Temperature-Responsive Competitive Inhibition of CRISPR-Cas9
Temperature-Responsive Competitive Inhibition of CRISPR-Cas9
复制标题
DOI：
10.1016/j.molcel.2018.11.016
复制
发表时间：
2019-02-07
期刊：
影响因子：
16
通讯作者：
Doudna, Jennifer A.
中科院分区：
文献类型：
--
作者：
Jiang, Fuguo;Liu, Jun-Jie;Doudna, Jennifer A.
CRISPR-Cas免疫系统利用RNA引导的核酸酶来保护细菌免受噬菌体感染。噬菌体反过来又进化出抑制性“抗CRISPR”（Acr）蛋白，包括六种抑制剂（AcrIIA 1-AcrIIA 6），可以阻断II-A型CRISPR-Cas9酶的DNA切割和基因组编辑。我们在这里表明，AcrIIA 2及其更有效的同源物AcrIIA 2b通过封闭DNA结合所需的蛋白质残基来阻止Cas9与DNA结合。Cryo-EM确定的与S结合的AcrIIA 2或AcrIIA 2b的结构。化脓性链球菌Cas9揭示了与其他已知Acr不同的DNA结合的竞争性抑制模式。AcrIIA 2和AcrIIA 2b对Cas9抑制的温度依赖性的差异源于抑制剂结构和Cas9上的局部底物结合环境的差异。这些发现扩展了在时间、空间和条件上调节CRISPR-Cas9基因组编辑的自然工具箱。
CRISPR-Cas immune systems utilize RNA-guided nucleases to protect bacteria from bacteriophage infection. Bacteriophages have in turn evolved inhibitory "anti-CRISPR'' (Acr) proteins, including six inhibitors (AcrIIA1-AcrIIA6) that can block DNA cutting and genome editing by type II-A CRISPR-Cas9 enzymes. We show here that AcrIIA2 and its more potent homolog, AcrIIA2b, prevent Cas9 binding to DNA by occluding protein residues required for DNA binding. Cryo-EM-determined structures of AcrIIA2 or AcrIIA2b bound to S. pyogenes Cas9 reveal a mode of competitive inhibition of DNA binding that is distinct from other known Acrs. Differences in the temperature dependence of Cas9 inhibition by AcrIIA2 and AcrIIA2b arise from differences in both inhibitor structure and the local inhibitor-binding environment on Cas9. These findings expand the natural toolbox for regulating CRISPR-Cas9 genome editing temporally, spatially, and conditionally.