KR20230158531A - 신규 crispr 효소, 방법, 시스템 및 그 용도 - Google Patents

신규 crispr 효소, 방법, 시스템 및 그 용도 Download PDF

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KR20230158531A
KR20230158531A KR1020237034367A KR20237034367A KR20230158531A KR 20230158531 A KR20230158531 A KR 20230158531A KR 1020237034367 A KR1020237034367 A KR 1020237034367A KR 20237034367 A KR20237034367 A KR 20237034367A KR 20230158531 A KR20230158531 A KR 20230158531A
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cas9
sequence
seq
protein
nucleic acid
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Korean (ko)
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베른트 제츠
루이스 바레라
데이비드 에이. 본
밍 쑨
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빔 테라퓨틱스, 인크.
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    • C12N9/22—Ribonucleases [RNase]; Deoxyribonucleases [DNase]
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    • A61K38/465—Hydrolases (3) acting on ester bonds (3.1), e.g. lipases, ribonucleases
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KR1020237034367A 2021-03-23 2022-03-23 신규 crispr 효소, 방법, 시스템 및 그 용도 Pending KR20230158531A (ko)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
US202163164798P 2021-03-23 2021-03-23
US63/164,798 2021-03-23
PCT/US2022/021523 WO2022204268A2 (en) 2021-03-23 2022-03-23 Novel crispr enzymes, methods, systems and uses thereof

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KR20230158531A true KR20230158531A (ko) 2023-11-20

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US (1) US20240167008A1 (https=)
EP (1) EP4314265A2 (https=)
JP (1) JP2024511621A (https=)
KR (1) KR20230158531A (https=)
CN (1) CN117529555A (https=)
AU (1) AU2022245243A1 (https=)
CA (1) CA3211495A1 (https=)
WO (1) WO2022204268A2 (https=)

Families Citing this family (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN121079412A (zh) 2023-04-28 2025-12-05 比姆医疗股份有限公司 经修饰的指导rna
AU2024274409A1 (en) * 2023-05-12 2025-11-27 Vertex Pharmaceuticals Incorporated Compact proteins
WO2025024493A1 (en) 2023-07-25 2025-01-30 Flagship Pioneering Innovations Vii, Llc Cas endonucleases and related methods
KR20260044217A (ko) 2023-07-25 2026-04-01 플래그쉽 파이어니어링 이노베이션스 Vii, 엘엘씨 Cas 엔도뉴클레아제 및 관련 방법
WO2025072331A1 (en) 2023-09-26 2025-04-03 Flagship Pioneering Innovations Vii, Llc Cas nucleases and related methods
WO2025117877A2 (en) 2023-12-01 2025-06-05 Flagship Pioneering Innovations Vii, Llc Cas nucleases and related methods
CN117866926B (zh) * 2024-03-07 2024-08-16 珠海舒桐医疗科技有限公司 一种CRISPR-FrCas9蛋白突变体及应用

Family Cites Families (18)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4880635B1 (en) 1984-08-08 1996-07-02 Liposome Company Dehydrated liposomes
US4797368A (en) 1985-03-15 1989-01-10 The United States Of America As Represented By The Department Of Health And Human Services Adeno-associated virus as eukaryotic expression vector
US4921757A (en) 1985-04-26 1990-05-01 Massachusetts Institute Of Technology System for delayed and pulsed release of biologically active substances
US4920016A (en) 1986-12-24 1990-04-24 Linear Technology, Inc. Liposomes with enhanced circulation time
JPH0825869B2 (ja) 1987-02-09 1996-03-13 株式会社ビタミン研究所 抗腫瘍剤包埋リポソ−ム製剤
US4917951A (en) 1987-07-28 1990-04-17 Micro-Pak, Inc. Lipid vesicles formed of surfactants and steroids
US4911928A (en) 1987-03-13 1990-03-27 Micro-Pak, Inc. Paucilamellar lipid vesicles
US5173414A (en) 1990-10-30 1992-12-22 Applied Immune Sciences, Inc. Production of recombinant adeno-associated virus vectors
US5587308A (en) 1992-06-02 1996-12-24 The United States Of America As Represented By The Department Of Health & Human Services Modified adeno-associated virus vector capable of expression from a novel promoter
US5846946A (en) 1996-06-14 1998-12-08 Pasteur Merieux Serums Et Vaccins Compositions and methods for administering Borrelia DNA
AU2005274948B2 (en) 2004-07-16 2011-09-22 Genvec, Inc. Vaccines against aids comprising CMV/R-nucleic acid constructs
US8404658B2 (en) 2007-12-31 2013-03-26 Nanocor Therapeutics, Inc. RNA interference for the treatment of heart failure
US9405700B2 (en) 2010-11-04 2016-08-02 Sonics, Inc. Methods and apparatus for virtualization in an integrated circuit
CN105139759B (zh) 2015-09-18 2017-10-10 京东方科技集团股份有限公司 一种拼接屏
IL310721B2 (en) 2015-10-23 2025-11-01 Harvard College Nucleobase editors and uses thereof
CN110214183A (zh) 2016-08-03 2019-09-06 哈佛大学的校长及成员们 腺苷核碱基编辑器及其用途
EP3592853A1 (en) * 2017-03-09 2020-01-15 President and Fellows of Harvard College Suppression of pain by gene editing
US20220307001A1 (en) * 2018-02-27 2022-09-29 President And Fellows Of Harvard College Evolved cas9 variants and uses thereof

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US20240167008A1 (en) 2024-05-23
WO2022204268A3 (en) 2022-10-20
WO2022204268A2 (en) 2022-09-29
CN117529555A (zh) 2024-02-06
AU2022245243A1 (en) 2023-09-28
JP2024511621A (ja) 2024-03-14
EP4314265A2 (en) 2024-02-07
CA3211495A1 (en) 2022-09-29

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