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JPWO2020168291A5 - - Google Patents

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Publication number
JPWO2020168291A5
JPWO2020168291A5 JP2021547336A JP2021547336A JPWO2020168291A5 JP WO2020168291 A5 JPWO2020168291 A5 JP WO2020168291A5 JP 2021547336 A JP2021547336 A JP 2021547336A JP 2021547336 A JP2021547336 A JP 2021547336A JP WO2020168291 A5 JPWO2020168291 A5 JP WO2020168291A5
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Prior art keywords
ribonucleic acid
sequence
engineered
engineered nuclease
acid sequence
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JP2021547336A
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Japanese (ja)
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JP2022520428A (en
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Priority claimed from PCT/US2020/018432 external-priority patent/WO2020168291A1/en
Publication of JP2022520428A publication Critical patent/JP2022520428A/en
Publication of JPWO2020168291A5 publication Critical patent/JPWO2020168291A5/ja
Priority to JP2023146414A priority Critical patent/JP7502537B2/en
Priority to JP2024091759A priority patent/JP2024133476A/en
Ceased legal-status Critical Current

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Claims (16)

操作されたヌクレアーゼ組成物であって、
（ａ）配列番号：２２４２と少なくとも９０％の配列同一性を有する配列を含むＲｕｖＣ＿ＩＩＩドメインを含むエンドヌクレアーゼと、
（ｂ）前記エンドヌクレアーゼと複合体を形成するように構成される、操作されたガイドリボ核酸構造であって、前記操作されたガイドリボ核酸構造は、
（ｉ）標的デオキシリボ核酸配列にハイブリダイズするように構成されたガイドリボ核酸配列と、
（ｉｉ）前記エンドヌクレアーゼに結合するように構成されたｔｒａｃｒリボ核酸配列であって、前記エンドヌクレアーゼが配列番号：４２１と少なくとも９０％の配列同一性を有する配列を含む、ｔｒａｃｒリボ核酸配列と、を含む、操作されたガイドリボ核酸構造と、
を含む、
操作されたヌクレアーゼ組成物。 An engineered nuclease composition comprising
(a) an endonuclease comprising a RuvC_III domain comprising a sequence having at least 90% sequence identity with SEQ ID NO:2242;
(b) an engineered guide ribonucleic acid structure configured to form a complex with said endonuclease, said engineered guide ribonucleic acid structure comprising:
(i) a guide ribonucleic acid sequence configured to hybridize to a target deoxyribonucleic acid sequence;
(ii) a tracr ribonucleic acid sequence configured to bind to said endonuclease, said endonuclease comprising a sequence having at least 90% sequence identity with SEQ ID NO:421; an engineered guide ribonucleic acid structure comprising
including,
Engineered Nuclease Compositions . 前記エンドヌクレアーゼはＨＮＨドメインをさらに含む、請求項１に記載の操作されたヌクレアーゼ組成物。2. The engineered nuclease composition of claim 1, wherein said endonuclease further comprises an HNH domain. 前記ｔｒａｃｒリボ核酸配列は、配列番号：５４９５からの約６０～９０の連続するヌクレオチドに対して少なくとも８０％の配列同一性を有する配列を含む、請求項１に記載の操作されたヌクレアーゼ組成物。2. The engineered nuclease composition of claim 1, wherein said tracr ribonucleic acid sequence comprises a sequence having at least 80% sequence identity to about 60-90 contiguous nucleotides from SEQ ID NO:5495. 前記エンドヌクレアーゼは、配列番号：５５１７のプロトスペーサー隣接モチーフ（ＰＡＭ）配列に結合するように構成される、請求項１に記載の操作されたヌクレアーゼ組成物。2. The engineered nuclease composition of claim 1, wherein said endonuclease is configured to bind to the protospacer adjacent motif (PAM) sequence of SEQ ID NO:5517. 前記操作されたガイドリボ核酸構造は、少なくとも２つのリボ核酸ポリヌクレオチドを含む、請求項１に記載の操作されたヌクレアーゼ組成物。2. The engineered nuclease composition of claim 1, wherein said engineered guide ribonucleic acid structure comprises at least two ribonucleic acid polynucleotides. 前記操作されたガイドリボ核酸構造は、前記ガイドリボ核酸配列と前記ｔｒａｃｒリボ核酸配列とを含む１つのリボ核酸ポリヌクレオチドを含む、請求項１に記載の操作されたヌクレアーゼ組成物。2. The engineered nuclease composition of claim 1, wherein said engineered guide ribonucleic acid structure comprises one ribonucleic acid polynucleotide comprising said guide ribonucleic acid sequence and said tracr ribonucleic acid sequence. 前記ガイドリボ核酸配列は、原核生物、細菌、古細菌、真核生物、真菌、植物、哺乳動物、またはヒトのゲノム配列に相補的である、請求項１に記載の操作されたヌクレアーゼ組成物。2. The engineered nuclease composition of claim 1, wherein the guide ribonucleic acid sequence is complementary to a prokaryotic, bacterial, archaeal, eukaryotic, fungal, plant, mammalian, or human genomic sequence. 前記エンドヌクレアーゼは、前記エンドヌクレアーゼのＮ末端またはＣ末端の近位にある１つ以上の核局在化配列（ＮＬＳ）を含む、請求項１に記載の操作されたヌクレアーゼ組成物。2. The engineered nuclease composition of claim 1, wherein said endonuclease comprises one or more nuclear localization sequences (NLS) proximal to the N-terminus or C-terminus of said endonuclease. 前記１つ以上のＮＬＳは、配列番号：５５９７の配列を含む、請求項８に記載の操作されたヌクレアーゼ組成物。9. The engineered nuclease composition of claim 8, wherein said one or more NLSs comprise the sequence of SEQ ID NO:5597. ５’から３’に、前記標的デオキシリボ核酸配列の５’に少なくとも２０のヌクレオチドの配列を含む第１の相同性アームと、少なくとも１０のヌクレオチドの合成ＤＮＡ配列と、前記標的デオキシリボ核酸配列の３’に少なくとも２０のヌクレオチドの配列を含む第２の相同性アームとを含む、一本鎖または二本鎖のＤＮＡ修復鋳型をさらに含む、請求項１に記載の操作されたヌクレアーゼ組成物。5′ to 3′, a first homology arm comprising a sequence of at least 20 nucleotides 5′ of said target deoxyribonucleic acid sequence, a synthetic DNA sequence of at least 10 nucleotides, and 3′ of said target deoxyribonucleic acid sequence. 2. The engineered nuclease composition of claim 1, further comprising a single-stranded or double-stranded DNA repair template comprising a second homology arm comprising a sequence of at least 20 nucleotides in . 前記操作されたヌクレアーゼシステムは、ＭｇThe engineered nuclease system comprises Mg ＋+ の供給源をさらに含む、請求項１に記載の操作されたヌクレアーゼ組成物。2. The engineered nuclease composition of claim 1, further comprising a source of 前記エンドヌクレアーゼおよび前記ｔｒａｃｒリボ核酸配列は、同じ門内の別個の細菌種に由来する、請求項１に記載の操作されたヌクレアーゼ組成物。2. The engineered nuclease composition of claim 1, wherein said endonuclease and said tracr ribonucleic acid sequence are from separate bacterial species within the same phylum. 前記ガイドＲＮＡ構造は、ステムおよびループからなるヘアピンを含むと予想されるＲＮＡ配列を含み、ここで、前記ステムは、少なくとも１０の塩基対のリボヌクレオチド、および前記ループの４つの塩基対以内の非対称バルジを含む、請求項１に記載の操作されたヌクレアーゼ組成物。Said guide RNA structure comprises an RNA sequence predicted to contain a hairpin consisting of a stem and a loop, wherein said stem comprises at least 10 base pairs of ribonucleotides and an asymmetric within 4 base pairs of said loop. 2. The engineered nuclease composition of claim 1, comprising a bulge. 前記操作されたガイドリボ核酸構造の前記ｔｒａｃｒリボ核酸配列は、少なくとも８の塩基対のリボヌクレオチドを含むヘアピンを含む、請求項１に記載の操作されたヌクレアーゼ組成物。2. The engineered nuclease composition of claim 1, wherein the tracr ribonucleic acid sequence of the engineered guide ribonucleic acid structure comprises a hairpin comprising at least 8 base pairs of ribonucleotides. 前記操作されたガイドリボ核酸構造の前記ガイドリボ核酸配列は、ガイドリボ核酸配列の少なくとも８つのヌクレオチドおよびｔｒａｃｒリボ核酸配列の少なくとも８つのヌクレオチドを含む中断されていない塩基対領域を有するヘアピンを含むと予想され、ここで、前記ｔｒａｃｒリボ核酸配列は、５’から３’に、第１のヘアピンと第２のヘアピンとを含み、ここで、前記第１のヘアピンは前記第２のヘアピンよりも長いステムを有する、請求項１に記載の操作されたヌクレアーゼ組成物。said guide ribonucleic acid sequence of said engineered guide ribonucleic acid structure is expected to comprise a hairpin having an uninterrupted base pair region comprising at least 8 nucleotides of a guide ribonucleic acid sequence and at least 8 nucleotides of a tracr ribonucleic acid sequence; wherein said tracr ribonucleic acid sequence comprises, from 5′ to 3′, a first hairpin and a second hairpin, wherein said first hairpin has a longer stem than said second hairpin The engineered nuclease composition of claim 1. 前記エンドヌクレアーゼは、配列番号：４２１、または配列番号：４２１と少なくとも９０％の同一性を有する保存的に置換された配列を含む、請求項１に記載の操作されたヌクレアーゼ組成物。2. The engineered nuclease composition of claim 1, wherein said endonuclease comprises SEQ ID NO:421, or a conservatively substituted sequence having at least 90% identity to SEQ ID NO:421.
JP2021547336A 2019-02-14 2020-02-14 Enzyme with RUVC domain Ceased JP2022520428A (en)

