WO2020098793A1 - Enzyme crispr-cas12a et système - Google Patents

Enzyme crispr-cas12a et système Download PDF

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WO2020098793A1
WO2020098793A1 PCT/CN2019/118871 CN2019118871W WO2020098793A1 WO 2020098793 A1 WO2020098793 A1 WO 2020098793A1 CN 2019118871 W CN2019118871 W CN 2019118871W WO 2020098793 A1 WO2020098793 A1 WO 2020098793A1
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sequence
nucleic acid
protein
cell
acid molecule
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PCT/CN2019/118871
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Chinese (zh)
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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
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中国农业大学
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Priority to CN201980074517.6A priority Critical patent/CN113015798B/zh
Publication of WO2020098793A1 publication Critical patent/WO2020098793A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • 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
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    • 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
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C—CHEMISTRY; METALLURGY
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    • 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
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • 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
    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases RNAses, DNAses

Definitions

  • the invention relates to the field of nucleic acid editing, in particular to the field of regularly clustered short palindrome repeat (CRISPR) technology.
  • CRISPR regularly clustered short palindrome repeat
  • 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.
  • 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.
  • target sequences such as Cpf1, C2c1, CasX, CasY.
  • 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.
  • 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.
  • Cpf1 enzyme also known as Cas12a or SmCpf1
  • the inventors developed a new CRISPR / SmCpf1 system and a gene editing method based on the system.
  • 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.
  • 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.
  • 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.
  • 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).
  • 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.
  • the protein of the invention comprises or consists of a sequence selected from the following:
  • the protein of the present invention has the amino acid sequence shown in SEQ ID NO: 1.
  • the protein of the present invention can be derivatized, for example, linked to another molecule (eg, another polypeptide or protein).
  • another molecule eg, another polypeptide or protein
  • derivatization eg, labeling
  • the protein of the present invention is also intended to include such derivatized forms.
  • 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.
  • the protein of the present invention can be linked to other functional units.
  • 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.
  • NLS nuclear localization signal
  • it can be linked to a targeting moiety to make the protein of the present invention targeted.
  • it can be linked to a detectable label to facilitate detection of the protein of the invention.
  • it can be linked to an epitope tag to facilitate expression, detection, tracing and / or purification of the protein of the invention.
  • the present invention provides a conjugate comprising a protein and a modified moiety as described above.
  • the modified moiety is selected from additional proteins or polypeptides, detectable labels, or any combination thereof.
  • 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.
  • the conjugates of the invention comprise one or more NLS sequences, such as the NLS of the SV40 viral large T antigen.
  • the NLS sequence is shown in SEQ ID NO: 7.
  • the NLS sequence is located at, near, or near the terminus (eg, N-terminus or C-terminus) of the protein of the present invention.
  • the NLS sequence is located at, near, or near the C-terminus of the protein of the present invention.
  • the conjugates of the present invention comprise epitope tags.
  • 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.
  • the conjugate of the present invention comprises a reporter gene sequence.
  • 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.
  • 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. .
  • MBP maltose binding protein
  • DBD Lex A's DNA binding domain
  • GAL4's DBD GAL4's DBD
  • the conjugates of the invention contain a detectable label, such as a fluorescent dye, such as FITC or DAPI.
  • a detectable label such as a fluorescent dye, such as FITC or DAPI.
  • the protein of the invention is optionally coupled, conjugated, or fused to the modified moiety via a linker.
  • the modified portion is directly connected to the N-terminus or C-terminus of the protein of the present invention.
  • the modified portion is connected to the N-terminus or C-terminus of the protein of the present invention through a linker.
  • 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, ⁇ -Ala, GABA, or Ava), or PEG.
  • the invention provides a fusion protein comprising the protein of the invention and additional protein or polypeptide.
  • 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.
  • the 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 located at, near, or near the terminus (eg, N-terminus or C-terminus) of the protein of the present invention.
  • the NLS sequence is located at, near, or near the C-terminus of the protein of the present invention.
  • the fusion protein of the present invention contains an epitope tag.
  • the fusion protein of the invention comprises a reporter gene sequence.
  • the fusion protein of the present invention comprises a domain capable of binding to DNA molecules or intracellular molecules.
  • the 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 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.
  • the present invention provides an isolated nucleic acid molecule comprising or consisting of a sequence selected from the following:
  • 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.
  • the isolated nucleic acid molecule is a repeat sequence in the CRISPR-Cas system.
  • the nucleic acid molecule comprises or consists of a sequence selected from:
  • the isolated nucleic acid molecule is RNA.
  • the invention provides a composite comprising:
  • a protein component selected from: the protein, conjugate or fusion protein of the present 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 with a target sequence
  • the protein component and the nucleic acid component combine with each other to form a complex.
