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- JPWO2019183150A5 JPWO2019183150A5 JP2020550714A JP2020550714A JPWO2019183150A5 JP WO2019183150 A5 JPWO2019183150 A5 JP WO2019183150A5 JP 2020550714 A JP2020550714 A JP 2020550714A JP 2020550714 A JP2020550714 A JP 2020550714A JP WO2019183150 A5 JPWO2019183150 A5 JP WO2019183150A5
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- 229920001184 polypeptide Polymers 0.000 claims description 119
- 102000004196 processed proteins & peptides Human genes 0.000 claims description 119
- 108090000765 processed proteins & peptides Proteins 0.000 claims description 119
- 125000003275 alpha amino acid group Chemical group 0.000 claims description 90
- 108020005004 Guide RNA Proteins 0.000 claims description 48
- 108091033409 CRISPR Proteins 0.000 claims description 44
- 102000039446 nucleic acids Human genes 0.000 claims description 44
- 108020004707 nucleic acids Proteins 0.000 claims description 44
- 150000007523 nucleic acids Chemical class 0.000 claims description 44
- 102000040430 polynucleotide Human genes 0.000 claims description 36
- 108091033319 polynucleotide Proteins 0.000 claims description 36
- 239000002157 polynucleotide Substances 0.000 claims description 36
- 241000191940 Staphylococcus Species 0.000 claims description 20
- 108020004414 DNA Proteins 0.000 claims description 16
- 102000053602 DNA Human genes 0.000 claims description 16
- 241000937219 Staphylococcus microti Species 0.000 claims description 16
- 229920002477 rna polymer Polymers 0.000 claims description 12
- FWMNVWWHGCHHJJ-SKKKGAJSSA-N 4-amino-1-[(2r)-6-amino-2-[[(2r)-2-[[(2r)-2-[[(2r)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid Chemical compound C([C@H](C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N1CCC(N)(CC1)C(O)=O)NC(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FWMNVWWHGCHHJJ-SKKKGAJSSA-N 0.000 claims description 10
- 150000002632 lipids Chemical class 0.000 claims description 8
- 239000002502 liposome Substances 0.000 claims description 8
- 239000002105 nanoparticle Substances 0.000 claims description 8
- 102000004389 Ribonucleoproteins Human genes 0.000 claims description 6
- 108010081734 Ribonucleoproteins Proteins 0.000 claims description 6
- 241000702421 Dependoparvovirus Species 0.000 claims description 4
- 108020004999 messenger RNA Proteins 0.000 claims description 4
- 101710163270 Nuclease Proteins 0.000 claims description 2
- 230000008685 targeting Effects 0.000 claims description 2
- 239000002773 nucleotide Substances 0.000 claims 1
- 125000003729 nucleotide group Chemical group 0.000 claims 1
Description
別の態様では、（ａ）請求項１～１１のいずれか一項に記載のｓＲＧＮポリペプチド、またはｓＲＧＮポリペプチドをコードする核酸と、（ｂ）ガイドＲＮＡ（ｇＲＮＡ）、またはｇＲＮＡをコードする核酸とを含み、ｇＲＮＡがｓＲＧＮポリペプチドまたはそのバリアントを標的ポリヌクレオチド配列へとガイドすることができる、キットが、本明細書に提供される。一部の実施形態では、キットは、異種ポリヌクレオチド配列を含むドナー鋳型をさらに含み、異種ポリヌクレオチド配列は、標的ポリヌクレオチド配列に挿入され得る。
特定の実施形態では、例えば以下の項目が提供される。
（項目１）
Ｎ末端からＣ末端に向かって：
１）配列番号３４～３７のいずれか１つのアミノ酸配列を含むかまたは配列番号３４～３７のいずれか１つに対して少なくとも約９０％配列同一性を有するそのバリアントを含むミニドメイン１と、
２）配列番号３８～４１のいずれか１つのアミノ酸配列を含むかまたは配列番号３８～４１のいずれか１つに対して少なくとも約９０％配列同一性を有するそのバリアントを含むミニドメイン２と、
３）配列番号４２～４５のいずれか１つのアミノ酸配列を含むかまたは配列番号４２～４５のいずれか１つに対して少なくとも約９０％配列同一性を有するそのバリアントを含むミニドメイン３と、
４）配列番号４６～４９のいずれか１つのアミノ酸配列を含むかまたは配列番号４６～４９のいずれか１つに対して少なくとも約９０％配列同一性を有するそのバリアントを含むミニドメイン４と、
５）配列番号５０～５３のいずれか１つのアミノ酸配列を含むかまたは配列番号５０～５３のいずれか１つに対して少なくとも約９０％配列同一性を有するそのバリアントを含むミニドメイン５と、
６）配列番号５４～５７のいずれか１つのアミノ酸配列を含むかまたは配列番号５４～５７のいずれか１つに対して少なくとも約９０％配列同一性を有するそのバリアントを含むミニドメイン６と、
７）配列番号５８～６１のいずれか１つのアミノ酸配列を含むかまたは配列番号５８～６１のいずれか１つに対して少なくとも約９０％配列同一性を有するそのバリアントを含むミニドメイン７と、
