WO2019206233A1 - Protéine effectrice crispr/cas éditée par arn et système - Google Patents

Protéine effectrice crispr/cas éditée par arn et système Download PDF

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WO2019206233A1
WO2019206233A1 PCT/CN2019/084340 CN2019084340W WO2019206233A1 WO 2019206233 A1 WO2019206233 A1 WO 2019206233A1 CN 2019084340 W CN2019084340 W CN 2019084340W WO 2019206233 A1 WO2019206233 A1 WO 2019206233A1
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sequence
protein
nucleic acid
seq
composition
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PCT/CN2019/084340
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English (en)
Chinese (zh)
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赖锦盛
张湘博
周英思
朱金洁
吕梦璐
赵海铭
宋伟彬
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中国农业大学
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Priority to CN201980028197.0A priority Critical patent/CN112020560B/zh
Publication of WO2019206233A1 publication Critical patent/WO2019206233A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/46—Hydrolases (3)
    • A61K38/47—Hydrolases (3) acting on glycosyl compounds (3.2), e.g. cellulases, lactases
    • 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

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-mediated immunity mainly includes three stages: (1) adaptive stage, cas1-cas2 protein complex inserts the target DNA fragment into the CRISPR repeat region; (2) expression and processing stage, CRISPR sequence transcription and cas The effector protein processes the mature crRNA; (3) the interference phase, the cas protein and the crRNA assemble into a complex to process the target DNA or RNA.
  • the CRISPR-cas system has evolved into a variety of defense mechanisms, including RNA-mediated DNA or RNA editing mechanisms.
  • the CRISPR-cas system is divided into class1 and class2.
  • Class1 is a complex composed of multiple proteins and is relatively complex.
  • the Class 2 system is relatively simple in structure and has only one effector protein, so it is widely used.
  • Type II and typeV edit DNA Type VI CRISPR-Cas system has cleavage activity on the target RNA.
  • the RNA-edited CRISPR-Cas system can regulate genes at the level of gene transcription, as well as detect live viruses, RNA interference, gene-selective splicing, and fluorescence in situ hybridization. Therefore, it is especially important to mine new RNA editing systems.
  • the inventors of the present application have unexpectedly discovered a novel RNA-directed ribonuclease endonuclease after extensive experimentation and repeated exploration. Based on this finding, the inventors have developed a new RNA editing 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: 1-7, 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 biological functions include, but are not limited to, activity binding to a targeting RNA, endoribonuclease activity, binding to a specific site of a target sequence under the guidance of a targeting RNA, and cleavage.
  • the ortholog, homolog, variant has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least compared to the sequence from which it is derived. 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
  • the ortholog, homolog, variant has at least 80%, at least 85%, at least 90%, compared to the sequence set forth in any one of SEQ ID NOs: 1-7, 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
  • the biological function of the sequence eg, activity binding to the targeting RNA, endoribonuclease activity, activity that binds to a specific site of the target sequence and cleaves under the guidance of a targeting RNA).
  • the protein is an effector protein in the CRISPR/Cas system.
  • the protein of the invention comprises a sequence selected from the group consisting of: or consists of a sequence selected from the group consisting of:
  • the protein of the invention comprises a sequence selected from the group consisting of: or consists of a sequence selected from the group consisting of:
  • the protein of the invention has the amino acid sequence set forth in SEQ ID NO:1.
  • the protein of the invention comprises a sequence selected from the group consisting of: or consists of a sequence selected from the group consisting of:
  • the protein of the invention has the amino acid sequence set forth in SEQ ID NO:2.
  • the protein of the invention comprises a sequence selected from the group consisting of: or consists of a sequence selected from the group consisting of:
  • the protein of the invention has the amino acid sequence set forth in SEQ ID NO:3.
  • 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: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.
  • 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: 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 SEQ ID NO:7.
  • 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:22.
  • the NLS sequence is located 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: 23-29.
  • 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 isolated nucleic acid molecule comprises one or more stem loops or an optimized secondary structure.
  • the sequence of any of (ii)-(v) retains the secondary structure of the sequence from which it is derived.
