National Institutes of Health/National Human Genome Research Institute (NIH/NHGRI)
RM1HG009490
米国
引用
ジャーナル: Science / 年: 2020 タイトル: DNA capture by a CRISPR-Cas9-guided adenine base editor. 著者: Audrone Lapinaite / Gavin J Knott / Cody M Palumbo / Enrique Lin-Shiao / Michelle F Richter / Kevin T Zhao / Peter A Beal / David R Liu / Jennifer A Doudna / 要旨: CRISPR-Cas-guided base editors convert A•T to G•C, or C•G to T•A, in cellular DNA for precision genome editing. To understand the molecular basis for DNA adenosine deamination by adenine base ...CRISPR-Cas-guided base editors convert A•T to G•C, or C•G to T•A, in cellular DNA for precision genome editing. To understand the molecular basis for DNA adenosine deamination by adenine base editors (ABEs), we determined a 3.2-angstrom resolution cryo-electron microscopy structure of ABE8e in a substrate-bound state in which the deaminase domain engages DNA exposed within the CRISPR-Cas9 R-loop complex. Kinetic and structural data suggest that ABE8e catalyzes DNA deamination up to ~1100-fold faster than earlier ABEs because of mutations that stabilize DNA substrates in a constrained, transfer RNA-like conformation. Furthermore, ABE8e's accelerated DNA deamination suggests a previously unobserved transient DNA melting that may occur during double-stranded DNA surveillance by CRISPR-Cas9. These results explain ABE8e-mediated base-editing outcomes and inform the future design of base editors.
タンパク質・ペプチド: t-RNA adenine deaminase A v8e (TadA-8e)
リガンド: MAGNESIUM ION
リガンド: ZINC ION
-
超分子 #1: CRISPR-Cas9 ABE8e in a substrate-bound state
超分子
名称: CRISPR-Cas9 ABE8e in a substrate-bound state / タイプ: complex / ID: 1 / 親要素: 0 / 含まれる分子: #1-#5 詳細: ABE8e in a substrate-bound state where the deaminase (TadA-8e) engages DNA exposed within the CRISPR-Cas9 R-loop complex.
凍結剤: ETHANE / チャンバー内湿度: 100 % / チャンバー内温度: 281.15 K / 装置: FEI VITROBOT MARK IV 詳細: The grids were rapidly plunged into liquid ethane using a FEI Vitrobot MarkIV maintained at 8C and 100% humidity, after being blotted for 4.5s with blot force 8..