Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, United States.
Gladstone Institutes, San Francisco, United States.
Elife. 2019 Aug 9;8:e49110. doi: 10.7554/eLife.49110.
CRISPR-Cas systems provide bacteria and archaea with programmable immunity against mobile genetic elements. Evolutionary pressure by CRISPR-Cas has driven bacteriophage to evolve small protein inhibitors, anti-CRISPRs (Acrs), that block Cas enzyme function by wide-ranging mechanisms. We show here that the inhibitor AcrVA4 uses a previously undescribed strategy to recognize the Cas12a (LbCas12a) pre-crRNA processing nuclease, forming a Cas12a dimer, and allosterically inhibiting DNA binding. The Cas12a (AsCas12a) enzyme, widely used for genome editing applications, contains an ancestral helical bundle that blocks AcrVA4 binding and allows it to escape anti-CRISPR recognition. Using biochemical, microbiological, and human cell editing experiments, we show that Cas12a orthologs can be rendered either sensitive or resistant to AcrVA4 through rational structural engineering informed by evolution. Together, these findings explain a new mode of CRISPR-Cas inhibition and illustrate how structural variability in Cas effectors can drive opportunistic co-evolution of inhibitors by bacteriophage.
CRISPR-Cas 系统为细菌和古菌提供了可编程的免疫能力，以抵御移动遗传元件。CRISPR-Cas 的进化压力促使噬菌体进化出小的蛋白质抑制剂，即抗 CRISPR 蛋白 (Acrs)，通过广泛的机制来阻断 Cas 酶的功能。我们在这里表明，抑制剂 AcrVA4 采用了一种以前未知的策略来识别 Cas12a (LbCas12a) 前 crRNA 加工核酸酶，形成 Cas12a 二聚体，并别构抑制 DNA 结合。Cas12a 酶（AsCas12a）广泛用于基因组编辑应用，它包含一个原始的螺旋束，阻止 AcrVA4 的结合，并使其能够逃避抗 CRISPR 的识别。通过基于进化的理性结构工程的生化、微生物学和人类细胞编辑实验，我们表明 Cas12a 同源物可以通过合理的结构工程设计变得对 AcrVA4 敏感或耐药。总之，这些发现解释了一种新的 CRISPR-Cas 抑制模式，并说明了 Cas 效应子的结构变异性如何通过噬菌体驱动抑制剂的机会性共同进化。