８）配列番号６２～６５のいずれか１つのアミノ酸配列を含むかまたは配列番号６２～６５のいずれか１つに対して少なくとも約９０％配列同一性を有するそのバリアントを含むミニドメイン８と
を含み、
前記ミニドメインのうちの少なくとも２つが、Ｓｔａｐｈｙｌｏｃｏｃｃｕｓ ｌｕｇｄｕｎｅｎｓｉｓ Ｃａｓ９（配列番号３０）、Ｓｔａｐｈｙｌｏｃｏｃｃｕｓ ｐａｓｔｅｕｒｉ Ｃａｓ９（配列番号３１）、Ｓｔａｐｈｙｌｏｃｏｃｃｕｓ ｍｉｃｒｏｔｉ Ｃａｓ９（配列番号３３）およびＳｔａｐｈｙｌｏｃｏｃｃｕｓ ｈｙｉｃｕｓ Ｃａｓ９（配列番号３２）からなる群から選択される異なる親Ｃａｓ９エンドヌクレアーゼに由来する、
合成ＲＮＡガイドヌクレアーゼ（ｓＲＧＮ）ポリペプチド。
（項目２）
前記ミニドメインのうちの少なくとも３つが、Ｓｔａｐｈｙｌｏｃｏｃｃｕｓ ｌｕｇｄｕｎｅｎｓｉｓ Ｃａｓ９（配列番号３０）、Ｓｔａｐｈｙｌｏｃｏｃｃｕｓ ｐａｓｔｅｕｒｉ Ｃａｓ９（配列番号３１）、Ｓｔａｐｈｙｌｏｃｏｃｃｕｓ ｍｉｃｒｏｔｉ Ｃａｓ９（配列番号３３）およびＳｔａｐｈｙｌｏｃｏｃｃｕｓ ｈｙｉｃｕｓ Ｃａｓ９（配列番号３２）からなる群から選択される異なる親Ｃａｓ９エンドヌクレアーゼに由来する、項目１に記載のｓＲＧＮポリペプチド。
（項目３）
ミニドメイン８が、配列番号６２のアミノ酸配列、または配列番号６２に対して少なくとも約９０％配列同一性を有するそのバリアントを含む、項目１または２に記載のｓＲＧＮポリペプチド。
（項目４）
ミニドメイン８が、配列番号６３のアミノ酸配列、または配列番号６３に対して少なくとも約９０％配列同一性を有するそのバリアントを含む、項目１または２に記載のｓＲＧＮポリペプチド。
（項目５）
ミニドメイン１が、配列番号３４のアミノ酸配列、または配列番号３４に対して少なくとも約９０％配列同一性を有するそのバリアントを含む、項目１～４のいずれか一項に記載のｓＲＧＮポリペプチド。
（項目６）
Ｎ末端からＣ末端に向かって：
１）配列番号６６～６９のいずれか１つのアミノ酸配列を含むかまたは配列番号６６～６９のいずれか１つに対して少なくとも約９０％配列同一性を有するそのバリアントを含むミニドメイン１と、
２）配列番号７０～７３のいずれか１つのアミノ酸配列を含むかまたは配列番号７０～７３のいずれか１つに対して少なくとも約９０％配列同一性を有するそのバリアントを含むミニドメイン２と、
３）配列番号７４～７７のいずれか１つのアミノ酸配列を含むかまたは配列番号７４～７７のいずれか１つに対して少なくとも約９０％配列同一性を有するそのバリアントを含むミニドメイン３と、
４）配列番号７８～８１のいずれか１つのアミノ酸配列を含むかまたは配列番号７８～８１のいずれか１つに対して少なくとも約９０％配列同一性を有するそのバリアントを含むミニドメイン４と、
５）配列番号８２～８５のいずれか１つのアミノ酸配列を含むかまたは配列番号８２～８５のいずれか１つに対して少なくとも約９０％配列同一性を有するそのバリアントを含むミニドメイン５と、
６）配列番号８６～８９のいずれか１つのアミノ酸配列を含むかまたは配列番号８６～８９のいずれか１つに対して少なくとも約９０％配列同一性を有するそのバリアントを含むミニドメイン６と、
７）配列番号９０～９３のいずれか１つのアミノ酸配列を含むかまたは配列番号９０～９３のいずれか１つに対して少なくとも約９０％配列同一性を有するそのバリアントを含むミニドメイン７と、
８）配列番号９４～９７のいずれか１つのアミノ酸配列を含むかまたは配列番号９４～９７のいずれか１つに対して少なくとも約９０％配列同一性を有するそのバリアントを含むミニドメイン８と、
９）配列番号９８～１０１のいずれか１つのアミノ酸配列を含むかまたは配列番号９８～１０１のいずれか１つに対して少なくとも約９０％配列同一性を有するそのバリアントを含むミニドメイン９と、
１０）配列番号１０２～１０５のいずれか１つのアミノ酸配列を含むかまたは配列番号１０２～１０５のいずれか１つに対して少なくとも約９０％配列同一性を有するそのバリアントを含むミニドメイン１０と、
１１）配列番号１０６～１０９のいずれか１つのアミノ酸配列を含むかまたは配列番号１０６～１０９のいずれか１つに対して少なくとも約９０％配列同一性を有するそのバリアントを含むミニドメイン１１と、
１２）配列番号１１０～１１３のいずれか１つのアミノ酸配列を含むかまたは配列番号１１０～１１３のいずれか１つに対して少なくとも約９０％配列同一性を有するそのバリアントを含むミニドメイン１２と
を含み、
前記ミニドメインのうちの少なくとも２つが、Ｓｔａｐｈｙｌｏｃｏｃｃｕｓ ｌｕｇｄｕｎｅｎｓｉｓ Ｃａｓ９（配列番号３０）、Ｓｔａｐｈｙｌｏｃｏｃｃｕｓ ｐａｓｔｅｕｒｉ Ｃａｓ９（配列番号３１）、Ｓｔａｐｈｙｌｏｃｏｃｃｕｓ ｍｉｃｒｏｔｉ Ｃａｓ９（配列番号３３）およびＳｔａｐｈｙｌｏｃｏｃｃｕｓ ｈｙｉｃｕｓ Ｃａｓ９（配列番号３２）からなる群から選択される異なる親Ｃａｓ９エンドヌクレアーゼに由来する、
ｓＲＧＮポリペプチド。
（項目７）
前記ミニドメインのうちの少なくとも３つが、Ｓｔａｐｈｙｌｏｃｏｃｃｕｓ ｌｕｇｄｕｎｅｎｓｉｓ Ｃａｓ９（配列番号３０）、Ｓｔａｐｈｙｌｏｃｏｃｃｕｓ ｐａｓｔｅｕｒｉ Ｃａｓ９（配列番号３１）、Ｓｔａｐｈｙｌｏｃｏｃｃｕｓ ｍｉｃｒｏｔｉ Ｃａｓ９（配列番号３３）およびＳｔａｐｈｙｌｏｃｏｃｃｕｓ ｈｙｉｃｕｓ Ｃａｓ９（配列番号３２）からなる群から選択される異なる親Ｃａｓ９エンドヌクレアーゼに由来する、項目６に記載のｓＲＧＮポリペプチド。
（項目８）
ミニドメイン１２が、配列番号１１０のアミノ酸配列、または配列番号１１０に対して少なくとも約９０％配列同一性を有するそのバリアントを含む、項目６または７に記載のｓＲＧＮポリペプチド。
（項目９）
ミニドメイン１２が、配列番号１１１のアミノ酸配列、または配列番号１１１に対して少なくとも約９０％配列同一性を有するそのバリアントを含む、項目６または７に記載のｓＲＧＮポリペプチド。
（項目１０）
（ｉ）ミニドメイン１が、配列番号６６のアミノ酸配列、または配列番号６６に対して少なくとも約９０％配列同一性を有するそのバリアントを含み、（ｉｉ）ミニドメイン２が、配列番号７０のアミノ酸配列、または配列番号７０に対して少なくとも約９０％配列同一性を有するそのバリアントを含む、項目６～９のいずれか一項に記載のｓＲＧＮポリペプチド。
（項目１１）
ａ）配列番号１のアミノ酸配列を含むかまたは配列番号１に対して少なくとも約９０％配列同一性を有するそのバリアントを含むＧｉｂ１１ポリペプチド、
ｂ）配列番号２のアミノ酸配列を含むかまたは配列番号２に対して少なくとも約９０％配列同一性を有するそのバリアントを含むＧｉｂ１１Ｓｐａ－１ポリペプチド、
ｃ）配列番号３のアミノ酸配列を含むかまたは配列番号３に対して少なくとも約９０％配列同一性を有するそのバリアントを含むＧｉｂ１１Ｓｐａ－２ポリペプチド、
ｄ）配列番号４のアミノ酸配列を含むかまたは配列番号４に対して少なくとも約９０％配列同一性を有するそのバリアントを含むＧｉｂ１１Ｓｐａ－３ポリペプチド、
ｅ）配列番号５のアミノ酸配列を含むかまたは配列番号５に対して少なくとも約９０％配列同一性を有するそのバリアントを含むＰ２Ｈ１２ポリペプチド、
ｆ）配列番号６のアミノ酸配列を含むかまたは配列番号６に対して少なくとも約９０％配列同一性を有するそのバリアントを含むＥ２ポリペプチド、
ｇ）配列番号７のアミノ酸配列を含むかまたは配列番号７に対して少なくとも約９０％配列同一性を有するそのバリアントを含むＥ２＋Ｋ７４１Ｄ＋Ｌ７４３Ｋポリペプチド、
ｈ）配列番号８のアミノ酸配列を含むかまたは配列番号８に対して少なくとも約９０％配列同一性を有するそのバリアントを含むＥ２＋Ｓ６７０Ｔ＋Ｎ６７５Ｄポリペプチド、
ｉ）配列番号９のアミノ酸配列を含むかまたは配列番号９に対して少なくとも約９０％配列同一性を有するそのバリアントを含むＥ２＋Ｋ７４１Ｎ＋Ｌ７４３Ｎポリペプチド、
ｊ）配列番号１０のアミノ酸配列を含むかまたは配列番号１０に対して少なくとも約９０％配列同一性を有するそのバリアントを含むＦ８ポリペプチド；
ｋ）配列番号１１のアミノ酸配列を含むかまたは配列番号１１に対して少なくとも約９０％配列同一性を有するそのバリアントを含むＦ８＋Ｋ７３７Ｄ＋Ｌ７３９Ｋポリペプチド、および