  • the nucleic acid molecule comprises a sequence selected from the group consisting of: or consists of a sequence selected from the group consisting of:
  • the isolated nucleic acid molecule is RNA.
  • the isolated nucleic acid molecule comprises a sequence selected from the group consisting of: or consists of:
  • the isolated nucleic acid molecule comprises a sequence selected from the group consisting of: or consists of:
  • the isolated nucleic acid molecule comprises a sequence selected from the group consisting of: or consists of:
  • the isolated nucleic acid molecule comprises a sequence selected from the group consisting of: or consists of:
  • the isolated nucleic acid molecule comprises a sequence selected from the group consisting of: or consists of:
  • the isolated nucleic acid molecule comprises a sequence selected from the group consisting of: or consists of:
  • the isolated nucleic acid molecule comprises a sequence selected from the group consisting of: or consists of:
  • the isolated nucleic acid molecule comprises a sequence selected from the group consisting of: or consists of:
  • the isolated nucleic acid molecule comprises a sequence selected from the group consisting of: or consists of:
  • the isolated nucleic acid molecule comprises a sequence selected from the group consisting of: or consists of:
  • the isolated nucleic acid molecule comprises a sequence selected from the group consisting of: or consists of:
  • the isolated nucleic acid molecule comprises a sequence selected from the group consisting of: or consists of:
  • the 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 an isolated nucleic acid molecule as described above and a targeting sequence capable of hybridizing to the target sequence
  • the targeting sequence is linked to the 3' or 5' 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 tracrRNA.
  • 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.
  • the host cell is a prokaryotic cell.
  • 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, 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' or 5' end of the homologous 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 is an RNA sequence from a prokaryotic or eukaryotic cell. In certain embodiments, the target sequence is a non-naturally occurring RNA sequence.
  • the target sequence is present in a cell. In certain embodiments, the target sequence is present within the nucleus or within the cytoplasm (eg, an organelle). In certain embodiments, the cell is a prokaryotic cell. In certain embodiments, the cell is a eukaryotic cell.
  • the protein is linked to one or more NLS sequences.
  • the conjugate or fusion protein comprises one or more NLS sequences.
  • the NLS sequence is linked to the N-terminus or C-terminus of the protein.
  • the NLS sequence is fused to the N-terminus or C-terminus of the protein.
  • the present invention also provides a composition comprising one or more carriers, the one or more carriers comprising:
  • a first nucleic acid which is a nucleotide sequence encoding a protein or fusion protein of the invention; optionally the first nucleic acid is operably linked to a first regulatory element;
  • a second nucleic acid encoding a nucleotide sequence comprising a targeting RNA; optionally the second nucleic acid is operably linked to a second regulatory element;
  • the first nucleic acid and the second nucleic acid are present on the same or different carrier;
  • the targeting RNA comprises a homologous repeat sequence and a targeting sequence 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' or 5' end of the homologous 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 is an RNA sequence from a prokaryotic or eukaryotic cell. In certain embodiments, the target sequence is a non-naturally occurring RNA sequence.
  • the target sequence is present in a cell. In certain embodiments, the target sequence is present within the nucleus or within the cytoplasm (eg, an organelle). In certain embodiments, the cell is a prokaryotic cell. In certain embodiments, the cell is a eukaryotic cell.
  • the protein is linked to one or more NLS sequences.
  • the conjugate or fusion protein comprises one or more NLS sequences.
  • the NLS sequence is linked to the N-terminus or C-terminus of the protein.
  • the NLS sequence is fused to the N-terminus or C-terminus of the protein.
  • 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 further comprises an RNA template.
  • the RNA template comprises an RNA sequence encoding a protein or a non-coding RNA sequence (eg, a microRNA).
  • the invention provides a method of modifying a target sequence, comprising: the complex of the fifth aspect, the composition of the ninth aspect, the composition of the tenth aspect Or the delivery composition as described herein is contacted with the target sequence or delivered to a cell comprising the target sequence; wherein the target sequence is associated with or is present in the gene of interest And the target sequence is RNA.
  • the target sequence 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 sequence is present in a nucleic acid molecule (eg, a plasmid) in vitro. In certain embodiments, the target sequence is present in a plasmid.
