JP2022113799A5 - - Google Patents

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JP2022113799A5
JP2022113799A5 JP2022095742A JP2022095742A JP2022113799A5 JP 2022113799 A5 JP2022113799 A5 JP 2022113799A5 JP 2022095742 A JP2022095742 A JP 2022095742A JP 2022095742 A JP2022095742 A JP 2022095742A JP 2022113799 A5 JP2022113799 A5 JP 2022113799A5
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host cell
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cas9 protein
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  1. エンジニアリングされたＣＲＩＳＰＲ－Ｃａｓ系を含む、単離された真核宿主細胞であって、
    ＣＲＩＳＰＲ－Ｃａｓ系がＣａｓ９タンパク質およびＣＲＩＳＰＲ－Ｃａｓ系キメラＲＮＡを含み、
    キメラＲＮＡが、真核宿主細胞におけるプロトスペーサー隣接モチーフ（ＰＡＭ）に隣接する標的配列にハイブリダイズし、ＣＲＩＳＰＲ－Ｃａｓ複合体の標的配列への配列特異的結合を指向することができるガイド配列、ｔｒａｃｒ配列にハイブリダイズすることができるｔｒａｃｒメイト配列、および少なくとも３０ヌクレオチド長を含むｔｒａｃｒ配列を含む、
    単離された真核宿主細胞。
  2. ｔｒａｃｒ配列が少なくとも４０のヌクレオチド長を含む、請求項１に記載の単離された真核宿主細胞。
  3. ｔｒａｃｒ配列が少なくとも５０のヌクレオチド長を含む、請求項１に記載の単離された真核宿主細胞。
  4. ガイド配列が１５～２５のヌクレオチド長を含む、請求項１から３の何れか一項に記載の単離された真核宿主細胞。
  5. ガイド配列が少なくとも２０ヌクレオチド長を含む、請求項１から３の何れか一項に記載の単離された真核宿主細胞。
  6. キメラＲＮＡが、
    (a) NNNNNNNNNNNNNNNNNNNNGUUUUAGAGCUAGAAAUagcaaguuaaaauaaggcuaguccguuaucaacuugaaaaaguggcaccgagucggugcuuu;
    (b) NNNNNNNNNNNNNNNNNNNNGUUUUAGAGCUAGAAAUagcaaguuaaaauaaggcuaguccguuaucaacuugaaaaagug;
    (c) NNNNNNNNNNNNNNNNNNNNGUUUUAGAGCUAGAAAUagcaaguuaaaauaaggcuaguccguuauca
    から選択される、ＲＮＡ配列を含む、請求項１から５の何れか一項に記載の単離された真核宿主細胞。
  7. キメラＲＮＡが、
    (a) NNNNNNNNNNNNNNNNNNNNgttttagagctaGAAAtagcaagttaaaataaggctagtccgttatcaacttgaaaaagtggcaccgagtcggtgcTTTTTT;
    (b) NNNNNNNNNNNNNNNNNNNNgttttagagctaGAAATAGcaagttaaaataaggctagtccgttatcaacttgaaaaagtgTTTTTTT;
    (c) NNNNNNNNNNNNNNNNNNNNgttttagagctagAAATAGcaagttaaaataaggctagtccgttatcaTTTTTTTT
    から選択される、ＤＮＡ配列によってコードされる、請求項１から５の何れか一項に記載の単離された真核宿主細胞。
  8. Ｃａｓ９タンパク質が少なくとも２つのＮＬＳに融合している、請求項１～７のいずれか一項に記載の単離された真核宿主細胞。
  9. 第１のＮＬＳがＣａｓ９タンパク質のアミノ末端に融合し、第２のＮＬＳがＣａｓ９タンパク質のカルボキシ末端に融合している、請求項８に記載の単離された真核宿主細胞。
  10. Ｃａｓ９タンパク質が、Ｓ．ｐｙｏｇｅｎｅｓ Ｃａｓ９である、請求項１～９のいずれか一項に記載の単離された真核宿主細胞。
  11. Ｃａｓ９タンパク質が、両ＤＮＡ鎖を切断することができるヌクレアーゼである、請求項１～１０のいずれか一項に記載の単離された真核宿主細胞。
  12. Ｃａｓ９タンパク質が、Ｓ．ｐｙｏｇｅｎｅｓ Ｃａｓ９のＤ１０Ａ、Ｈ８４０Ａ、Ｎ８５４Ａ、またはＮ８６３Ａに対応する変異を含み、Ｃａｓ９タンパク質が、１本のＤＮＡ鎖を切断する能力を欠くニッカーゼである、請求項１～１０のいずれか一項に記載の単離された真核宿主細胞。
  13. Ｃａｓ９タンパク質が、Ｓ．ｐｙｏｇｅｎｅｓ Ｃａｓ９のＤ１０Ａに対応する変異、およびＳ．ｐｙｏｇｅｎｅｓ Ｃａｓ９のＨ８４０Ａ、Ｎ８５４Ａ、またはＮ８６３Ａに対応する変異を含み、Ｃａｓ９タンパク質が全てのＤＮＡ切断活性を実質的に欠いている、請求項１～１０のいずれか一項に記載の単離された真核宿主細胞。
  14. Ｃａｓ９タンパク質が異種タンパク質ドメインに融合している、請求項１～１３のいずれか一項に記載の単離された真核宿主細胞。
  15. 異種タンパク質ドメインが、メチラーゼ活性、デメチラーゼ活性、転写活性化活性、転写抑制活性、転写放出因子活性、ヒストン修飾活性、ＲＮＡ切断活性及び核酸結合活性のうちの１つ以上の活性を有する、請求項１４に記載の単離された真核宿主細胞。
  16. 真核宿主細胞が、真核宿主細胞の核内に位置するＣａｓ９タンパク質とキメラＲＮＡによって形成されるＣＲＩＳＰＲ複合体を含む、請求項１～１５のいずれか一項に記載の単離された真核宿主細胞。
  17. 真核宿主細胞が、真核宿主細胞の核内において標的配列においてＣＲＩＳＰＲ複合体によってなされたＤＮＡ切断を修復することによって形成されたインデルを含む、請求項１～１６のいずれか一項に記載の単離された真核宿主細胞。
  18. 真核細胞が哺乳類細胞またはヒト細胞である、請求項１～１７のいずれか一項に記載の単離された真核宿主細胞。
JP2022095742A 2012-12-12 2022-06-14 遺伝子産物の発現を変更するためのＣＲＩＳＰＲ－Ｃａｓ系および方法 Active JP7463436B2 (ja)

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US201361791409P 2013-03-15 2013-03-15
US61/791,409 2013-03-15
US201361835931P 2013-06-17 2013-06-17
US61/835,931 2013-06-17
US201361842322P 2013-07-02 2013-07-02
US61/842,322 2013-07-02
US14/054,414 2013-10-15
US14/054,414 US8697359B1 (en) 2012-12-12 2013-10-15 CRISPR-Cas systems and methods for altering expression of gene products
JP2019035911A JP6870015B2 (ja) 2012-12-12 2019-02-28 遺伝子産物の発現を変更するためのＣＲＩＳＰＲ−Ｃａｓ系および方法
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