WO2019214604A1 - Protéine effectrice crispr/cas et système associé - Google Patents

Protéine effectrice crispr/cas et système associé Download PDF

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WO2019214604A1
WO2019214604A1 PCT/CN2019/085826 CN2019085826W WO2019214604A1 WO 2019214604 A1 WO2019214604 A1 WO 2019214604A1 CN 2019085826 W CN2019085826 W CN 2019085826W WO 2019214604 A1 WO2019214604 A1 WO 2019214604A1
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sequence
nucleic acid
protein
cell
acid molecule
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PCT/CN2019/085826
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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 CN201980030881.2A priority Critical patent/CN112105728B/zh
Publication of WO2019214604A1 publication Critical patent/WO2019214604A1/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
    • C12N15/11—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
    • 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
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • 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 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: 1-18, or an ortholog, homolog, variant or functional fragment thereof; Wherein the ortholog, homologue, variant or functional fragment substantially retains the biological function of the sequence from which it is derived.
  • the protein has Cas effector activity. In certain embodiments, the protein is an effector protein in the CRISPR/Cas system.
  • the biological function of the above sequence refers to Cas effector activity, including but not limited to, 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 Activity.
  • 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, and substantially retains the Cas effector activity of the sequence from which it is derived (eg, Activity that binds 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).
  • Activity that binds to the targeting RNA eg, Endonuclease activity, activity that binds to a specific site of the target sequence and cleaves under the guidance of a targeting RNA.
  • 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: 1-18, 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 the origin of the sequence Sequence of Cas effector activity (eg, activity binding to targeting RNA, endonuclease activity, activity that binds to a specific site of the target sequence and cleaves under the guidance of a targeting RNA).
  • Cas effector activity eg, activity binding to 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 from a species selected from the group consisting of Sulfuricurvum sp, Omnitrophica WOR_2, Smithella sp, and Agrobacterium sp. And X.
  • the protein has the amino acid sequence set forth in any one of SEQ ID NOs: 5-18, or an ortholog, homolog, variant or functional fragment thereof; wherein the homologous The source, homologue, variant or functional fragment substantially retains the biological function of the sequence from which it is derived.
  • the ortholog, homolog, variant has at least 80%, at least 85%, at least 90% compared to the amino acid sequence set forth in any one of SEQ ID NOs: 5-18 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 origin
  • the Cas effector activity of the sequence eg, activity binding 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 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 or 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 or 4.
  • 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: 5 or 6.
  • 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 any one of SEQ ID NOs: 7-18.
  • 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:73.
  • 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: 74-91.
  • 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 a sequence selected from the group consisting 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 a sequence selected from the group consisting 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 a sequence selected from the group consisting 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 a sequence selected from the group consisting 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 a sequence selected from the group consisting 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 a sequence selected from the group consisting 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 a sequence selected from the group consisting 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 a sequence selected from the group consisting 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 a sequence selected from the group consisting 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, in the 5' to 3' direction, the isolated nucleic acid molecule of the fourth aspect 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 5'-NTN or 5'-TNN The sequence shown, wherein N is selected from the group consisting of 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 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 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 5'-NTN or 5'-TNN The sequence shown, wherein N is selected from the group consisting of 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 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 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.
  • 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 microvesicle, a gene gun, or a viral vector (eg, replication defective reverse transcription) Virus, lentivirus, adenovirus or adeno-associated virus).
  • a viral vector eg, replication defective reverse transcription
  • 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 includes an editing template for homologous recombination.
  • 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 the editing template, wherein the repair results in a mutation comprising one or more nucleotides of the target gene Insert, delete, or replace.
  • 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 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 Use of a kit or delivery composition of the invention in the preparation of a formulation for:
  • 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 said cell contains a modification which 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.
  • Cas12g means a Cas effector protein first discovered and identified by the present inventors having an amino acid sequence selected from the group consisting of:
  • the Cas12g 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.
  • Cas12h 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 Cas12h 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.
  • Cas12w 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 Cas12w 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.
  • Cas12j 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 Cas12j 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 23 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 as 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 EXPRE SSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San 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 the 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 Cas proteins and systems of the invention have significant advantages over the prior art.
  • the 5'-TG structure of the PAM domain of the Cas effector of the present invention can complement each other with a system in which the PAM is 5'-TTN, expanding the recognition range.
  • the Cas effector of the present invention is capable of DNA cleavage in eukaryotes and is about 200-400 amino acids smaller in size than Cpf1 and Cas9 proteins, and thus is significantly more efficient than Cpf1 and Cas9 in transfection efficiency.
  • Figure 1 is a graph showing the results of PAM domain analysis of Cas12h.1 in Example 3.
  • 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.
  • 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, we only consider macromolecular proteins 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.
  • the CRISPR/Cas protein family was obtained by annotating the CRISPR-rich 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.
  • Cas12g which includes two active homolog sequences, named Cas12g.1 (SEQ ID NO: 1) and Cas12g. 2 (SEQ. ID NO: 2), the coding DNAs of the two homologs are shown in SEQ ID NOs: 19, 20, respectively.
  • the prototype homologous repeat sequences (repeat sequences contained in the pre-crRNA) corresponding to Cas12g.1 and Cas12g.2 are shown in SEQ ID NOs: 37 and 38, respectively.
  • 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, we only consider macromolecular proteins 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.
  • 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.
  • Cas12h which includes two active homolog sequences, named Cas12h.1 (SEQ ID NO: 3) and Cas12h.2 (SEQ. ID NO: 4), the coding DNAs of the two homologs are shown in SEQ ID NOs: 21, 22, respectively.
  • the prototype homologous repeat sequences (repeat sequences contained in the pre-crRNA) corresponding to Cas12h.1 and Cas12h.2 are shown in SEQ ID NOs: 39 and 40, respectively.
  • the recombinant plasmid pACYC-Duet-1+CRISPR/Cas12h expresses the Cas12h.1 protein shown in SEQ ID NO: 3 and the Cas12h.1 targeting RNA shown in SEQ ID NO:39.
  • the recombinant plasmid pACYC-Duet-1+CRISPR/Cas12h.1 contains an expression cassette, and the nucleotide sequence of the expression cassette is shown in SEQ ID NO:94.
  • SEQ ID NO: 94 the nucleotide sequence of the pLacZ promoter from positions 1 to 44 from the 5' end, and the nucleotide sequence of the Cas12h.1 gene from positions 45 to 2708, from page 2709 to The 2794 position is the nucleotide sequence of the terminator (used to terminate transcription).
  • positions 2795 to 2834 are the nucleotide sequence of the J23119 promoter
  • positions 2835 to 3007 are the nucleotide sequence of the CRISPR array
  • positions 3008 to 3093 are the nucleotide sequence of the rrnB-T1 terminator. (used to terminate transcription).
  • SEQ ID NO: 93 The sequence shown in SEQ ID NO: 93 was artificially synthesized and ligated into the pUC19 vector, wherein the sequence set forth in SEQ ID NO: 93 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 Cas12h.1 locus and E. coli containing no Cas.12h.1 locus. After 1 hour of treatment at 37 ° C, we extracted the plasmid and PCR amplification and sequencing of the PAM region sequence.
  • PAM library domain The number of occurrences of 65,536 combinations of PAM sequences in the experimental and control groups were separately counted and normalized by the number of all PAM sequences in each group. For any one PAM sequence, when log2 (control group normalized value / experimental group normalized value) is greater than 2, this PAM is considered to be significantly consumed. The significantly consumed PAM sequence was predicted by Weblogo. The results are shown in Figure 1.
  • the PAM domain of Cas12h.1 is a 5'-TG structure. This PAM can complement other systems with PAM 5'-TTN. Recognition range.