  • a nucleic acid molecule eg, a plasmid
  • the modification refers to a cleavage of the target sequence, such as a double strand break or a single strand break.
  • the target sequence is a ssRNA.
  • the method further comprises contacting the RNA template with the target sequence or delivering to a cell comprising the target sequence.
  • the modification further comprises inserting an RNA template (eg, an exogenous nucleic acid) into the fragment.
  • 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 for RNA interference or modulation of gene expression.
  • the methods modulate gene expression by modulating RNA processing or RNA activation (RNAa).
  • RNA processing can include RNA splicing (including alternative splicing), viral replication (eg, satellite viruses, phage or retroviruses, eg, HBV, HCV, HIV, etc.) or tRNA biosynthesis.
  • the RNAa promotes gene expression.
  • the method inhibits gene expression by interfering with or reducing RNAa.
  • 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 one or more of the following, or in the preparation of a formulation for one or more selected from the group consisting of:
  • the invention provides a method of detecting a target sequence, comprising the complex of the fifth aspect, the composition of the ninth aspect, the composition of the tenth aspect or A delivery composition as described herein is contacted with the target sequence or delivered to a cell comprising the target sequence; wherein the target sequence is RNA.
  • a targeting sequence contained in the complex, composition or delivery composition is capable of hybridizing to the target sequence.
  • the target sequence is present in a nucleic acid molecule in vitro.
  • the target sequence is present in a cell.
  • the cell is a prokaryotic cell.
  • the cell is a living cell.
  • the protein component comprised by the complex, composition or delivery composition carries a detectable label.
  • the protein component comprised by the complex, composition or delivery composition is fused with a fluorescent protein (eg, GFP).
  • a fluorescent protein eg, GFP
  • the method is northern blotting.
  • the northern blotting method involves isolating RNA samples by size by electrophoresis.
  • the complexes or compositions of the invention can be used to specifically bind and detect target RNA sequences.
  • the method is fluorescence in situ hybridization.
  • the protein component of the complex, composition or delivery composition of the invention carries a detectable label (eg, a fluorescent label).
  • 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 detecting a target sequence, or in the preparation of a preparation for detecting a target sequence.
  • Cas13e in the complex when the CRISPR/Cas complex of the invention binds to a target RNA, Cas13e in the complex is activated, followed by cleavage of any nearby ssRNA sequence (ie, collateral cleavage) ). Once Cas13e is primed by the target RNA, other (non-complementary) RNA molecules can be cleaved. This confounding RNA cleavage can cause cytotoxicity or otherwise affect cell physiology or cellular status.
  • the invention provides a method of modulating the state of a target cell, comprising the composition of the fifth aspect, the composition of the ninth aspect, as described in the tenth aspect
  • the composition or delivery composition as described herein is introduced into the target cell.
  • a target sequence that hybridizes to a targeting sequence contained in the complex, composition, or delivery composition is present in the target cell.
  • the modulating the state of the target cell comprises:
  • the cell is a prokaryotic cell.
  • 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 one or more of the following, or in the preparation of a formulation for one or more selected from the group consisting of:
  • the methods as described above may be therapeutic or prophylactic, and may target a particular target cell, cell (sub)pop, or cell/tissue type.
  • the particular cell, cell (sub)pop, or cell/tissue type expresses one or more target sequences, such as one or more specific target RNAs.
  • target cells include tumor cells that express a particular transcript, a given class of neurons, cells that cause autoimmunity, or cells that are infected with a particular pathogen (eg, a virus).
  • the present invention also provides a method of treating a pathological condition characterized by the presence of a defective cell, comprising administering to a subject in need thereof a complex as described in the fifth aspect
  • a method of treating a pathological condition characterized by the presence of a defective cell comprising administering to a subject in need thereof a complex as described in the fifth aspect
  • 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 of the fourth aspect
  • the nucleic acid molecule, the complex according to the fifth aspect, the isolated nucleic acid molecule according to the sixth aspect, the carrier according to the seventh aspect, the composition according to the ninth aspect, according to the tenth aspect Use of the composition, kit of the invention or delivery composition for the treatment of a pathological condition characterized by the presence of defective cells. It will be appreciated that in the above embodiments, the complex or composition of the invention preferably targets a specific target sequence of the defective cell.
