Abstract：
The CRISPR system is an adaptive immune system found in prokaryotes that defends host cells against the invasion of foreign DNA1. As part of the ongoing struggle between phages and the bacterial immune system, the CRISPR system has evolved into various types, each with distinct functionalities2. Type II Cas9 is the most extensively studied of these systems and has diverse subtypes. It remains uncertain whether members of this family can evolve additional mechanisms to counter viral invasions3,4. Here we identify 2,062 complete Cas9 loci, predict the structures of their associated proteins and reveal three structural growth trajectories for type II-C Cas9. We found that novel associated genes (NAGs) tended to be present within the loci of larger II-C Cas9s. Further investigation revealed that CbCas9 from Chryseobacterium species contains a novel β-REC2 domain, and forms a heterotetrameric complex with an NAG-encoded CRISPR-Cas-system-promoting (pro-CRISPR) protein of II-C Cas9 (PcrIIC1). The CbCas9-PcrIIC1 complex exhibits enhanced DNA binding and cleavage activity, broader compatibility for protospacer adjacent motif sequences, increased tolerance for mismatches and improved anti-phage immunity, compared with stand-alone CbCas9. Overall, our work sheds light on the diversity and \'growth evolutionary\' trajectories of II-C Cas9 proteins at the structural level, and identifies many NAGs-such as PcrIIC1, which serves as a pro-CRISPR factor to enhance CRISPR-mediated immunity.
摘要：
CRISPR系统是在原核生物中发现的适应性免疫系统,其保护宿主细胞免受外源DNA1的侵袭。作为噬菌体和细菌免疫系统之间持续斗争的一部分，CRISPR系统已经演变为各种类型，每个都有不同的功能2。II型Cas9是这些系统中研究最广泛的，具有不同的亚型。尚不确定该家族的成员是否可以进化出其他机制来抵抗病毒侵袭3,4。这里我们确定了2062个完整的Cas9基因座，预测其相关蛋白的结构，并揭示II-C型Cas9的三个结构生长轨迹。我们发现，新的相关基因（NAGs）倾向于存在于较大的II-CCas9s的基因座内。进一步的研究表明，来自金黄杆菌属物种的CbCas9含有一个新的β-REC2结构域，并与II-CCas9（PcrIIC1）的NAG编码的CRISPR-Cas系统促进（pro-CRISPR）蛋白形成异四聚体复合物。CbCas9-PcrIIC1复合物表现出增强的DNA结合和切割活性，前间隔区相邻基序序列的更广泛兼容性，增加对错配的耐受性和改善的抗噬菌体免疫，与独立的CbCas9相比。总的来说,我们的工作揭示了II-CCas9蛋白在结构水平上的多样性和“生长进化”轨迹，并鉴定了许多NAG，例如PcrIIC1，它作为pro-CRISPR因子来增强CRISPR介导的免疫。