ｌ）配列番号１２のアミノ酸配列を含むかまたは配列番号１２に対して少なくとも約９０％配列同一性を有するそのバリアントを含むＦ８＋Ｋ７３７Ｎ＋Ｌ７３９Ｎポリペプチド
からなる群から選択されるｓＲＧＮポリペプチド。
（項目１２）
ａ）配列番号１のアミノ酸配列を含むかまたは配列番号１に対して少なくとも約９０％配列同一性を有するそのバリアントを含むＧｉｂ１１ポリペプチド、
ｂ）配列番号２のアミノ酸配列を含むかまたは配列番号２に対して少なくとも約９０％配列同一性を有するそのバリアントを含むＧｉｂ１１Ｓｐａ－１ポリペプチド、および
ｃ）配列番号４のアミノ酸配列を含むかまたは配列番号４に対して少なくとも約９０％配列同一性を有するそのバリアントを含むＧｉｂ１１Ｓｐａ－３ポリペプチド
からなる群から選択される、項目１１に記載のｓＲＧＮポリペプチド。
（項目１３）
ａ）配列番号１のアミノ酸配列を含むＧｉｂ１１ポリペプチド、
ｂ）配列番号２のアミノ酸配列を含むＧｉｂ１１Ｓｐａ－１ポリペプチド、および
ｃ）配列番号４のアミノ酸配列を含むＧｉｂ１１Ｓｐａ－３ポリペプチド
からなる群から選択される、項目１２に記載のｓＲＧＮポリペプチド。
（項目１４）
項目１～１３のいずれか一項に記載のｓＲＧＮポリペプチドをコードする核酸。
（項目１５）
宿主細胞における発現のためにコドン最適化されている、項目１４に記載の核酸。
（項目１６）
配列番号１３～２９のいずれか１つのヌクレオチド配列を含むかまたは配列番号１３～２９のいずれか１つに対して少なくとも約９０％配列同一性を有するそのバリアントを含む、項目１４または１５に記載の核酸。
（項目１７）
（ａ）項目１～１３のいずれか一項に記載のｓＲＧＮポリペプチド、または前記ｓＲＧＮポリペプチドをコードする核酸と、
（ｂ）ガイドＲＮＡ（ｇＲＮＡ）、または前記ｇＲＮＡをコードする核酸と
を含み、前記ｇＲＮＡが、前記ｓＲＧＮポリペプチドまたはそのバリアントを標的ポリヌクレオチド配列へとガイドすることができる、系。
（項目１８）
異種ポリヌクレオチド配列を含むドナー鋳型をさらに含み、前記異種ポリヌクレオチド配列が、前記標的ポリヌクレオチド配列に挿入され得る、項目１７に記載の系。
（項目１９）
前記ｓＲＧＮポリペプチドをコードする核酸が、宿主細胞における発現のためにコドン最適化されている、および／または前記異種ポリヌクレオチド配列が、宿主細胞における発現のためにコドン最適化されている、項目１７または１８に記載の系。
（項目２０）
前記ｓＲＧＮポリペプチドをコードする核酸が、デオキシリボ核酸（ＤＮＡ）である、項目１７～１９のいずれか一項に記載の系。
（項目２１）
前記ｓＲＧＮポリペプチドをコードする核酸が、リボ核酸（ＲＮＡ）である、項目１７～１９のいずれか一項に記載の系。
（項目２２）
前記ｓＲＧＮポリペプチドをコードするＲＮＡが、ｍＲＮＡである、項目２１に記載の系。
（項目２３）
前記ドナー鋳型が、アデノ随伴ウイルス（ＡＡＶ）ベクターにコードされる、項目１７～２２のいずれか一項に記載の系。
（項目２４）
前記ｓＲＧＮポリペプチド、または前記ｓＲＧＮポリペプチドをコードする核酸が、リポソームまたは脂質ナノ粒子内に製剤化されている、項目１７～２３のいずれか一項に記載の系。
（項目２５）
前記リポソームまたは脂質ナノ粒子が、前記ｇＲＮＡ、または前記ｇＲＮＡをコードする核酸も含む、項目２４に記載の系。
（項目２６）
前記ｇＲＮＡと予め複合体化されてリボヌクレオタンパク質（ＲＮＰ）複合体を形成しているｓＲＧＮポリペプチドを含む、項目１７～２３のいずれか一項に記載の系。
（項目２７）
標的遺伝子座における標的ＤＮＡを標的化、編集、改変または操作する方法であって、
（ａ）項目１～１３のいずれか一項に記載のｓＲＧＮポリペプチド、または前記ｓＲＧＮポリペプチドをコードする核酸と、
（ｂ）ガイドＲＮＡ（ｇＲＮＡ）、または前記ｇＲＮＡをコードする核酸と
を前記標的ＤＮＡに提供するステップを含み、
前記ｇＲＮＡが前記ｓＲＧＮポリペプチドを前記標的遺伝子座へとガイドすることができる、方法。
（項目２８）
異種ポリヌクレオチド配列を含むドナー鋳型を前記標的ＤＮＡに提供するステップをさらに含み、前記異種ポリヌクレオチド配列が、前記標的遺伝子座に挿入され得る、項目２７に記載の方法。
（項目２９）
前記ｓＲＧＮポリペプチドをコードする核酸が、宿主細胞における発現のためにコドン最適化されている、および／または前記異種ポリヌクレオチド配列が、宿主細胞における発現のためにコドン最適化されている、項目２７または２８に記載の方法。
（項目３０）
前記ｓＲＧＮポリペプチドをコードする核酸が、デオキシリボ核酸（ＤＮＡ）である、項目２７～２９のいずれか一項に記載の方法。
（項目３１）
前記ｓＲＧＮポリペプチドをコードする核酸が、リボ核酸（ＲＮＡ）である、項目２７～２９のいずれか一項に記載の方法。
（項目３２）
前記ｓＲＧＮポリペプチドをコードするＲＮＡが、ｍＲＮＡである、項目３１に記載の方法。
（項目３３）
前記ドナー鋳型が、アデノ随伴ウイルス（ＡＡＶ）ベクターにコードされる、項目２７～３２のいずれか一項に記載の方法。
（項目３４）
前記ｓＲＧＮポリペプチド、または前記ｓＲＧＮポリペプチドをコードする核酸が、リポソームまたは脂質ナノ粒子内に製剤化されている、項目２７～３３のいずれか一項に記載の方法。
（項目３５）
前記リポソームまたは脂質ナノ粒子が、前記ｇＲＮＡ、または前記ｇＲＮＡをコードする核酸も含む、項目３４に記載の方法。
（項目３６）
ＲＮＰ複合体として前記ｇＲＮＡと予め複合体化されたｓＲＧＮポリペプチドを標的ＤＮＡに提供するステップを含む、項目２７～３３のいずれか一項に記載の方法。
（項目３７）
（ａ）項目１～１３のいずれか一項に記載のｓＲＧＮポリペプチド、または前記ｓＲＧＮポリペプチドをコードする核酸と、
（ｂ）ガイドＲＮＡ（ｇＲＮＡ）、またはｇＲＮＡをコードする核酸と
を含み、前記ｇＲＮＡが、前記ｓＲＧＮポリペプチドまたはそのバリアントを標的ポリヌクレオチド配列へとガイドすることができる、改変された細胞。
（項目３８）
異種ポリヌクレオチド配列を含むドナー鋳型をさらに含み、前記異種ポリヌクレオチド配列が、前記標的ポリヌクレオチド配列に挿入され得る、項目３７に記載の改変された細胞。
（項目３９）
細胞のゲノムが、項目２７～３６のいずれか一項に記載の方法により編集されている、遺伝子改変された細胞。
（項目４０）
（ａ）項目１～１３のいずれか一項に記載のｓＲＧＮポリペプチド、または前記ｓＲＧＮポリペプチドをコードする核酸と、
（ｂ）ガイドＲＮＡ（ｇＲＮＡ）、またはｇＲＮＡをコードする核酸と
を含むキットであって、
前記ｇＲＮＡが、前記ｓＲＧＮポリペプチドまたはそのバリアントを標的ポリヌクレオチド配列へとガイドすることができる、キット。
（項目４１）
異種ポリヌクレオチド配列を含むドナー鋳型をさらに含み、前記異種ポリヌクレオチド配列が、前記標的ポリヌクレオチド配列に挿入され得る、項目４０に記載のキット。
In another embodiment, (a) a nucleic acid encoding the sRGN polypeptide or sRGN polypeptide according to any one of claims 1 to 11, and (b) a nucleic acid encoding a guide RNA (gRNA) or gRNA. A kit is provided herein in which a gRNA can guide an sRGN polypeptide or variant thereof to a target polynucleotide sequence. In some embodiments, the kit further comprises a donor template comprising a heterologous polynucleotide sequence, the heterologous polynucleotide sequence can be inserted into the target polynucleotide sequence.