  • the pathological condition characterized by the presence of a defective cell is a tumor.
  • the present invention also provides a method of treating a tumor comprising administering to a subject in need thereof a complex according to the fifth aspect, as described in the ninth aspect A composition, a composition as described in the tenth aspect, or a delivery composition as described herein.
  • 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 of the fourth aspect
  • the nucleic acid molecule, the complex according to the fifth aspect, the isolated nucleic acid molecule according to the sixth aspect, the carrier according to the seventh aspect, the composition according to the ninth aspect, according to the tenth aspect Use of the composition, kit of the invention or delivery composition for the treatment of a tumor.
  • the complex or composition of the invention preferably targets a tumor cell specific target sequence.
  • the pathological condition characterized by the presence of a defective cell is a disease caused by a pathogen infection or infection by a pathogen.
  • the present invention also provides a method of treating a pathogen infection or a disease caused by a pathogen infection, comprising administering to a subject in need thereof a compound as described in the fifth aspect
  • 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 of the fourth aspect
  • the complex or composition of the invention preferably targets a specific target sequence (e.g., a target sequence derived from the pathogen) of a cell infected by the pathogen.
  • the pathological condition characterized by the presence of a defective cell is an autoimmune disease.
  • the present invention also provides a method of treating an autoimmune disease, comprising administering to a subject in need thereof a complex according to the fifth aspect, as in the ninth aspect A composition, a composition as described in the tenth aspect, or a delivery composition as described herein.
  • 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 of the fourth aspect
  • the nucleic acid molecule, the complex according to the fifth aspect, the isolated nucleic acid molecule according to the sixth aspect, the carrier according to the seventh aspect, the composition according to the ninth aspect, according to the tenth aspect Use of the composition, kit of the invention or delivery composition for the treatment of an autoimmune disease.
  • the complex or composition of the invention preferably targets a specific target sequence of a cell (eg, a particular immune cell) that causes the autoimmune disease.
  • 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 of the fourth aspect
  • Such methods of treatment include gene editing, transcriptome editing, or gene therapy.
  • modifications introduced to cells by the methods of the invention can cause the cells and their progeny to be altered to improve the production of their biological products, such as antibodies, starch, ethanol, or other desired cellular output.
  • modifications introduced into the cell by the methods of the invention can cause the cell and its progeny to include changes that result in a change in the produced biological product.
  • the invention relates to a cell or a progeny thereof obtained by the method as described above, wherein the cell contains a modification that is not found in its wild type.
  • the modification results in a change in transcription or translation of at least one RNA product. In certain embodiments, the modification results in increased expression of at least one RNA product. In certain embodiments, the modification results in decreased expression of at least one RNA product.
  • the cell is a prokaryotic cell.
  • the cell is a eukaryotic cell. In certain embodiments, the cell is a mammalian cell, such as a human cell.
  • 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.
  • Cas13e 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 Cas13e of the present invention is an endonuclease which binds to a specific site of a target RNA sequence and cleaves under the guidance of a guide RNA.
  • 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 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. , 55, 50, 45, 40, 35, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 15 , 10 or fewer 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 is RNA. Therefore, “target sequence” and “target RNA” are used interchangeably in the present invention.
  • the target sequence can be any suitable form of RNA, such as mRNA, tRNA, rRNA, miRNA, siRNA or shRNA.
  • the expression "target sequence” may be any endogenous or exogenous RNA sequence to a cell (eg, a eukaryotic cell).
  • 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.
  • RNA template A sequence or template that can be used to integrate into an RNA sequence comprising the target sequence is referred to as an "RNA template.”
  • the RNA template is an exogenous nucleic acid.
  • the target RNA can be a sequence encoding a gene product (eg, a protein) (eg, mRNA or pre-mRNA) or a non-coding sequence (eg, ncRNA, lncRNA, tRNA or rRNA).
  • a gene product eg, a protein
  • mRNA or pre-mRNA eg, mRNA or pre-mRNA
  • a non-coding sequence eg, ncRNA, lncRNA, tRNA or rRNA
  • Non-limiting examples of target RNA include sequences associated with signaling biochemical pathways (eg, signaling biochemical pathway-associated RNA) or disease-associated RNA.
