripe is truncated to 17 nt, the activity of Cas12i.1 is greatly weakly reduced.
  • the repetition was truncated to 15 nt, the activity of Cas12i.1 was barely detectable.
  • Cas12i.1 when a targeting RNA targeting double-stranded DNA is present, Cas12i.1 is activated by the target double-stranded DNA, whereby M13 single-stranded DNA can be efficiently and non-specifically cleaved (lane 1); when no target exists When targeting RNA to double-stranded DNA, Cas12i.1 is not activated by target DNA and does not non-specifically cleave M13 single-stranded DNA (lane 2).
  • the non-specific cleavage properties of Cas12i.1 against single-stranded DNA after target DNA recognition can be applied to the field of DNA detection, such as detection of tumor-labeled nucleic acids, Ebola, avian influenza, African pigs. ⁇ and other viruses.
  • Cas12i.3 when a targeting RNA targeting double-stranded DNA is present, Cas12i.3 is activated by the target double-stranded DNA, and then the M13 single-stranded DNA can be efficiently and non-specifically cleaved (lane 1); when there is no targeting When targeting RNA to double-stranded DNA, Cas12i.3 is not activated by target DNA and does not non-specifically cleave M13 single-stranded DNA (lane 2).
  • the non-specific cleavage properties of Cas12i against single-stranded DNA after target DNA recognition can be applied to the field of DNA detection, such as detection of tumor-labeled nucleic acids, Ebola, avian influenza, African swine fever and other viruses.

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Abstract

L'invention concerne une protéine effectrice Cas, comprenant une protéine de fusion de ladite protéine et des molécules d'acide nucléique codant pour celle-ci. L'invention concerne également un complexe et un composé pour l'édition d'acide nucléique (par exemple, gène ou édition de gène), comprenant la protéine effectrice Cas ou la protéine de fusion, ou les molécules d'acide nucléique codant pour celle-ci. L'invention concerne également un procédé d'édition d'acide nucléique (par exemple, gène ou édition de gène), à l'aide de la protéine effectrice Cas ou de la protéine de fusion.
PCT/CN2019/083418 2018-04-20 2019-04-19 Protéine effectrice crispr/cas et système WO2019201331A1 (fr)

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WO2020098772A1 (fr) * 2018-11-15 2020-05-22 中国农业大学 Enzyme crispr-cas12j et système
EP4158020A4 (fr) * 2020-05-29 2024-06-26 Arbor Biotechnologies, Inc. Compositions comprenant un polypeptide de cas12i2 et leurs utilisations
CN114277015A (zh) * 2021-03-16 2022-04-05 山东舜丰生物科技有限公司 新型crispr酶以及应用
CN114277015B (zh) * 2021-03-16 2023-12-15 山东舜丰生物科技有限公司 Crispr酶以及应用
WO2022247873A1 (fr) 2021-05-27 2022-12-01 中国科学院动物研究所 Nucléase cas12i modifiée, protéine effectrice et utilisation de celle-ci
CN114480383A (zh) * 2021-06-08 2022-05-13 山东舜丰生物科技有限公司 一种具有碱基突变的同向重复序列及其应用
CN114480383B (zh) * 2021-06-08 2023-06-30 山东舜丰生物科技有限公司 一种具有碱基突变的同向重复序列及其应用
CN116218816A (zh) * 2021-10-28 2023-06-06 山东舜丰生物科技有限公司 新型的Cas12酶和系统以及应用
WO2024198961A1 (fr) * 2023-03-24 2024-10-03 尧唐(上海)生物科技有限公司 Protéine cas et son mutant, système d'édition génique correspondant et utilisation associée

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