In certain embodiments, for example, the following items are provided.
(Item 1)
From N-terminus to C-terminus:
1) A minidomain 1 containing the amino acid sequence of any one of SEQ ID NOs: 34-37 or containing a variant thereof having at least about 90% sequence identity to any one of SEQ ID NOs: 34-37.
2) A minidomain 2 containing the amino acid sequence of any one of SEQ ID NOs: 38-41 or containing a variant thereof having at least about 90% sequence identity to any one of SEQ ID NOs: 38-41.
3) A mini-domain 3 containing the amino acid sequence of any one of SEQ ID NOs: 42-45 or containing a variant thereof having at least about 90% sequence identity to any one of SEQ ID NOs: 42-45.
4) A minidomain 4 comprising any one of the amino acid sequences of SEQ ID NOs: 46-49 or containing a variant thereof having at least about 90% sequence identity to any one of SEQ ID NOs: 46-49.
5) A mini-domain 5 containing the amino acid sequence of any one of SEQ ID NOs: 50-53 or containing a variant thereof having at least about 90% sequence identity to any one of SEQ ID NOs: 50-53.
6) A minidomain 6 containing the amino acid sequence of any one of SEQ ID NOs: 54-57 or containing a variant thereof having at least about 90% sequence identity to any one of SEQ ID NOs: 54-57.
7) A minidomain 7 containing the amino acid sequence of any one of SEQ ID NOs: 58-61 or containing a variant thereof having at least about 90% sequence identity to any one of SEQ ID NOs: 58-61.
8) With a minidomain 8 containing the amino acid sequence of any one of SEQ ID NOs: 62-65 or containing a variant thereof having at least about 90% sequence identity to any one of SEQ ID NOs: 62-65.
Including
At least two of the minidomains consist of Staphylococcus rugdunensis Cas9 (SEQ ID NO: 30), Staphylococcus pasteri Cas9 (SEQ ID NO: 31), Staphylococcus microti Cas9 (SEQ ID NO: 33) and Staphylococcus microti Cas9 (SEQ ID NO: 33) and Staphylococcus Cas9 (SEQ ID NO: 33). Derived from different parent Cas9 endonucleases
Synthetic RNA-guided nuclease (sRGN) polypeptide.
(Item 2)
At least three of the minidomains consist of Staphylococcus rugdunensis Cas9 (SEQ ID NO: 30), Staphylococcus pasteri Cas9 (SEQ ID NO: 31), Staphylococcus microti Cas9 (SEQ ID NO: 33) and Staphylococcus microti Cas9 (SEQ ID NO: 33) The sRGN polypeptide according to item 1, which is derived from a different parent Cas9 endonuclease.
(Item 3)
The sRGN polypeptide according to item 1 or 2, wherein the minidomain 8 comprises the amino acid sequence of SEQ ID NO: 62, or a variant thereof having at least about 90% sequence identity to SEQ ID NO: 62.
(Item 4)
The sRGN polypeptide according to item 1 or 2, wherein the minidomain 8 comprises the amino acid sequence of SEQ ID NO: 63, or a variant thereof having at least about 90% sequence identity to SEQ ID NO: 63.
(Item 5)
The sRGN polypeptide according to any one of items 1 to 4, wherein the minidomain 1 comprises the amino acid sequence of SEQ ID NO: 34, or a variant thereof having at least about 90% sequence identity to SEQ ID NO: 34.
(Item 6)
From N-terminus to C-terminus:
1) A minidomain 1 comprising any one of the amino acid sequences of SEQ ID NOs: 66-69 or containing a variant thereof having at least about 90% sequence identity to any one of SEQ ID NOs: 66-69.
2) A minidomain 2 containing the amino acid sequence of any one of SEQ ID NOs: 70-73 or containing a variant thereof having at least about 90% sequence identity to any one of SEQ ID NOs: 70-73.
3) A minidomain 3 comprising any one of the amino acid sequences of SEQ ID NOs: 74-77 or a variant thereof having at least about 90% sequence identity to any one of SEQ ID NOs: 74-77.
4) A minidomain 4 comprising any one of the amino acid sequences of SEQ ID NOs: 78-81 or containing a variant thereof having at least about 90% sequence identity to any one of SEQ ID NOs: 78-81.
5) A minidomain 5 comprising any one of the amino acid sequences of SEQ ID NOs: 82-85 or containing a variant thereof having at least about 90% sequence identity to any one of SEQ ID NOs: 82-85.
6) A minidomain 6 containing the amino acid sequence of any one of SEQ ID NOs: 86-89 or containing a variant thereof having at least about 90% sequence identity to any one of SEQ ID NOs: 86-89.
7) A minidomain 7 containing the amino acid sequence of any one of SEQ ID NOs: 90-93 or containing a variant thereof having at least about 90% sequence identity to any one of SEQ ID NOs: 90-93.
8) A minidomain 8 comprising any one of the amino acid sequences of SEQ ID NOs: 94-97 or containing a variant thereof having at least about 90% sequence identity to any one of SEQ ID NOs: 94-97.
9) A minidomain 9 comprising any one of the amino acid sequences of SEQ ID NOs: 98-101 or containing a variant thereof having at least about 90% sequence identity to any one of SEQ ID NOs: 98-101.
10) A minidomain 10 comprising the amino acid sequence of any one of SEQ ID NOs: 102-105 or containing a variant thereof having at least about 90% sequence identity to any one of SEQ ID NOs: 102-105.
11) A minidomain 11 comprising any one of the amino acid sequences of SEQ ID NOs: 106-109 or containing a variant thereof having at least about 90% sequence identity to any one of SEQ ID NOs: 106-109.
12) With a minidomain 12 containing the amino acid sequence of any one of SEQ ID NOs: 110-113 or containing a variant thereof having at least about 90% sequence identity to any one of SEQ ID NOs: 110-113.
Including
At least two of the minidomains consist of Staphylococcus rugdunensis Cas9 (SEQ ID NO: 30), Staphylococcus pasteri Cas9 (SEQ ID NO: 31), Staphylococcus microti Cas9 (SEQ ID NO: 33) and Staphylococcus microti Cas9 (SEQ ID NO: 33) and Staphylococcus Cas9 (SEQ ID NO: 33). Derived from different parent Cas9 endonucleases
sRGN polypeptide.