  • the "disease-associated RNA” refers to an RNA sequence produced by the RNA sequence in an abnormal level or abnormal form in a tissue or cell affected by the disease, compared to a tissue or a cell not having a disease control. presence.
  • the "disease-associated RNA” may be transcribed from a gene having an abnormally elevated expression level, or may be an RNA transcribed from a gene having abnormally decreased expression, wherein the altered expression level is related to the occurrence and/or development of the disease.
  • Disease-associated RNA also refers to RNA transcribed from a gene having a mutation or gene mutation that is directly responsible for or is in linkage disequilibrium with the gene causing the disease.
  • the translated product may be known or unknown and may be at a normal or abnormal level.
  • the target RNA can comprise interfering RNA (ie, RNA present in the RNA interference pathway, eg, shRNA, siRNA, etc.).
  • the target RNA is a microRNA (miRNA).
  • wild type has the meaning commonly understood by those skilled in the art to mean a typical form of a organism, a strain, a gene, or a feature that distinguishes it from a mutant or variant when it exists in nature. It can be isolated from sources in nature and not intentionally modified.
  • nucleic acid molecule or polypeptide As used herein, the terms “non-naturally occurring” or “engineered” are used interchangeably and refer to artificial participation. When these terms are used to describe a nucleic acid molecule or polypeptide, it is meant that the nucleic acid molecule or polypeptide is at least substantially freed from at least one other component of its association in nature or as found in nature.
  • an "ortholog" of a protein as referred to herein refers to a protein belonging to a different species that performs the same or similar function as a protein that is an ortholog thereof.
  • identity is used to mean the matching of sequences between two polypeptides or between two nucleic acids.
  • a position in the two sequences being compared is occupied by the same base or amino acid monomer subunit (for example, a position in each of the two DNA molecules is occupied by adenine, or two
  • Each position in each of the polypeptides is occupied by lysine, and then each molecule is identical at that position.
  • the "percent identity" between the two sequences is a function of the number of matching positions shared by the two sequences divided by the number of positions to be compared x 100. For example, if 6 of the 10 positions of the two sequences match, then the two sequences have 60% identity.
  • the DNA sequences CTGACT and CAGGTT share 50% identity (3 out of a total of 6 positions match).
  • the comparison is made when the two sequences are aligned to produce maximum identity.
  • Such alignment can be achieved by, for example, the method of Needleman et al. (1970) J. Mol. Biol. 48: 443-453, which can be conveniently performed by a computer program such as the Align program (DNAstar, Inc.). It is also possible to use the algorithm of E. Meyers and W. Miller (Comput. Appl Biosci., 4: 11-17 (1988)) integrated into the ALIGN program (version 2.0), using the PAM 120 weight residue table.
  • the gap length penalty of 12 and the gap penalty of 4 were used to determine the percent identity between the two amino acid sequences.
  • the Needleman and Wunsch (J MoI Biol. 48: 444-453 (1970)) algorithms in the GAP program integrated into the GCG software package can be used, using the Blossum 62 matrix or The PAM250 matrix and the gap weight of 16, 14, 12, 10, 8, 6 or 4 and the length weight of 1, 2, 3, 4, 5 or 6 to determine the percent identity between two amino acid sequences .
  • vector refers to a nucleic acid vehicle into which a polynucleotide can be inserted.
  • a vector is referred to as an expression vector when the vector enables expression of the protein encoded by the inserted polynucleotide.
  • the vector can be introduced into the host cell by transformation, transduction or transfection, and the genetic material element carried thereby can be expressed in the host cell.
  • Vectors are well known to those skilled in the art and include, but are not limited to, plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or P1 derived artificial chromosomes (PAC).
  • Phage such as lambda phage or M13 phage and animal virus.
  • Animal viruses useful as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, nipples Multi-tumor vacuolar virus (such as SV40).
  • a vector may contain a variety of elements that control expression, including, but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. In addition, the vector may also contain an origin of replication.
