WO2022266849A1 - Criblage de nouvelle protéine crispr-cas13 et son utilisation - Google Patents

Criblage de nouvelle protéine crispr-cas13 et son utilisation Download PDF

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Publication number
WO2022266849A1
WO2022266849A1 PCT/CN2021/101596 CN2021101596W WO2022266849A1 WO 2022266849 A1 WO2022266849 A1 WO 2022266849A1 CN 2021101596 W CN2021101596 W CN 2021101596W WO 2022266849 A1 WO2022266849 A1 WO 2022266849A1
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Prior art keywords
rna
protein
crispr
cas13
sequence
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PCT/CN2021/101596
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English (en)
Chinese (zh)
Inventor
周海波
许争争
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中国科学院脑科学与智能技术卓越创新中心
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Priority to PCT/CN2021/101596 priority Critical patent/WO2022266849A1/fr
Priority to CN202280042114.5A priority patent/CN117480251A/zh
Priority to PCT/CN2022/100531 priority patent/WO2022268135A1/fr
Publication of WO2022266849A1 publication Critical patent/WO2022266849A1/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
    • 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
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the nucleic acid molecule is codon-optimized for expression in a particular host cell.
  • the gRNA sequence comprises a direct repeat (DR) sequence and a sequence targeting a spacer region of the target RNA portion.
  • DR direct repeat
  • the DR sequence may be a derivative corresponding to any of the following, wherein the derivative (i) has one or more (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) nucleotide additions, deletions, or substitutions; (ii) any one of the sequences shown in Table 1 has at least 20 %, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 97% sequence identity; (iii) any one of the sequences shown in Table 1 under stringent conditions, or hybridize to any one of (i) and (ii); or (iv) is the complement of any one of (i)-(iii), provided that the derivative is not any of the sequences shown in Table 1 One, and said derivative encodes, or is itself an RNA, said RNA substantially maintaining the same secondary structure as any RNA encoded by SEQ ID NO: 199-397.
  • the derivative has one or more (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) nucleotide additions, deletions, or substitution
  • the target cells are ex vivo cells, in vitro cells or in vivo cells.
  • the prokaryotic microorganism is a DNA virus or nucleic acid thereof, an RNA virus or nucleic acid thereof.
  • the negative control is the control group that only expresses mcherry protein (red light) and DZ109 protein (green light);
  • PS397-PS399 is the experimental group containing sgRNA targeting different regions of mcherry, and the DR used is DZ109b.
  • R1 and R2 represent two different experimental replicates.
  • Eukaryotic cells such as mammalian cells, including human cells (human primary cells or established human cell lines).
  • the cells may be non-human mammalian cells, e.g., from non-human primates (e.g. monkeys), cows/bulls/cattle, sheep, goats, pigs, horses, dogs, cats, rodents (e.g. rabbits, small, rats, hamsters) etc.
  • the cells are from fish (eg, salmon), birds (eg, birds, including chickens, ducks, geese), reptiles, shellfish (eg, oysters, clams, lobsters, shrimps), insects, worms, yeast, and the like.
  • the cells may be from a plant, such as a monocot or a dicot.
  • the plants may be food crops such as barley, cassava, cotton, peanuts, corn, millet, oil palm fruit, potatoes, beans, rapeseed or canola, rice, rye, sorghum, soybeans, sugar cane, sugar Beets, sunflowers and wheat.
  • the plants may be cereals (eg barley, corn, millet, rice, rye, sorghum and wheat).
  • the plants may be tubers (eg cassava and potatoes).
  • the plant may be a sugar crop (eg, sugar beet and sugar cane).
  • the plant may be an oleaginous crop (such as soybean, peanut, rapeseed or canola, sunflower and oil palm fruit).
  • the plant may be a fiber crop (eg cotton).
  • These signals can be received by detection instruments and converted into electrical signals before being read out, so that the detection of target nucleic acids can be achieved.
  • further integration of machine learning algorithm models can further quantify and predict target nucleic acids. Therefore, it can be widely used in virus detection, such as the detection of new coronavirus; it can also be widely used in the non-invasive diagnosis of diseases (such as tumors), such as liquid biopsy.