(Item 7)
At least three of the minidomains consist of Staphylococcus rugdunensis Cas9 (SEQ ID NO: 30), Staphylococcus pasteri Cas9 (SEQ ID NO: 31), Staphylococcus microti Cas9 (SEQ ID NO: 33) and Staphylococcus microti Cas9 (SEQ ID NO: 33) Item 6. The sRGN polypeptide according to item 6, which is derived from a different parent Cas9 endonuclease.
(Item 8)
The sRGN polypeptide according to item 6 or 7, wherein the minidomain 12 comprises the amino acid sequence of SEQ ID NO: 110, or a variant thereof having at least about 90% sequence identity to SEQ ID NO: 110.
(Item 9)
The sRGN polypeptide according to item 6 or 7, wherein the minidomain 12 comprises the amino acid sequence of SEQ ID NO: 111, or a variant thereof having at least about 90% sequence identity to SEQ ID NO: 111.
(Item 10)
(I) Minidomain 1 comprises the amino acid sequence of SEQ ID NO: 66, or a variant thereof having at least about 90% sequence identity to SEQ ID NO: 66, and (ii) minidomain 2 contains the amino acid sequence of SEQ ID NO: 70. , Or the sRGN polypeptide according to any one of items 6-9, comprising a variant thereof having at least about 90% sequence identity to SEQ ID NO: 70.
(Item 11)
A Gib11 polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or a variant thereof having at least about 90% sequence identity to SEQ ID NO: 1.
b) A Gib11Spa-1 polypeptide comprising the amino acid sequence of SEQ ID NO: 2 or a variant thereof having at least about 90% sequence identity to SEQ ID NO: 2.
c) A Gib11Spa-2 polypeptide comprising the amino acid sequence of SEQ ID NO: 3 or a variant thereof having at least about 90% sequence identity to SEQ ID NO: 3.
d) A Gib11Spa-3 polypeptide comprising the amino acid sequence of SEQ ID NO: 4 or a variant thereof having at least about 90% sequence identity to SEQ ID NO: 4.
e) A P2H12 polypeptide comprising the amino acid sequence of SEQ ID NO: 5 or a variant thereof having at least about 90% sequence identity to SEQ ID NO: 5.
f) An E2 polypeptide comprising the amino acid sequence of SEQ ID NO: 6 or a variant thereof having at least about 90% sequence identity to SEQ ID NO: 6.
g) An E2 + K741D + L743K polypeptide comprising the amino acid sequence of SEQ ID NO: 7 or comprising a variant thereof having at least about 90% sequence identity to SEQ ID NO: 7.
h) An E2 + S670T + N675D polypeptide comprising the amino acid sequence of SEQ ID NO: 8 or comprising a variant thereof having at least about 90% sequence identity to SEQ ID NO: 8.
i) An E2 + K741N + L743N polypeptide comprising the amino acid sequence of SEQ ID NO: 9 or comprising a variant thereof having at least about 90% sequence identity to SEQ ID NO: 9.
j) An F8 polypeptide comprising the amino acid sequence of SEQ ID NO: 10 or a variant thereof having at least about 90% sequence identity to SEQ ID NO: 10;
k) An F8 + K737D + L739K polypeptide comprising the amino acid sequence of SEQ ID NO: 11 or comprising a variant thereof having at least about 90% sequence identity to SEQ ID NO: 11 and
l) F8 + K737N + L739N polypeptide comprising the amino acid sequence of SEQ ID NO: 12 or comprising a variant thereof having at least about 90% sequence identity to SEQ ID NO: 12.
An sRGN polypeptide selected from the group consisting of.
(Item 12)
A Gib11 polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or a variant thereof having at least about 90% sequence identity to SEQ ID NO: 1.
b) A Gib11Spa-1 polypeptide comprising the amino acid sequence of SEQ ID NO: 2 or containing a variant thereof having at least about 90% sequence identity to SEQ ID NO: 2.
c) Gib11Spa-3 polypeptide comprising the amino acid sequence of SEQ ID NO: 4 or a variant thereof having at least about 90% sequence identity to SEQ ID NO: 4.
The sRGN polypeptide according to item 11, selected from the group consisting of.
(Item 13)
a) Gib11 polypeptide containing the amino acid sequence of SEQ ID NO: 1,
b) The Gib11Spa-1 polypeptide containing the amino acid sequence of SEQ ID NO: 2 and
c) Gib11Spa-3 polypeptide containing the amino acid sequence of SEQ ID NO: 4
Item 12. The sRGN polypeptide selected from the group consisting of.
(Item 14)
A nucleic acid encoding the sRGN polypeptide according to any one of items 1 to 13.
(Item 15)
14. The nucleic acid of item 14, which is codon-optimized for expression in a host cell.
(Item 16)
14 or 15, wherein the variant comprises any one of SEQ ID NOs: 13-29 or has at least about 90% sequence identity to any one of SEQ ID NOs: 13-29. Nucleic acid.
(Item 17)
(A) The sRGN polypeptide according to any one of items 1 to 13, or the nucleic acid encoding the sRGN polypeptide.
(B) With a guide RNA (gRNA) or a nucleic acid encoding the gRNA.
A system in which the gRNA can guide the sRGN polypeptide or variant thereof to a target polynucleotide sequence.
(Item 18)
17. The system of item 17, further comprising a donor template comprising a heterologous polynucleotide sequence, wherein the heterologous polynucleotide sequence can be inserted into the target polynucleotide sequence.
(Item 19)
Item 17: The nucleic acid encoding the sRGN polypeptide is codon-optimized for expression in a host cell and / or the heterologous polynucleotide sequence is codon-optimized for expression in a host cell. Or the system according to 18.
(Item 20)
The system according to any one of items 17 to 19, wherein the nucleic acid encoding the sRGN polypeptide is deoxyribonucleic acid (DNA).
(Item 21)
The system according to any one of items 17 to 19, wherein the nucleic acid encoding the sRGN polypeptide is ribonucleic acid (RNA).
(Item 22)
The system according to item 21, wherein the RNA encoding the sRGN polypeptide is mRNA.
(Item 23)
The system according to any one of items 17 to 22, wherein the donor template is encoded by an adeno-associated virus (AAV) vector.
(Item 24)
The system according to any one of items 17 to 23, wherein the sRGN polypeptide or the nucleic acid encoding the sRGN polypeptide is formulated in a liposome or lipid nanoparticles.
(Item 25)
24. The system of item 24, wherein the liposome or lipid nanoparticles also comprises the gRNA, or nucleic acid encoding the gRNA.
(Item 26)
The system according to any one of items 17 to 23, comprising an sRGN polypeptide that is pre-complexed with the gRNA to form a ribonucleoprotein (RNP) complex.
(Item 27)
A method of targeting, editing, modifying or manipulating target DNA at a target locus.
(A) The sRGN polypeptide according to any one of items 1 to 13, or the nucleic acid encoding the sRGN polypeptide.
(B) With a guide RNA (gRNA) or a nucleic acid encoding the gRNA.
Includes the step of providing the target DNA
A method in which the gRNA can guide the sRGN polypeptide to the target locus.
(Item 28)
27. The method of item 27, further comprising providing a donor template comprising a heterologous polynucleotide sequence to the target DNA, wherein the heterologous polynucleotide sequence can be inserted into the target locus.
(Item 29)
The nucleic acid encoding the sRGN polypeptide is codon-optimized for expression in a host cell and / or the heterologous polynucleotide sequence is codon-optimized for expression in a host cell, item 27. Or the method according to 28.
(Item 30)
The method according to any one of items 27 to 29, wherein the nucleic acid encoding the sRGN polypeptide is deoxyribonucleic acid (DNA).
(Item 31)
The method according to any one of items 27 to 29, wherein the nucleic acid encoding the sRGN polypeptide is ribonucleic acid (RNA).