  • the term "host cell” refers to a cell that can be used to introduce a vector, including, but not limited to, a prokaryotic cell such as Escherichia coli or Bacillus subtilis, such as a fungal cell such as a yeast cell or an Aspergillus.
  • a prokaryotic cell such as Escherichia coli or Bacillus subtilis
  • a fungal cell such as a yeast cell or an Aspergillus.
  • S2 Drosophila cells or insect cells such as Sf9
  • animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells or human cells.
  • a vector can be introduced into a host cell to thereby produce a transcript, protein, or peptide, including a protein, fusion protein, isolated nucleic acid molecule, etc. as described herein (eg, a CRISPR transcript, such as a nucleic acid transcript) , protein, or enzyme).
  • a CRISPR transcript such as a nucleic acid transcript
  • regulatory element is intended to include promoters, enhancers, internal ribosome entry sites (IRES), and other expression control elements (eg, transcription termination signals, such as polyadenylation signals and Poly U sequence), a detailed description can be found in Goeddel, GENE 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.
  • 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.
  • rodent eg, a mouse or rat
  • non-human primate eg, a macaque or a cynomolgus monkey
  • the Cas effector protein and system of the invention can efficiently perform RNA editing, detecting living virus, RNA interference, gene selective scission, fluorescence in situ hybridization, etc., and realizing regulation of genes at the level of gene transcription. This will provide important resources for new applications in genomic engineering and biotechnology.
  • Figure 1 shows the two HEPN domains of the cas13e protein.
  • Figure 2 shows the secondary structure of the repeat sequence of cas13e.
  • A, B, C, D, E, F, and G are the repeat secondary structures of cas13e1.1, cas13e1.2, cas13e1.3, cas13e1.4, cas13e1.5, cas13e2.1, and cas13e2.2, respectively.
  • Figure 3 shows the activity of cas13e1.3 protein on the processing of pre-crRNA in bacteria.
  • Figure 4 shows the in vitro processing activity of cas13e1.3 protein on pre-crRNA.
  • SEQ ID NO: description 1 Amino acid sequence of Cas13e1.1 2 Amino acid sequence of Cas13e1.2 3 Amino acid sequence of Cas13e1.3 4 Amino acid sequence of Cas13e1.4 5 Amino acid sequence of Cas13e1.5 6 Amino acid sequence of Cas13e2.1 7 Amino acid sequence of Cas13e2.2 8 Encoding nucleic acid sequence of Cas13e1.1 9 Encoding nucleic acid sequence of Cas13e1.2 10 Encoding nucleic acid sequence of Cas13e1.3 11 Encoding nucleic acid sequence of Cas13e1.4
  • Cas13e a novel family of Cas effector proteins, namely Cas13e, which can be divided into two subfamilies, namely cas13e.1 and cas13e.2.
  • the family protein sequences are designated Cas13e1.1 (SEQ ID NO: 1), Cas13e1.2 (SEQ ID NO: 2), Cas13e1.3 (SEQ ID NO: 3), Cas13e1.4 (SEQ ID NO: 4), Cas13e1.5 (SEQ ID NO: 5), Cas13e2.1 (SEQ ID NO: 6), Cas13e2.2 (SEQ ID NO: 7), the DNA encoding these seven proteins are as shown in SEQ ID NOs: 8-14, respectively. Show.
  • the prototype homologous repeat sequences corresponding to the seven proteins are shown in SEQ ID NOs: 15-21.
  • the two HEPN domains of the above seven cas13e proteins were obtained by performing multiple sequence alignment of the proteins to analyze the functional domains containing "RxxxxH” (Fig. 1). Further, the secondary structure of the prototype repeat sequence of the above seven cas13e proteins was obtained by analysis by Vienna RNA software (Fig. 2).
  • a double-stranded DNA molecule of SEQ ID NO: 10 is artificially synthesized, and a double-stranded DNA molecule encoding SEQ ID NO: 32 (CRISPR array: repeat + spacer 1 + repeat + spacer + repeat) is artificially synthesized.
  • the double-stranded DNA molecule synthesized in the step 1 was ligated to the prokaryotic expression vector pACYC-Duet-1 to obtain a recombinant plasmid pACYC-Duet-1-CRISPR/cas13e1.3.