  • the targeting sequence is attached 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 guide RNA in the CRISPR-Cas system.
  • the nucleic acid molecule is RNA.
  • the complex does not contain trans-acting crRNA (tracrRNA).
  • 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.
  • 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 present invention provides an isolated nucleic acid molecule, comprising:
  • 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.
  • 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.
  • the vector of the present invention is, for example, a plasmid, cosmid, bacteriophage, cosmid, and the like.
  • 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.
  • the present invention also provides a host cell comprising the 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 (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.
  • 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: the protein, conjugate, fusion protein, nucleotide sequence encoding the protein or fusion protein of the invention, and any combination thereof;
  • a second component which is a nucleotide sequence comprising a guide RNA, or a nucleotide sequence encoding the nucleotide sequence comprising a guide RNA;
  • 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).
  • the direct repeat sequence is an isolated nucleic acid molecule as defined in the fourth aspect.
  • 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.
  • the composition does not include tracrRNA.
  • 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.
  • the target sequence 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.
  • the target sequence when the target sequence is RNA, the target sequence does not have PAM domain restrictions.
  • 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.
  • 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.
  • 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 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 present invention; optionally the first nucleic acid is operably linked to a first regulatory element;
  • 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;
  • 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).
  • the direct repeat sequence is an isolated nucleic acid molecule as defined in the fourth aspect.
  • 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.
  • the composition does not include tracrRNA.
  • the composition is non-naturally occurring or modified. In certain embodiments, at least one component in 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 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.
  • PAM motif
  • the target sequence when the target sequence is RNA, the target sequence does not have PAM domain restrictions.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • vectors eg, bacterial vectors with an origin of bacterial replication and episomal mammalian vectors
  • Other vectors eg, non-episomal mammalian vectors
  • 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.
  • 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.
  • the present invention provides a delivery composition
  • a delivery vehicle 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 delivery vehicle is a particle.
  • 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).
  • viral vectors eg, replication-deficient reverse transcription
  • Virus lentivirus, adenovirus or adeno-associated virus
  • the present 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 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.
  • 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.
  • the kit of the invention comprises the composition of the tenth aspect. In some embodiments, the kit also includes instructions for using the composition.
  • the components included in the kit of the present invention may be provided in any suitable container.
  • 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.
  • the buffer is alkaline. In certain embodiments, the buffer has a pH of from about 7 to about 10.
  • 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.
  • the kit includes homologous recombination template polynucleotides.
  • 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.
  • the target gene is present in the 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 non-human primate, bovine, porcine, or rodent cells.
  • the cell is a non-mammalian eukaryotic cell, such as poultry or fish.
  • 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.
  • 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.
  • a nucleic acid molecule eg, plasmid
  • 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.
  • the break results in reduced transcription of the target gene.
  • the method further comprises: contacting the editing template with the target gene, or delivering to the cell containing the target gene.
  • 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.
  • the mutation results in one or more amino acid changes in the protein expressed from the gene containing the target sequence.
  • the modification further includes inserting an editing template (eg, exogenous nucleic acid) into the break.
  • an editing template eg, exogenous nucleic acid
  • the protein, conjugate, fusion protein, isolated nucleic acid molecule, complex, carrier, or composition is contained in a delivery vehicle.
  • 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).
  • viral vectors eg, replication-defective retroviruses, lentiviruses, adenoviruses
  • adeno-associated virus eg, viral vectors (eg, replication-defective retroviruses, lentiviruses, adenoviruses) Or adeno-associated virus).
  • 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.
  • 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.
  • the nucleic acid molecule is present in the 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 non-human primate, bovine, porcine, or rodent cells.
  • the cell is a non-mammalian eukaryotic cell, such as poultry or fish.
  • 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.
  • 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.
  • a nucleic acid molecule eg, 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, carrier, or composition is contained in a delivery vehicle.
  • 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).
  • viral vectors eg, replication-defective retroviruses, lentiviruses, adenoviruses
  • adeno-associated virus eg, viral vectors (eg, replication-defective retroviruses, lentiviruses, adenoviruses) Or adeno-associated virus).
  • 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.
  • 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.
  • 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.
  • 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 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.
  • 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.
  • 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 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.