(Item 32)
31. The method of item 31, wherein the RNA encoding the sRGN polypeptide is mRNA.
(Item 33)
The method of any one of items 27-32, wherein the donor template is encoded by an adeno-associated virus (AAV) vector.
(Item 34)
The method according to any one of items 27 to 33, wherein the sRGN polypeptide or the nucleic acid encoding the sRGN polypeptide is formulated in a liposome or lipid nanoparticles.
(Item 35)
34. The method of item 34, wherein the liposome or lipid nanoparticles also comprises the gRNA, or nucleic acid encoding the gRNA.
(Item 36)
28. The method of any one of items 27-33, comprising providing the target DNA with an sRGN polypeptide precomplexed with the gRNA as an RNP complex.
(Item 37)
(A) The sRGN polypeptide according to any one of items 1 to 13, or the nucleic acid encoding the sRGN polypeptide.
(B) Guide RNA (gRNA) or nucleic acid encoding gRNA
A modified cell comprising, wherein the gRNA can guide the sRGN polypeptide or variant thereof to a target polynucleotide sequence.
(Item 38)
37. The modified cell of item 37, further comprising a donor template comprising a heterologous polynucleotide sequence, wherein the heterologous polynucleotide sequence can be inserted into the target polynucleotide sequence.
(Item 39)
A genetically modified cell in which the genome of the cell has been edited by the method according to any one of items 27-36.
(Item 40)
(A) The sRGN polypeptide according to any one of items 1 to 13, or the nucleic acid encoding the sRGN polypeptide.
(B) Guide RNA (gRNA) or nucleic acid encoding gRNA
Is a kit that includes
A kit in which the gRNA can guide the sRGN polypeptide or variant thereof to a target polynucleotide sequence.
(Item 41)
40. The kit of item 40, further comprising a donor template comprising a heterologous polynucleotide sequence, wherein the heterologous polynucleotide sequence can be inserted into the target polynucleotide sequence.
Claims (41)
１）配列番号３４～３７のいずれか１つのアミノ酸配列を含むかまたは配列番号３４～３７のいずれか１つに対して少なくとも約９０％配列同一性を有するそのバリアントを含むミニドメイン１と、
２）配列番号３８～４１のいずれか１つのアミノ酸配列を含むかまたは配列番号３８～４１のいずれか１つに対して少なくとも約９０％配列同一性を有するそのバリアントを含むミニドメイン２と、
３）配列番号４２～４５のいずれか１つのアミノ酸配列を含むかまたは配列番号４２～４５のいずれか１つに対して少なくとも約９０％配列同一性を有するそのバリアントを含むミニドメイン３と、
４）配列番号４６～４９のいずれか１つのアミノ酸配列を含むかまたは配列番号４６～４９のいずれか１つに対して少なくとも約９０％配列同一性を有するそのバリアントを含むミニドメイン４と、
５）配列番号５０～５３のいずれか１つのアミノ酸配列を含むかまたは配列番号５０～５３のいずれか１つに対して少なくとも約９０％配列同一性を有するそのバリアントを含むミニドメイン５と、
６）配列番号５４～５７のいずれか１つのアミノ酸配列を含むかまたは配列番号５４～５７のいずれか１つに対して少なくとも約９０％配列同一性を有するそのバリアントを含むミニドメイン６と、
７）配列番号５８～６１のいずれか１つのアミノ酸配列を含むかまたは配列番号５８～６１のいずれか１つに対して少なくとも約９０％配列同一性を有するそのバリアントを含むミニドメイン７と、
８）配列番号６２～６５のいずれか１つのアミノ酸配列を含むかまたは配列番号６２～６５のいずれか１つに対して少なくとも約９０％配列同一性を有するそのバリアントを含むミニドメイン８と
を含み、
前記ミニドメインのうちの少なくとも２つが、Ｓｔａｐｈｙｌｏｃｏｃｃｕｓ ｌｕｇｄｕｎｅｎｓｉｓ Ｃａｓ９（配列番号３０）、Ｓｔａｐｈｙｌｏｃｏｃｃｕｓ ｐａｓｔｅｕｒｉ Ｃａｓ９（配列番号３１）、Ｓｔａｐｈｙｌｏｃｏｃｃｕｓ ｍｉｃｒｏｔｉ Ｃａｓ９（配列番号３３）およびＳｔａｐｈｙｌｏｃｏｃｃｕｓ ｈｙｉｃｕｓ Ｃａｓ９（配列番号３２）からなる群から選択される異なる親Ｃａｓ９エンドヌクレアーゼに由来する、
合成ＲＮＡガイドヌクレアーゼ（ｓＲＧＮ）ポリペプチド。 From N-terminus to C-terminus:
1) A minidomain 1 containing the amino acid sequence of any one of SEQ ID NOs: 34-37 or containing a variant thereof having at least about 90% sequence identity to any one of SEQ ID NOs: 34-37.
2) A minidomain 2 containing the amino acid sequence of any one of SEQ ID NOs: 38-41 or containing a variant thereof having at least about 90% sequence identity to any one of SEQ ID NOs: 38-41.
3) A mini-domain 3 containing the amino acid sequence of any one of SEQ ID NOs: 42-45 or containing a variant thereof having at least about 90% sequence identity to any one of SEQ ID NOs: 42-45.
4) A minidomain 4 comprising any one of the amino acid sequences of SEQ ID NOs: 46-49 or containing a variant thereof having at least about 90% sequence identity to any one of SEQ ID NOs: 46-49.
5) A mini-domain 5 containing the amino acid sequence of any one of SEQ ID NOs: 50-53 or containing a variant thereof having at least about 90% sequence identity to any one of SEQ ID NOs: 50-53.
6) A minidomain 6 containing the amino acid sequence of any one of SEQ ID NOs: 54-57 or containing a variant thereof having at least about 90% sequence identity to any one of SEQ ID NOs: 54-57.
7) A minidomain 7 containing the amino acid sequence of any one of SEQ ID NOs: 58-61 or containing a variant thereof having at least about 90% sequence identity to any one of SEQ ID NOs: 58-61.
8) Containing a minidomain 8 comprising any one of the amino acid sequences of SEQ ID NOs: 62-65 or a variant thereof having at least about 90% sequence identity to any one of SEQ ID NOs: 62-65. ,
At least two of the minidomains consist of Staphylococcus rugdunensis Cas9 (SEQ ID NO: 30), Staphylococcus pasteri Cas9 (SEQ ID NO: 31), Staphylococcus microti Cas9 (SEQ ID NO: 33) and Staphylococcus microti Cas9 (SEQ ID NO: 33) and Staphylococcus Cas9 (SEQ ID NO: 33). Derived from different parent Cas9 endonucleases
Synthetic RNA-guided nuclease (sRGN) polypeptide.