  • the recombinant plasmid pACYC-Duet-1-CRISPR/cas13e1.3 was subjected to one-generation sequencing to confirm the DNA sequence described in the step 1.
  • the recombinant plasmid pACYC-Duet-1-CRISPR/cas13e1.3 was introduced into Escherichia coli EC100 to obtain a recombinant strain, and the recombinant strain was named EC100-CRISPR/cas13e1.3.
  • the monoclonal clone of EC100-CRISPR/cas13e1.3 was taken, inoculated into 100 mL of LB liquid medium (containing 50 ⁇ g/mL ampicillin), and cultured at 37 ° C, shaking at 200 rpm for 12 hours to obtain a culture bacterial solution.
  • Extraction of bacterial RNA Transfer 1.5 mL of the bacterial culture to a pre-chilled microcentrifuge tube, centrifuge at 6,000 x g for 5 minutes at 4 °C. After centrifugation, the supernatant was discarded, and the cell pellet was resuspended in 200 ⁇ L MaxBacterial Enhancement Reagent preheated to 95 ° C, and mixed by pipetting. Incubate at 95 ° C for 4 minutes. Add 1 mL to the lysate Mix well by pipetting and incubate for 5 minutes at room temperature. Add 0.2 mL of cold chloroform, mix by shaking the tube for 15 seconds, and incubate for 2-3 minutes at room temperature.
  • RNA pellet was dissolved in 50 ⁇ L of RNase-free water and incubated at 60 ° C for 10 minutes.
  • RNAI Digestion of DNA: 20 ug of RNA was dissolved in 39.5 ⁇ L dH 2 O at 65 ° C for 5 min. On ice for 5 min, 0.5 ⁇ L of RNAI, 5 ⁇ L of buffer, 5 ⁇ L of DNase I, and 37 ° C for 45 min (50 ⁇ L system) were added. Add 50 ⁇ L dH 2 O and adjust the volume to 100 ⁇ L.
  • the precipitate was washed by adding 350 ⁇ L of 75% ethanol, centrifuged at 16000 g for 10 min at 4 ° C, and the supernatant was discarded. Dry, add 20 ⁇ L of RNase-free water, 65 ° C, and dissolve the precipitate for 5 min. NanoDrop measures the concentration and runs the glue.
  • the precipitate was washed by adding 350 ⁇ L of 75% ethanol, centrifuged at 16000 g for 10 min at 4 ° C, and the supernatant was discarded. Dry, add 21 ⁇ L of RNase-free water, 65 ° C, dissolve the precipitate for 5 min, and measure the concentration by NanoDrop.
  • RNA monophosphorylation 20 ⁇ L of RNA, 1 min at 90 ° C, and cooled on ice for 5 min. 2 ⁇ L of RNA 5'Polphosphatase 10 ⁇ Reaction buffer, 0.5 ⁇ L of Inhibitor, 1 ⁇ L of RNA 5'Polphosphatase (20 Units), RNase-free water to 20 ⁇ L, and 37 ° C for 60 min were added. Add 80 ⁇ L dH 2 O and adjust the volume to 100 ⁇ L.
  • cDNA library 16.5 ⁇ L RNase-free water. 5 ⁇ L Poly(A) Polymerase 10 ⁇ Reaction buffer. 5 ⁇ L of 10 mM ATP. 1.5 ⁇ L RiboGuard RNase Inhibitor. 20 ⁇ L RNA Substrate. 2 ⁇ L Poly(A) Polymerase (4 Units). 50 ⁇ L total volume. 37 ° C for 20 min. Add 50 ⁇ L dH 2 O and adjust the volume to 100 ⁇ L.
  • the cDNA library was added to the sequencing linker and sent to Beijing Berry Hekang for sequencing.
  • RNA sequence of 25 nt to 50 nt was retained and aligned to the reference sequence of the CRISPR array using bowtie.
  • the results are shown in Fig. 3.
  • the peak shape is the structure of the second-generation sequencing sequence alignment of the CRISPR block
  • the vertical line shows the enzyme cleavage site
  • the gray rectangle is the Repeat structure diagram
  • the dark gray diamond shape is the spacer sequence structure diagram.