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Abstract

La présente invention concerne le domaine de l'édition d'acides nucléiques et, en particulier, le domaine technique des répétitions palindromiques groupées, courtes et régulièrement espacées (CRISPR). 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 un complexe et un composé pour l'édition d'acide nucléique (par exemple, l'édition de gène ou de génome), 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, édition de gène ou de génome), qui utilise la protéine effectrice Cas ou la protéine de fusion.
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WO2020098772A1 (fr) * 2018-11-15 2020-05-22 中国农业大学 Enzyme crispr-cas12j et système
GB2595606B (en) * 2019-03-07 2022-09-21 Univ California CRISPR-Cas effector polypeptides and methods of use thereof
US11578313B2 (en) 2019-03-07 2023-02-14 The Regents Of The University Of California CRISPR-Cas effector polypeptides and methods of use thereof
CN113811607A (zh) * 2019-03-07 2021-12-17 加利福尼亚大学董事会 CRISPR-Cas效应子多肽和其使用方法
WO2020181101A1 (fr) * 2019-03-07 2020-09-10 The Regents Of The University Of California Polypeptides effecteurs crispr-cas et procédés d'utilisation associés
JP2022521771A (ja) * 2019-03-07 2022-04-12 ザ リージェンツ オブ ザ ユニバーシティ オブ カリフォルニア ＣＲＩＳＰＲ－Ｃａｓエフェクターポリペプチド及びその使用方法
US11377646B2 (en) 2019-03-07 2022-07-05 The Regents Of The University Of California Modified cells comprising CRISPR systems
US11739309B2 (en) 2019-03-07 2023-08-29 The Regents Of The University Of California CRISPR-Cas12J effector polypeptides and methods of use thereof
US11685909B2 (en) 2019-03-07 2023-06-27 The Regents Of The University Of California CRISPR-Cas effector polypeptides and methods of use thereof
US11530398B2 (en) 2019-03-07 2022-12-20 The Regents Of The University Of California CRISPR-Cas effector polypeptides and methods of use thereof
GB2595606A (en) * 2019-03-07 2021-12-01 Univ California CRISPR-Cas effector polypeptides and methods of use thereof
JP7239725B2 (ja) 2019-03-07 2023-03-14 ザ リージェンツ オブ ザ ユニバーシティ オブ カリフォルニア ＣＲＩＳＰＲ－Ｃａｓエフェクターポリペプチド及びその使用方法
WO2021113522A1 (fr) * 2019-12-04 2021-06-10 Arbor Biotechnologies, Inc. Compositions comprenant une nucléase et leurs utilisations
US20230159943A1 (en) * 2020-04-20 2023-05-25 The Regents Of The University Of California Crispr systems in plants
US11946077B2 (en) 2020-06-04 2024-04-02 Emendobio Inc. OMNI-59, 61, 67, 76, 79, 80, 81, and 82 CRISPR nucleases
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
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
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

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