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  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Biomedical Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Analytical Chemistry (AREA)
  • Plant Pathology (AREA)
  • Immunology (AREA)
  • Medicinal Chemistry (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

Criblage d'une nouvelle protéine CRISPR-Cas13 et son utilisation. L'invention concerne également une nouvelle protéine de la famille Cas13, un procédé de criblage de la nouvelle protéine de la famille Cas13, et des systèmes de détection d'ARN, des systèmes d'édition d'ARN et d'autres systèmes correspondants, et leur utilisation. L'invention concerne la protéine Cas13 et les systèmes de détection d'ARN, les systèmes d'édition d'ARN et autres systèmes connexes. La nouvelle protéine Cas13 contient des domaines HEPN plus étendus. Grâce au procédé de recherche rapide de la protéine Cas13, on obtient diverses nouvelles protéines Cas13 présentant de larges perspectives d'application et une énorme valeur marchande.
PCT/CN2021/101596 2021-06-22 2021-06-22 Criblage de nouvelle protéine crispr-cas13 et son utilisation WO2022266849A1 (fr)

Priority Applications (3)

Application Number Priority Date Filing Date Title
PCT/CN2021/101596 WO2022266849A1 (fr) 2021-06-22 2021-06-22 Criblage de nouvelle protéine crispr-cas13 et son utilisation
CN202280042114.5A CN117480251A (zh) 2021-06-22 2022-06-22 新型crispr-cas13蛋白的筛选及其应用
PCT/CN2022/100531 WO2022268135A1 (fr) 2021-06-22 2022-06-22 Criblage et utilisation de protéines crispr-cas13 de type nouveau

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/CN2021/101596 WO2022266849A1 (fr) 2021-06-22 2021-06-22 Criblage de nouvelle protéine crispr-cas13 et son utilisation

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WO2022266849A1 true WO2022266849A1 (fr) 2022-12-29

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PCT/CN2021/101596 WO2022266849A1 (fr) 2021-06-22 2021-06-22 Criblage de nouvelle protéine crispr-cas13 et son utilisation
PCT/CN2022/100531 WO2022268135A1 (fr) 2021-06-22 2022-06-22 Criblage et utilisation de protéines crispr-cas13 de type nouveau

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WO (2) WO2022266849A1 (fr)

Cited By (2)

* Cited by examiner, † Cited by third party
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CN117844782A (zh) * 2024-03-06 2024-04-09 崖州湾国家实验室 靶向范围广的基因编辑核酸酶及其在核酸检测中的应用
CN117844782B (zh) * 2024-03-06 2024-05-31 崖州湾国家实验室 靶向范围广的基因编辑核酸酶及其在核酸检测中的应用

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* Cited by examiner, † Cited by third party
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WO2023076952A1 (fr) * 2021-10-27 2023-05-04 Metagenomi, Inc. Enzymes ayant des domaines hepn

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CN110312799A (zh) * 2016-08-17 2019-10-08 博德研究所 新型crispr酶和系统
CN112041444A (zh) * 2018-03-14 2020-12-04 阿伯生物技术公司 新型crispr dna靶向酶及系统
US20200392473A1 (en) * 2017-12-22 2020-12-17 The Broad Institute, Inc. Novel crispr enzymes and systems
CN112105728A (zh) * 2018-05-07 2020-12-18 中国农业大学 CRISPR/Cas效应蛋白及系统
CN112410377A (zh) * 2020-02-28 2021-02-26 中国科学院脑科学与智能技术卓越创新中心 VI-E型和VI-F型CRISPR-Cas系统及用途
US20210139890A1 (en) * 2017-06-30 2021-05-13 Arbor Biotechnologies, Inc. Novel crispr rna targeting enzymes and systems and uses thereof

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JP2021508460A (ja) * 2017-12-22 2021-03-11 ザ・ブロード・インスティテュート・インコーポレイテッド Ｃｒｉｓｐｒエフェクター系に基づくマルチプレックス診断
EP3999117A4 (fr) * 2019-07-12 2023-07-26 Duke University Systèmes de nanoparticules pour l'administration ciblée de crispr/cas13 et leurs procédés d'utilisation
US11767528B2 (en) * 2019-08-16 2023-09-26 Massachusetts Institute Of Technology Targeted trans-splicing using CRISPR/Cas13

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110312799A (zh) * 2016-08-17 2019-10-08 博德研究所 新型crispr酶和系统
US20210139890A1 (en) * 2017-06-30 2021-05-13 Arbor Biotechnologies, Inc. Novel crispr rna targeting enzymes and systems and uses thereof
US20200392473A1 (en) * 2017-12-22 2020-12-17 The Broad Institute, Inc. Novel crispr enzymes and systems
CN112041444A (zh) * 2018-03-14 2020-12-04 阿伯生物技术公司 新型crispr dna靶向酶及系统
CN112105728A (zh) * 2018-05-07 2020-12-18 中国农业大学 CRISPR/Cas效应蛋白及系统
CN112410377A (zh) * 2020-02-28 2021-02-26 中国科学院脑科学与智能技术卓越创新中心 VI-E型和VI-F型CRISPR-Cas系统及用途

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN117844782A (zh) * 2024-03-06 2024-04-09 崖州湾国家实验室 靶向范围广的基因编辑核酸酶及其在核酸检测中的应用
CN117844782B (zh) * 2024-03-06 2024-05-31 崖州湾国家实验室 靶向范围广的基因编辑核酸酶及其在核酸检测中的应用

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WO2022268135A1 (fr) 2022-12-29
CN117480251A (zh) 2024-01-30

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