１）配列番号６６～６９のいずれか１つのアミノ酸配列を含むかまたは配列番号６６～６９のいずれか１つに対して少なくとも約９０％配列同一性を有するそのバリアントを含むミニドメイン１と、
２）配列番号７０～７３のいずれか１つのアミノ酸配列を含むかまたは配列番号７０～７３のいずれか１つに対して少なくとも約９０％配列同一性を有するそのバリアントを含むミニドメイン２と、
３）配列番号７４～７７のいずれか１つのアミノ酸配列を含むかまたは配列番号７４～７７のいずれか１つに対して少なくとも約９０％配列同一性を有するそのバリアントを含むミニドメイン３と、
４）配列番号７８～８１のいずれか１つのアミノ酸配列を含むかまたは配列番号７８～８１のいずれか１つに対して少なくとも約９０％配列同一性を有するそのバリアントを含むミニドメイン４と、
５）配列番号８２～８５のいずれか１つのアミノ酸配列を含むかまたは配列番号８２～８５のいずれか１つに対して少なくとも約９０％配列同一性を有するそのバリアントを含むミニドメイン５と、
６）配列番号８６～８９のいずれか１つのアミノ酸配列を含むかまたは配列番号８６～８９のいずれか１つに対して少なくとも約９０％配列同一性を有するそのバリアントを含むミニドメイン６と、
７）配列番号９０～９３のいずれか１つのアミノ酸配列を含むかまたは配列番号９０～９３のいずれか１つに対して少なくとも約９０％配列同一性を有するそのバリアントを含むミニドメイン７と、
８）配列番号９４～９７のいずれか１つのアミノ酸配列を含むかまたは配列番号９４～９７のいずれか１つに対して少なくとも約９０％配列同一性を有するそのバリアントを含むミニドメイン８と、
９）配列番号９８～１０１のいずれか１つのアミノ酸配列を含むかまたは配列番号９８～１０１のいずれか１つに対して少なくとも約９０％配列同一性を有するそのバリアントを含むミニドメイン９と、
１０）配列番号１０２～１０５のいずれか１つのアミノ酸配列を含むかまたは配列番号１０２～１０５のいずれか１つに対して少なくとも約９０％配列同一性を有するそのバリアントを含むミニドメイン１０と、
１１）配列番号１０６～１０９のいずれか１つのアミノ酸配列を含むかまたは配列番号１０６～１０９のいずれか１つに対して少なくとも約９０％配列同一性を有するそのバリアントを含むミニドメイン１１と、
１２）配列番号１１０～１１３のいずれか１つのアミノ酸配列を含むかまたは配列番号１１０～１１３のいずれか１つに対して少なくとも約９０％配列同一性を有するそのバリアントを含むミニドメイン１２と
を含み、
前記ミニドメインのうちの少なくとも２つが、Ｓｔａｐｈｙｌｏｃｏｃｃｕｓ ｌｕｇｄｕｎｅｎｓｉｓ Ｃａｓ９（配列番号３０）、Ｓｔａｐｈｙｌｏｃｏｃｃｕｓ ｐａｓｔｅｕｒｉ Ｃａｓ９（配列番号３１）、Ｓｔａｐｈｙｌｏｃｏｃｃｕｓ ｍｉｃｒｏｔｉ Ｃａｓ９（配列番号３３）およびＳｔａｐｈｙｌｏｃｏｃｃｕｓ ｈｙｉｃｕｓ Ｃａｓ９（配列番号３２）からなる群から選択される異なる親Ｃａｓ９エンドヌクレアーゼに由来する、
ｓＲＧＮポリペプチド。 From N-terminus to C-terminus:
1) A minidomain 1 comprising any one of the amino acid sequences of SEQ ID NOs: 66-69 or containing a variant thereof having at least about 90% sequence identity to any one of SEQ ID NOs: 66-69.
2) A minidomain 2 containing the amino acid sequence of any one of SEQ ID NOs: 70-73 or containing a variant thereof having at least about 90% sequence identity to any one of SEQ ID NOs: 70-73.
3) A minidomain 3 comprising any one of the amino acid sequences of SEQ ID NOs: 74-77 or a variant thereof having at least about 90% sequence identity to any one of SEQ ID NOs: 74-77.
4) A minidomain 4 comprising any one of the amino acid sequences of SEQ ID NOs: 78-81 or containing a variant thereof having at least about 90% sequence identity to any one of SEQ ID NOs: 78-81.
5) A minidomain 5 comprising any one of the amino acid sequences of SEQ ID NOs: 82-85 or containing a variant thereof having at least about 90% sequence identity to any one of SEQ ID NOs: 82-85.
6) A minidomain 6 containing the amino acid sequence of any one of SEQ ID NOs: 86-89 or containing a variant thereof having at least about 90% sequence identity to any one of SEQ ID NOs: 86-89.
7) A minidomain 7 containing the amino acid sequence of any one of SEQ ID NOs: 90-93 or containing a variant thereof having at least about 90% sequence identity to any one of SEQ ID NOs: 90-93.
8) A minidomain 8 comprising any one of the amino acid sequences of SEQ ID NOs: 94-97 or containing a variant thereof having at least about 90% sequence identity to any one of SEQ ID NOs: 94-97.
9) A minidomain 9 comprising any one of the amino acid sequences of SEQ ID NOs: 98-101 or containing a variant thereof having at least about 90% sequence identity to any one of SEQ ID NOs: 98-101.
10) A minidomain 10 comprising the amino acid sequence of any one of SEQ ID NOs: 102-105 or containing a variant thereof having at least about 90% sequence identity to any one of SEQ ID NOs: 102-105.
11) A minidomain 11 comprising any one of the amino acid sequences of SEQ ID NOs: 106-109 or containing a variant thereof having at least about 90% sequence identity to any one of SEQ ID NOs: 106-109.
12) Containing a minidomain 12 comprising any one of the amino acid sequences of SEQ ID NOs: 110-113 or a variant thereof having at least about 90% sequence identity to any one of SEQ ID NOs: 110-113. ,
At least two of the minidomains consist of Staphylococcus rugdunensis Cas9 (SEQ ID NO: 30), Staphylococcus pasteri Cas9 (SEQ ID NO: 31), Staphylococcus microti Cas9 (SEQ ID NO: 33) and Staphylococcus microti Cas9 (SEQ ID NO: 33) and Staphylococcus Cas9 (SEQ ID NO: 33). Derived from different parent Cas9 endonucleases
sRGN polypeptide.
ｂ）配列番号２のアミノ酸配列を含むかまたは配列番号２に対して少なくとも約９０％配列同一性を有するそのバリアントを含むＧｉｂ１１Ｓｐａ－１ポリペプチド、
ｃ）配列番号３のアミノ酸配列を含むかまたは配列番号３に対して少なくとも約９０％配列同一性を有するそのバリアントを含むＧｉｂ１１Ｓｐａ－２ポリペプチド、
ｄ）配列番号４のアミノ酸配列を含むかまたは配列番号４に対して少なくとも約９０％配列同一性を有するそのバリアントを含むＧｉｂ１１Ｓｐａ－３ポリペプチド、
ｅ）配列番号５のアミノ酸配列を含むかまたは配列番号５に対して少なくとも約９０％配列同一性を有するそのバリアントを含むＰ２Ｈ１２ポリペプチド、
ｆ）配列番号６のアミノ酸配列を含むかまたは配列番号６に対して少なくとも約９０％配列同一性を有するそのバリアントを含むＥ２ポリペプチド、
ｇ）配列番号７のアミノ酸配列を含むかまたは配列番号７に対して少なくとも約９０％配列同一性を有するそのバリアントを含むＥ２＋Ｋ７４１Ｄ＋Ｌ７４３Ｋポリペプチド、
ｈ）配列番号８のアミノ酸配列を含むかまたは配列番号８に対して少なくとも約９０％配列同一性を有するそのバリアントを含むＥ２＋Ｓ６７０Ｔ＋Ｎ６７５Ｄポリペプチド、
ｉ）配列番号９のアミノ酸配列を含むかまたは配列番号９に対して少なくとも約９０％配列同一性を有するそのバリアントを含むＥ２＋Ｋ７４１Ｎ＋Ｌ７４３Ｎポリペプチド、
ｊ）配列番号１０のアミノ酸配列を含むかまたは配列番号１０に対して少なくとも約９０％配列同一性を有するそのバリアントを含むＦ８ポリペプチド；
ｋ）配列番号１１のアミノ酸配列を含むかまたは配列番号１１に対して少なくとも約９０％配列同一性を有するそのバリアントを含むＦ８＋Ｋ７３７Ｄ＋Ｌ７３９Ｋポリペプチド、および
ｌ）配列番号１２のアミノ酸配列を含むかまたは配列番号１２に対して少なくとも約９０％配列同一性を有するそのバリアントを含むＦ８＋Ｋ７３７Ｎ＋Ｌ７３９Ｎポリペプチド
からなる群から選択されるｓＲＧＮポリペプチド。 A Gib11 polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or a variant thereof having at least about 90% sequence identity to SEQ ID NO: 1.
