原核生物利用基于CRISPR-Cas的RNA引导的适应性免疫来抵御移动遗传元件,如噬菌体和质粒。在CRISPR适应期间,这是CRISPR免疫的第一阶段,Cas1-2整合酶复合物捕获入侵者衍生的前pacerDNA,并将其特异性整合在前导-重复连接处作为间隔区.对于这种整合,CRISPR-Cas系统的几种变体使用Cas4作为不可缺少的核酸酶,用于选择性地将含有前置序列的前间隔区相邻基序(PAM)加工至限定的长度。令人惊讶的是,然而,一些CRISPR-Cas系统,例如I-E型,失去了Cas4。尽管没有Cas4,但在I-E型中,预起搏器如何显示出无可挑剔的长度保护和PAM选择仍然很有趣。这里,使用体内和体外整合测定法,深度测序,和外切核酸酶足迹,我们表明Cas1-2/I-E-viaCas1的I-E型特异性延伸的C末端尾对大肠杆菌中含有可变长度的PAM的预起搏器表现出固有的亲和力尽管Cas1-2/I-E不修剪预起搏器,它的结合保护预起搏器边界免受外切核酸酶的作用。这确保通过外切核酸酶将暴露的末端修剪至适当大小的底物,以整合到CRISPR基因座中。总之,我们的工作表明,在一些CRISPR-Cas变体中,例如I-E型,PAM选择的特异性在于Cas1-2,而细胞非Cas核酸外切酶则选择了前pacer加工,从而抵消了对Cas4的需求。
Prokaryotes deploy CRISPR-Cas-based RNA-guided adaptive immunity to fend off mobile genetic elements such as phages and plasmids. During CRISPR adaptation, which is the first stage of CRISPR immunity, the Cas1-2 integrase complex captures invader-derived prespacer DNA and specifically integrates it at the leader-repeat junction as spacers. For this integration, several variants of CRISPR-Cas systems use Cas4 as an indispensable nuclease for selectively processing the protospacer adjacent motif (PAM) containing prespacers to a defined length. Surprisingly, however, a few CRISPR-Cas systems, such as type I-E, are bereft of Cas4. Despite the absence of Cas4, how the prespacers show impeccable conservation for length and PAM selection in type I-E remains intriguing. Here, using in vivo and in vitro integration assays, deep sequencing, and exonuclease footprinting, we show that Cas1-2/I-E-via the type I-E-specific extended C-terminal tail of Cas1-displays intrinsic affinity for PAM containing prespacers of variable length in Escherichia coli Although Cas1-2/I-E does not prune the prespacers, its binding protects the prespacer boundaries from exonuclease action. This ensures the pruning of exposed ends by exonucleases to aptly sized substrates for integration into the CRISPR locus. In summary, our work reveals that in a few CRISPR-Cas variants, such as type I-E, the specificity of PAM selection resides with Cas1-2, whereas the prespacer processing is co-opted by cellular non-Cas exonucleases, thereby offsetting the need for Cas4.