  • Primers for designing a precrRNA transcript template The structure of the transcription template is: T7 promoter + CRISPR array of Cas13e1.3 (SEQ ID NO: 32). The primers were designed using Primer 5.0 software to ensure that the upstream and downstream primers have overlapping sequences of at least 18 bp. A double stranded DNA template was obtained using a primer annealing procedure. The template was purified using a MinElute PCR Purifcation Kit, and the concentration was measured with Nanodrop, and stored at -20 ° C until use.
  • RNA transcription was performed using NEB's HiScribe T7 high-efficiency RNA synthesis kit, and the PCR reaction procedure was set to: 37 ° C / 3 h or 31 ° C / forever, DNAseI was added, 37 ° C / 45 min.
  • Cas13e1.3 is an EDTA-sensitive protein
  • an EDTA group was also designed, which added EDTA to the reaction system.
  • the SYBR-Gold nucleic acid gel dye was added to 1 ⁇ TBE running buffer, placed in a gel, and stained for 10 minutes at room temperature.
  • Cas13e1.3 is capable of cleaving pre-crRNA in vitro and is not an EDTA-sensitive protein.

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Abstract

L'invention se rapporte au domaine de l'édition d'acides nucléiques, en particulier au domaine de la technologie des courtes répétitions palindromiques groupées régulièrement espacées (CRISPR). En particulier, l'invention concerne une protéine effectrice Cas, une protéine de fusion comprenant la protéine effectrice Cas et des molécules d'acide nucléique codant pour celle-ci. L'invention concerne également un composé et une composition pour l'édition d'acides nucléiques (par exemple, l'édition génique ou génomique) comprenant la protéine ou la protéine de fusion de l'invention ou des 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) à l'aide de la protéine ou de la protéine de fusion de l'invention.
PCT/CN2019/084340 2018-04-25 2019-04-25 Protéine effectrice crispr/cas éditée par arn et système WO2019206233A1 (fr)

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WO2020098772A1 (fr) * 2018-11-15 2020-05-22 中国农业大学 Enzyme crispr-cas12j et système
WO2022098681A3 (fr) * 2020-11-03 2022-06-16 Caspr Biotech Corporation Nouvelles endonucléases guidées par un arn crispr-cas de classe 2
WO2023030340A1 (fr) * 2021-08-30 2023-03-09 Huigene Therapeutics Co., Ltd. Nouvelle conception d'arn guide et ses utilisations
EP4110933A4 (fr) * 2020-02-28 2024-02-21 HuidaGene Therapeutics Co., Ltd. Système crispr-cas de type vi-e et de type vi-f et ses utilisations

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CN116555227B (zh) * 2021-02-03 2024-08-06 山东舜丰生物科技有限公司 新型Cas酶以及应用
CN117050973B (zh) * 2021-10-29 2024-07-19 山东舜丰生物科技有限公司 新型Cas酶和系统以及应用
WO2024175016A1 (fr) * 2023-02-21 2024-08-29 中国农业大学 Protéine effectrice crispr/cas et système associé

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WINSTON X. YAN ET AL.: "Casl3d is a Compact RNA-targeting Type VI CRISPR Effector Positively Modulated by a WYL Domain-Containing Accessory Protein", MOL CELL, vol. 70, no. 2, 19 April 2018 (2018-04-19), pages 327 - 339, XP055529724, DOI: 10.1016/j.molcel.2018.02.028 *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2020098772A1 (fr) * 2018-11-15 2020-05-22 中国农业大学 Enzyme crispr-cas12j et système
EP4110933A4 (fr) * 2020-02-28 2024-02-21 HuidaGene Therapeutics Co., Ltd. Système crispr-cas de type vi-e et de type vi-f et ses utilisations
WO2022098681A3 (fr) * 2020-11-03 2022-06-16 Caspr Biotech Corporation Nouvelles endonucléases guidées par un arn crispr-cas de classe 2
WO2023030340A1 (fr) * 2021-08-30 2023-03-09 Huigene Therapeutics Co., Ltd. Nouvelle conception d'arn guide et ses utilisations

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