  • a poultry bird eg, chicken
  • fish or crustacean
  • 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.).
  • the cells are stem cells or stem cell lines.
  • 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:
  • 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.
  • CRISPR regularly clustered short palindrome repeat
  • Cas CRISPR-Cas system
  • CRISPR system CRISPR system
  • 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.
  • crRNA CRISPR RNA
  • tracrRNA trans-acting crRNA
  • 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.
  • the terms “guide RNA” and “mature crRNA” are used interchangeably and have the meaning commonly understood by those skilled in the art.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the targeting sequence is 10-30, or 15-25, or 15-22, or 19-25 or 19-22 nucleotides in length.
  • 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 .
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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”.
  • the editing template is an exogenous nucleic acid.
  • the recombination is homologous recombination.
  • target sequence or “target polynucleotide” may be any endogenous or exogenous polynucleotide for a cell (eg, eukaryotic cell).
  • 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).
  • a gene product eg, protein
  • non-coding sequence eg, regulatory polynucleotide or useless DNA.
  • PAM proximate motif
  • PAM protein kinase hydrolase
  • 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).
  • PAM sequence used with a given Cas effector protein.
  • 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.
  • target sequences or target polynucleotides include sequences related to signaling biochemical pathways, such as signaling biochemical pathway related genes or polynucleotides.
  • 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.
  • 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.
  • 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.
  • nucleic acid molecule or polypeptide 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.
  • ortholog 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.
  • identity is used to refer to the sequence matching between two polypeptides or between two nucleic acids.
  • 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)
  • 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.
  • the DNA sequences CTGACT and CAGGTT share 50% identity (3 out of 6 positions match).
  • 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.
  • the Needleman and Wunsch (JMoI Biol. 48: 444-453 (1970)) algorithms in the GAP program integrated into the GCG software package 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 .
  • the term "vector” refers to a nucleic acid vehicle into which a polynucleotide can be inserted.
  • the vector 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.
  • 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).
  • retroviruses including lentiviruses
  • adenoviruses such as herpes simplex virus
  • poxviruses such as herpes simplex virus
  • baculoviruses such as baculoviruses
  • papillomaviruses papillae Polyoma vacuolar virus
  • 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.
  • the vector may 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, 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.
  • 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
  • animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK293 cells or human cells.
  • 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).
  • 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).
  • 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).
  • 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.
  • the term "regulatory element” encompasses enhancer elements such as WPRE; CMV enhancer; R-U5 'fragment in the LTR of HTLV-I ((Mol. Cell.
  • 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.
  • 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).
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • treatment refers to treating or curing a disorder, delaying the onset of symptoms of the disorder, and / or delaying the development of the disorder.
  • 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.
  • the subject e.g., human
  • has a disorder e.g., a disorder caused by a disease-related genetic defect.
  • the SmCpf1 protein and system of the present invention have significant advantages.
  • the PAM domain of the SmCpf1 effector protein of the present invention has a 5'-TTN structure.
  • 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.
  • 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 4b is a schematic diagram of SmCpf1 cleaving DNMT1 in a human cell line.
  • SEQ ID: NO: description 1 Amino acid sequence of SmCpf1 2 SmCpf1 encoding nucleotide sequence 3 SmCpf1 Prototype Direct Repeat Sequence 4 SmCpf1 prototype direct repeat sequence encoding nucleic acid sequence 5 SmCpf1 mature direct repeat sequence 6 SmCpf1 mature direct repeat sequence encoding nucleic acid sequence 7 NLS sequence
  • LB liquid medium 10g Tryptone, 5g Yeast Extract, 10g NaCl, constant volume to 1L, sterilized. If antibiotics are needed, add 50 ⁇ g / 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 ⁇ g / 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
  • 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.
  • 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.
  • SmCpf1 also known as Cas12a
  • the protein sequence is shown in SEQ ID NO: 1
  • the coding DNA is As shown in SEQ ID NO: 2.
  • the prototype direct repeat sequence corresponding to SmCpf1 is shown in SEQ ID NO: 3.
  • the mature direct repeat sequence corresponding to SmCpf1 is shown in SEQ ID NO: 5.
  • the sequence alignment between SmCpf1 and other Cpf1 is shown in Figure 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.
  • step 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.
  • 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 ⁇ L 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.
  • RNA pellet was dissolved in 50 ⁇ L RNase-free water and incubated at 60 ° C for 10 minutes.
  • RNA monophosphorylation 20 ⁇ L RNA, 90 ° C for 1 min, and cooling on ice for 5 min.
  • 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 (4Units). 50 ⁇ L total volume. 37 ° C for 20min. Add 50 ⁇ L dH 2 O and adjust the volume to 100 ⁇ L.
  • 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.
  • 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.
  • 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).
  • positions 3863 to 3919 are the nucleotide sequence of the J23119 promoter
  • positions 3920 to 4081 are the nucleotide sequence of the CRISPR array
  • positions 4082 to 4108 are the nucleotide sequence of the rrnB-T1 terminator (Used to terminate transcription).
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • 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).

