JP2022523189A - Lachnospiraceae bacterium ND2006のCAS12A変異型遺伝子およびそれらによってコードされるポリペプチド - Google Patents
Lachnospiraceae bacterium ND2006のCAS12A変異型遺伝子およびそれらによってコードされるポリペプチド Download PDFInfo
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Abstract
Description
[0001]本出願は、2019年2月22日出願の、米国仮出願第62/808,984号の利益を主張するものであり、その開示は、参照によりその全体が本明細書に組み込まれる。
野生型および変異型LbCas12aタンパク質およびAsCas12aタンパク質のDNA配列およびアミノ酸配列
[0037]下のリストは、本発明に記載の異なる野生型(WT)および変異型Cas12aヌクレアーゼを示す。多くの場合、複数のコドンが同じアミノ酸をコードすることができるので、異なる多くのDNA配列が同じアミノ酸(AA)配列をコードする/発現することができることは、当業者であれば理解するであろう。下に示すDNA配列は例としてのみ利用するものであり、同じタンパク質(例えば、同じアミノ酸配列)をコードする他のDNA配列が企図される。さらに、NLSドメイン等などのさらなる特性、エレメント、またはタグを前記配列に付加することができることを理解されたい。WT LbCas12a(Cpf1)、WT AsCas12a、および変異型N527R LbCas12a、変異型D559P LbCas12a、変異型E759L LbCas12a、二重変異型N527R/D559P LbCas12a、二重変異型N527R/E795L LbCas12a、二重変異型D559P/E795L LbCas12a、三重変異型N527R/D559P/E795L LbCas12a、および二重変異型M537R/F870L AsCas12a、について例を示す。LbCas12aおよびAsCas12aの変異型について、アミノ酸配列およびDNA配列のみが提供されるが、NLSドメインおよびHisタグドメインが付加されて、哺乳動物細胞で使用するための組換えタンパク質の産生での使用を容易にすることができること、が企図される。
大腸菌細胞におけるLbCas12a変異型の過剰発現および精製
[0058]本実施例は、7つのCas12a変異型、N527R、D559P、E795L、N527R/D559P、D559P/E795L、N527R/E795LおよびN527R/D559P/E795Lの過剰発現および精製を実証する。LbCas12a変異型を、標準的なPCR条件とプライマーを使用して部位特異的変異誘発によって導入した(表1)。大腸菌BL21(DE3)細胞に形質転換した後、適当な株を有するコロニーを使用して、カナマイシン(0.05mg/mL)を含むTB培地に接種し、OD約0.9が達するまで37℃で増殖させ、次いでフラスコを18℃に30分間冷却した。1M IPTG(500μL)の添加を使用してタンパク質発現を誘導し、これに続いて18℃で19時間増殖させた。細胞を回収し、細胞ペレットを再懸濁し、15~20kpsiで4℃に予冷したAvestin Emulsiflex C3上で、3回パスで溶解させた。溶解物を16,000×gで、4℃で20分間、遠心分離して細胞デブリを除去した。
新規LbCas12a置換変異型は、リボ核タンパク質複合体を介してヒト細胞に送達される場合、ヒト細胞株ベースの活性アッセイにおいて切断活性を増強する。
[0061]以下の実施例が実証するところは、RNP複合体として送達される場合、ゲノム編集効率を改善するLbCas12a変異型の能力である。本例が実証するところは、エレクトロポレーショントランスフェクションによるヒト細胞へと、リボ核タンパク質(RNP)複合体による高用量で送達された場合に同等のゲノム編集効率を示し、それらによる低用量で送達された場合に向上したゲノム編集効率を示す、LbCas12a変異型の能力である。
単一LbCas12a置換変異型は、低用量でリボ核タンパク質複合体を介してヒト細胞に送達された場合、ヒト細胞株ベースの活性アッセイにおいて切断活性を増強する。
[0067]以下の実施例が実証するところは、エレクトロポレーショントランスフェクションによるヒト細胞へと、RNP複合体による低用量で送達された場合に、向上したゲノム編集効率を示す、変異型E795L LbCas12aの能力である。すなわち、本発明は、野生型または変異型Cas12aがRNP複合体としてヒト細胞へと送達される場合に、ゲノム編集効率を向上させる。
[0079] 1. Zetsche, B., et al., Cpf1 Is a Single RNA-Guided Endonuclease of a Class 2 CRISPR-Cas System. Cell, 2015. 163: p. 759.
[0080] 2. Hur, J. K., et al., Targeted mutagenesis in mice by electroporation of Cpf1 ribonucleoproteins. Nature Biotechnology, 2016. 34(8): p. 807.
[0081] 3. Kim, Y., et al., Generation of knockdown mice by Cpf1-mediated gene targeting. Nature Biotechnology, 2016. 34(8): p. 808.
