Cas9 specifies functional viral targets during CRISPR-Cas adaptation

摘要

认为细菌也许具有免疫记忆的观点曾经只是一种想象，但却因下面的发现而成为被接受的事实：CRISPR-Cas基因位点会迅速演变以获得短噬菌体序列或spacers,后者然后会在CRISPR重复段之前整合，构成噬菌体感染的一个记录。这些spacers被转录成小CRISPR RNAs(crRNAs),后者被用来定位入侵病毒的DNA。本期Nature上发表的两篇论文描述了关于细菌怎样形成入侵病毒的一个DNA记忆的分子层面的详细情况。Jennifer Doudna及同事发现，纯化的大肠杆菌Cas1-Cas2复合物能以与逆转录病毒整合酶和DNA转座酶相似的方式将寡核苷酸DNA基质整合到受体DNA内。Cas1是催化亚单元，而Cas2则会提高整合活性，它们一起形成"spacer之获取"所需的最小机构。Luciano Marraffini及同事发现，在酿脓链球菌的Ⅱ-型CRISPR-Cas系统中，以crRNA为引导使入侵病毒的DNA失活的Cas9核酸酶也是新spacer序列的整合所必需的，但其机制还有待确定。%Clustered regularly interspaced short palindromic repeat (CRISPR) loci and their associated (Cas) proteins provide adaptive immunity against viral infection in prokaryotes. Upon infection, short phage sequences known as spacers integrate between CRISPR repeats and are transcribed into small RNA molecules that guide the Cas9 nuclease to the viral targets (protospacers). Streptococcus pyogenes Cas9 cleavage of the viral genome requires the presence of a 5' -NGG-3' protospacer adjacent motif (PAM) sequence immediately downstream of the viral target. It is not known whether and how viral sequences flanked by the correct PAM are chosen as new spacers. Here we show that Cas9 selects functional spacers by recognizing their PAM during spacer acquisition. The replacement of cas9 with alleles that lack the PAM recognition motif or recognize an NGGNG PAM eliminated or changed PAM specificity during spacer acquisition, respectively. Cas9 associates with other proteins of the acquisition machinery (Cas1, Cas2 and Csn2), presumably to provide PAM-specificity to this process. These results establish a new function for Cas9 in the genesis of prokaryotic immunological memory.