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Abstract

L'invention concerne des protéines effectrices Cas, une protéine de fusion comprenant lesdites protéines, et des molécules d'acide nucléique les codant. L'invention concerne un complexe et un composé pour l'édition d'acide nucléique, telle que l'édition de gênes ou de génome, comprenant les protéines pertinentes ou la protéine de fusion, ou les molécules d'acide nucléique les codant. L'invention concerne également un procédé destiné à être utilisé dans l'édition d'acide nucléique, telle que l'édition de gènes ou de génome, le procédé utilisant les protéines pertinentes ou la protéine de fusion.
PCT/CN2019/118871 2018-11-15 2019-11-15 Enzyme crispr-cas12a et système WO2020098793A1 (fr)

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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
CN115261359A (zh) * 2021-05-21 2022-11-01 山东舜丰生物科技有限公司 一种新型crispr酶和系统以及应用
WO2023039346A1 (fr) * 2021-09-10 2023-03-16 The Regents Of The University Of California Systèmes crispr-cas régulés en température et leurs procédés d'utilisation
CN116286742A (zh) * 2022-09-29 2023-06-23 隆平生物技术(海南)有限公司 CasD蛋白、CRISPR/CasD基因编辑系统及其在植物基因编辑中的应用
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CN116179512A (zh) * 2023-03-16 2023-05-30 华中农业大学 靶标识别范围广的核酸内切酶及其应用
CN116179512B (zh) * 2023-03-16 2023-09-15 华中农业大学 靶标识别范围广的核酸内切酶及其应用
CN117568311A (zh) * 2023-11-16 2024-02-20 广西科学院 一种用于精准基因编辑的工程化crispr酶和系统及其应用

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