Broad-spectrum enzymatic inhibition of CRISPR-Cas12a
Broad-spectrum enzymatic inhibition of CRISPR-Cas12a
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DOI：
10.1038/s41594-019-0208-z
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发表时间：
2019-04-01
影响因子：
16.8
通讯作者：
Doudna, Jennifer A.
中科院分区：
文献类型：
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作者：
Knott, Gavin J.;Thornton, Brittney W.;Doudna, Jennifer A.
Cas 12 a是一种细菌RNA引导的核酸酶，广泛用于基因组编辑，最近还用作分子诊断。在细菌中，Cas 12 a酶可以被噬菌体衍生的蛋白质、抗CRISPR（Acr）抑制，以阻碍成簇的规则间隔短回文重复序列（CRISPR）适应性免疫系统。这些抑制剂如何通过阻止程序化DNA切割来禁用Cas 12 a尚不清楚。我们发现，三个这样的抑制剂（AcrVA 1，AcrVA 4和AcrVA 5）通过功能上不同的机制，包括以前未观察到的酶促策略阻断Cas 12 a活性。AcrVA 4和AcrVA 5抑制双链DNA（dsDNA）的识别，其中AcrVA 4驱动Cas 12 a的二聚化。相比之下，AcrVA 1是一种多重转换抑制剂，其触发Cas 12 a结合的引导RNA的靶识别序列的切割以不可逆地阻断Cas 12 a复合物。这些不同的机制为噬菌体提供了逃避CRISPR-Cas 12 a的工具，并支持生物技术应用，其中Cas 12 a的多重周转酶抑制是理想的。
Cas12a is a bacterial RNA-guided nuclease used widely for genome editing and, more recently, as a molecular diagnostic. In bacteria, Cas12a enzymes can be inhibited by bacteriophage-derived proteins, anti-CRISPRs (Acrs), to thwart clustered regularly interspaced short palindromic repeat (CRISPR) adaptive immune systems. How these inhibitors disable Cas12a by preventing programmed DNA cleavage is unknown. We show that three such inhibitors (AcrVA1, AcrVA4 and AcrVA5) block Cas12a activity via functionally distinct mechanisms, including a previously unobserved enzymatic strategy. AcrVA4 and AcrVA5 inhibit recognition of double-stranded DNA (dsDNA), with AcrVA4 driving dimerization of Cas12a. In contrast, AcrVA1 is a multiple-turnover inhibitor that triggers cleavage of the target-recognition sequence of the Cas12a-bound guide RNA to irreversibly inactivate the Cas12a complex. These distinct mechanisms equip bacteriophages with tools to evade CRISPR-Cas12a and support biotechnological applications for which multiple-turnover enzymatic inhibition of Cas12a is desirable.