[0082] 4. Kim, D., et al., Genome-wide analysis reveals specificities of Cpf1 endonucleases in human cells. Nature Biotechnology, 2016. 34(8): p. 863.
[0083] 5. Kleinstiver, B. P., et al., Genome-wide specificities of CRISPR-Cas Cpf1 nucleases in human cells. Nature Biotechnology, 2016. 34(8): p. 869.
[0084] 6. Kim, H. K., et al., In vivo high-throughput profiling of CRISPR-Cpf1 activity. Nature Methods, 2017. 14(2): p. 153.
[0085] 7. Zetsche, B., et al., Multiplex gene editing by CRISPR-Cpf1 using a single rRNA array. Nature Biotechnology, 2017. 35(1): p. 31.
[0086] 8. Kim, H., et al., CRISPR/Cpf1-mediated DNA-free plant genome editing. Nature Communications, 2017. 8(14406): p. 1.
[0087] 9. Yamano, T., et al., Crystal Structure of Cpf1 in Complex with Guide RNA and Target RNA. Cell, 2016. 65: p. 949.
[0088] 10. Yamano, T., et al., Structural Basis for the Canonical and Non-canonical PAM Recognition by CRISPR-Cpf1. Molecular Cell, 2017. 67: p. 633.
[0089] 11. Gao, L., et al., Engineered Cpf1 variants with altered PAM specificities. Nature Biotechnology, 2017. 35(8): p. 789.
[0090] 13. Robert, X. and Gouet, P., Deciphering key features in protein structures with the new ENDscript server. Nucleic Acids Research, 2014. 42(W1): p. W320.
Claims (12)
- a)以下の位置:N527、D559、およびE795から選択される、野生型LbCas12aタンパク質に導入された単一置換変異;または、
b)以下の位置:N527、D559、およびE795のうちの少なくとも2つから選択される、野生型LbCas12aタンパク質に導入された複数の置換変異、
からなる群から選択される置換変異を含む、単離された変異型LbCas12a。 - 配列番号3、配列番号4、および配列番号5からなる群から選択される、請求項1に記載の単離された変異型LbCas12aタンパク質。
- 配列番号6、配列番号7、配列番号8、および配列番号9からなる群から選択される、請求項1に記載の単離された変異型LbCas12aタンパク質。
- a)請求項1に記載の変異型LbCas12aタンパク質;および、
b)gRNA複合体、
を含む、単離されたリボ核タンパク質複合体であって、CRISPR/Cas12aエンドヌクレアーゼシステムとして活性であり、結果として生じるCRISPR/Cas12aエンドヌクレアーゼシステムは、野生型CRISPR/Cas12aエンドヌクレアーゼシステムと比べて、維持されたオンターゲット編集活性を提示する、
単離されたリボ核タンパク質複合体。 - 変異型LbCas12aタンパク質が、配列番号3、配列番号4、配列番号5、配列番号6、配列番号7、配列番号8、および配列番号9からなる群から選択される、請求項4に記載の単離されたリボ核タンパク質複合体。
- 変異型LbCas12aタンパク質およびgRNAを含むCRISPR/Cas12aエンドヌクレアーゼシステムであって、
野生型CRISPR/Cas12aエンドヌクレアーゼシステムと比べて、維持されたオンターゲット編集活性を提示する、
CRISPR/Cas12aエンドヌクレアーゼシステム。 - DNA発現ベクターによってコードされる、請求項6に記載のCRISPR/Cas12aエンドヌクレアーゼシステム。
- DNA発現ベクターが、プラスミド媒介性のベクターを含む、請求項7に記載のCRISPR/Cas12aエンドヌクレアーゼシステム。
- DNA発現ベクターが、細菌発現ベクターおよび真核生物発現ベクターから選択される、請求項8に記載のCRISPR/Cas12aエンドヌクレアーゼシステム。
- 変異型LbCas12aタンパク質をコードする単離された核酸であって、
変異型LbCas12aタンパク質がCRISPR/Cas12aエンドヌクレアーゼシステムにおいて活性であり、
CRISPR/Cas12aエンドヌクレアーゼシステムは、野生型CRISPR/Cas12aエンドヌクレアーゼシステムと比べて、維持されたオンターゲット編集活性を提示する、
単離された核酸。 - 変異型LbCas12aタンパク質が、
a)以下の位置:N527、D559、およびE795から選択される、野生型Cas12aタンパク質に導入された単一置換変異;または、
b)以下の位置:N527、D559、およびE795のうちの少なくとも2つから選択される、野生型Cas12aタンパク質に導入された複数の置換変異、
からなる群から選択される置換変異を含む、請求項10に記載の変異型LbCas12aタンパク質をコードする単離された核酸。 - 変異型Cas12aタンパク質が、
配列番号10、配列番号11、配列番号12、配列番号13、配列番号14、配列番号15、および配列番号16からなる群から選択される、
請求項10に記載の変異型Cas12aタンパク質をコードする単離された核酸。
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