b) A Gib11Spa-1 polypeptide comprising the amino acid sequence of SEQ ID NO: 2 or a variant thereof having at least about 90% sequence identity to SEQ ID NO: 2.
c) A Gib11Spa-2 polypeptide comprising the amino acid sequence of SEQ ID NO: 3 or a variant thereof having at least about 90% sequence identity to SEQ ID NO: 3.
d) A Gib11Spa-3 polypeptide comprising the amino acid sequence of SEQ ID NO: 4 or a variant thereof having at least about 90% sequence identity to SEQ ID NO: 4.
e) A P2H12 polypeptide comprising the amino acid sequence of SEQ ID NO: 5 or a variant thereof having at least about 90% sequence identity to SEQ ID NO: 5.
f) An E2 polypeptide comprising the amino acid sequence of SEQ ID NO: 6 or a variant thereof having at least about 90% sequence identity to SEQ ID NO: 6.
g) An E2 + K741D + L743K polypeptide comprising the amino acid sequence of SEQ ID NO: 7 or comprising a variant thereof having at least about 90% sequence identity to SEQ ID NO: 7.
h) An E2 + S670T + N675D polypeptide comprising the amino acid sequence of SEQ ID NO: 8 or comprising a variant thereof having at least about 90% sequence identity to SEQ ID NO: 8.
i) An E2 + K741N + L743N polypeptide comprising the amino acid sequence of SEQ ID NO: 9 or comprising a variant thereof having at least about 90% sequence identity to SEQ ID NO: 9.
j) An F8 polypeptide comprising the amino acid sequence of SEQ ID NO: 10 or a variant thereof having at least about 90% sequence identity to SEQ ID NO: 10;
k) F8 + K737D + L739K polypeptide comprising the amino acid sequence of SEQ ID NO: 11 or comprising a variant thereof having at least about 90% sequence identity to SEQ ID NO: 11 and l) comprising the amino acid sequence of SEQ ID NO: 12 or SEQ ID NO: An sRGN polypeptide selected from the group consisting of F8 + K737N + L739N polypeptides comprising the variant having at least about 90% sequence identity to 12.
ｂ）配列番号２のアミノ酸配列を含むかまたは配列番号２に対して少なくとも約９０％配列同一性を有するそのバリアントを含むＧｉｂ１１Ｓｐａ－１ポリペプチド、および
ｃ）配列番号４のアミノ酸配列を含むかまたは配列番号４に対して少なくとも約９０％配列同一性を有するそのバリアントを含むＧｉｂ１１Ｓｐａ－３ポリペプチド
からなる群から選択される、請求項１１に記載のｓＲＧＮポリペプチド。 A Gib11 polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or a variant thereof having at least about 90% sequence identity to SEQ ID NO: 1.
b) a Gib11Spa-1 polypeptide containing the amino acid sequence of SEQ ID NO: 2 or a variant thereof having at least about 90% sequence identity to SEQ ID NO: 2 and c) containing the amino acid sequence of SEQ ID NO: 4 or The sRGN polypeptide according to claim 11, selected from the group consisting of Gib11Spa-3 polypeptides comprising a variant thereof having at least about 90% sequence identity to SEQ ID NO: 4.
ｂ）配列番号２のアミノ酸配列を含むＧｉｂ１１Ｓｐａ－１ポリペプチド、および
ｃ）配列番号４のアミノ酸配列を含むＧｉｂ１１Ｓｐａ－３ポリペプチド
からなる群から選択される、請求項１２に記載のｓＲＧＮポリペプチド。 a) Gib11 polypeptide containing the amino acid sequence of SEQ ID NO: 1,
The sRGN polypeptide according to claim 12, which is selected from the group consisting of b) a Gib11Spa-1 polypeptide comprising the amino acid sequence of SEQ ID NO: 2 and c) a Gib11Spa-3 polypeptide comprising the amino acid sequence of SEQ ID NO: 4.
（ｂ）ガイドＲＮＡ（ｇＲＮＡ）、または前記ｇＲＮＡをコードする核酸と
を含み、前記ｇＲＮＡが、前記ｓＲＧＮポリペプチドまたはそのバリアントを標的ポリヌクレオチド配列へとガイドすることができる、系。 (A) The sRGN polypeptide according to any one of claims 1 to 13, or the nucleic acid encoding the sRGN polypeptide.
(B) A system comprising a guide RNA (gRNA), or a nucleic acid encoding the gRNA, wherein the gRNA can guide the sRGN polypeptide or variant thereof to a target polynucleotide sequence.
（ｂ）ガイドＲＮＡ（ｇＲＮＡ）、または前記ｇＲＮＡをコードする核酸と
を含む、標的遺伝子座における標的ＤＮＡを標的化、編集、改変または操作する方法において使用するための組成物であって、
前記ｇＲＮＡが前記ｓＲＧＮポリペプチドを前記標的遺伝子座へとガイドすることができ、前記方法は、前記組成物を前記標的ＤＮＡに提供するステップを含む、組成物。 (A) The sRGN polypeptide according to any one of claims 1 to 13, or the nucleic acid encoding the sRGN polypeptide.
(B) With a guide RNA (gRNA) or a nucleic acid encoding the gRNA.
A composition for use in a method of targeting, editing, modifying or manipulating a target DNA at a target locus, comprising.
A composition comprising the step of providing the composition to the target DNA, wherein the gRNA can guide the sRGN polypeptide to the target locus .
（ｂ）ガイドＲＮＡ（ｇＲＮＡ）、またはｇＲＮＡをコードする核酸と
を含み、前記ｇＲＮＡが、前記ｓＲＧＮポリペプチドまたはそのバリアントを標的ポリヌクレオチド配列へとガイドすることができる、改変された細胞。 (A) The sRGN polypeptide according to any one of claims 1 to 13, or the nucleic acid encoding the sRGN polypeptide.
(B) A modified cell comprising a guide RNA (gRNA), or a nucleic acid encoding the gRNA, wherein the gRNA can guide the sRGN polypeptide or variant thereof to a target polynucleotide sequence.
（ｂ）ガイドＲＮＡ（ｇＲＮＡ）、またはｇＲＮＡをコードする核酸と
を含むキットであって、
前記ｇＲＮＡが、前記ｓＲＧＮポリペプチドまたはそのバリアントを標的ポリヌクレオチド配列へとガイドすることができる、キット。 (A) The sRGN polypeptide according to any one of claims 1 to 13, or the nucleic acid encoding the sRGN polypeptide.
(B) A kit containing a guide RNA (gRNA) or a nucleic acid encoding gRNA.
A kit in which the gRNA can guide the sRGN polypeptide or variant thereof to a target polynucleotide sequence.
40. The kit of claim 40, further comprising a donor template comprising a heterologous polynucleotide sequence, wherein the heterologous polynucleotide sequence can be inserted into the target polynucleotide sequence.
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- 2019-03-19 CA CA3092497A patent/CA3092497A1/en active Pending
- 2019-03-19 AU AU2019239957A patent/AU2019239957A1/en not_active Abandoned
- 2019-03-19 WO PCT/US2019/023044 patent/WO2019183150A1/en unknown
- 2019-03-19 JP JP2020550714A patent/JP7550648B2/en active Active
- 2019-03-19 EP EP19712889.5A patent/EP3768834A1/en active Pending
- 2019-03-19 CN CN201980033565.0A patent/CN112424348A/en active Pending
- 2019-03-19 MA MA052074A patent/MA52074A/en unknown
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