Innovative Genomics Institute, University of California, Berkeley, Berkeley, CA 94720, USA.
Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720, USA.
Sci Adv. 2017 Jul 12;3(7):e1701620. doi: 10.1126/sciadv.1701620. eCollection 2017 Jul.
CRISPR (clustered regularly interspaced short palindromic repeats)-Cas9 gene editing technology is derived from a microbial adaptive immune system, where bacteriophages are often the intended target. Natural inhibitors of CRISPR-Cas9 enable phages to evade immunity and show promise in controlling Cas9-mediated gene editing in human cells. However, the mechanism of CRISPR-Cas9 inhibition is not known, and the potential applications for Cas9 inhibitor proteins in mammalian cells have not been fully established. We show that the anti-CRISPR protein AcrIIA4 binds only to assembled Cas9-single-guide RNA (sgRNA) complexes and not to Cas9 protein alone. A 3.9 Å resolution cryo-electron microscopy structure of the Cas9-sgRNA-AcrIIA4 complex revealed that the surface of AcrIIA4 is highly acidic and binds with a 1:1 stoichiometry to a region of Cas9 that normally engages the DNA protospacer adjacent motif. Consistent with this binding mode, order-of-addition experiments showed that AcrIIA4 interferes with DNA recognition but has no effect on preformed Cas9-sgRNA-DNA complexes. Timed delivery of AcrIIA4 into human cells as either protein or expression plasmid allows on-target Cas9-mediated gene editing while reducing off-target edits. These results provide a mechanistic understanding of AcrIIA4 function and demonstrate that inhibitors can modulate the extent and outcomes of Cas9-mediated gene editing.
CRISPR（成簇规律间隔短回文重复）-Cas9 基因编辑技术源自微生物适应性免疫系统，噬菌体通常是其靶向目标。CRISPR-Cas9 的天然抑制剂使噬菌体能够逃避免疫，并有望控制人类细胞中 Cas9 介导的基因编辑。然而，CRISPR-Cas9 抑制的机制尚不清楚，Cas9 抑制剂蛋白在哺乳动物细胞中的潜在应用尚未完全建立。我们表明，抗 CRISPR 蛋白 AcrIIA4 仅与组装的 Cas9-单指导 RNA（sgRNA）复合物结合，而不与单独的 Cas9 蛋白结合。Cas9-sgRNA-AcrIIA4 复合物的 3.9 Å 分辨率冷冻电镜结构表明，AcrIIA4 的表面高度酸性，并以 1:1 的化学计量与 Cas9 结合，该区域通常与 DNA 前间隔基序相邻基序结合。与这种结合模式一致，添加顺序实验表明，AcrIIA4 干扰 DNA 识别，但对预先形成的 Cas9-sgRNA-DNA 复合物没有影响。将 AcrIIA4 作为蛋白质或表达质粒递送到人类细胞中，可以在减少脱靶编辑的同时，实现靶向 Cas9 介导的基因编辑。这些结果提供了 AcrIIA4 功能的机制理解，并证明抑制剂可以调节 Cas9 介导的基因编辑的程度和结果。