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US201962805878P 2019-02-14 2019-02-14
US62/805,878 2019-02-14
US62/805,868 2019-02-14
US62/805,899 2019-02-14
US201962874414P 2019-07-15 2019-07-15
US62/874,414 2019-07-15
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KR (2) KR20240007322A (en)
CN (2) CN113728098A (en)
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MX2021009886A (en) * 2019-02-14 2021-10-13 Metagenomi Inc Enzymes with ruvc domains.
GB2594339B (en) * 2020-02-14 2023-02-08 Metagenomi Inc Enzymes with RUVC domains
MX2022011039A (en) 2020-03-06 2022-12-13 Metagenomi Inc Class ii, type v crispr systems.
KR20220161383A (en) * 2020-03-31 2022-12-06 메타지노미, 인크. Class II, type II CRISPR systems
WO2021226369A1 (en) * 2020-05-08 2021-11-11 Metagenomi Ip Technologies, Llc Enzymes with ruvc domains
WO2022056301A1 (en) * 2020-09-11 2022-03-17 Metagenomi Ip Technologies, Llc Base editing enzymes
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WO2022159742A1 (en) * 2021-01-22 2022-07-28 Metagenomi, Inc Novel engineered and chimeric nucleases
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JP2024517607A (en) * 2021-04-30 2024-04-23 メタゲノミ，インク． Enzymes containing RUVC domains
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