CRISPR-Cas systems, which provide adaptive immunity against foreign nucleic acids in prokaryotes, can serve as useful molecular tools for multiple applications in genome engineering. Diverse CRISPR-Cas systems originating from distinct prokaryotes function through a common mechanism involving the assembly of small crRNA molecules and Cas proteins into a ribonucleoprotein (RNP) effector complex, and formation of an R-loop structure upon binding to the target DNA. Extensive research on the I-E subtype established the prototypical mechanism of DNA interference in type I systems, where the coordinated action of a ribonucleoprotein Cascade complex and Cas3 protein destroys foreign DNA. However, diverse protein composition between type I subtypes suggests differences in the mechanism of DNA interference that could be exploited for novel practical applications that call for further exploration of these systems. Here we examined the mechanism of DNA interference provided by the type I-F1 system from Aggregatibacter actinomycetemcomitans D7S-1 (Aa). We show that functional Aa-Cascade complexes can be assembled not only with WT spacer of 32 nt but also with shorter or longer (14–176 nt) spacers. All complexes guided by the spacer bind to the target DNA sequence (protospacer) forming an R-loop when a C or CT protospacer adjacent motif (PAM) is present immediately upstream the protospacer (at −1 or −2,−1 position, respectively). The range of spacer and protospacer complementarity predetermine the length of the R-loop; however, only R-loops of WT length or longer trigger the nuclease/helicase Cas2/3, which initiates ATP-dependent unidirectional degradation at the PAM-distal end of the WT R-loop. Meanwhile, truncation of the WT R-loop at the PAM-distal end abolishes Cas2/3 cleavage. We provide a comprehensive characterisation of the DNA interference mechanism in the type I-F1 CRISPR-Cas system, which is different from the type I-E in a few aspects. First, DNA cleavage initiation, which usually happens at the PAM-proximal end in type I-E, is shifted to the PAM-distal end of WT R-loop in the type I-F1. Second, the R-loop length controls on/off switch of DNA interference in the type I-F1, while cleavage initiation is less restricted in the type I-E. These results indicate that DNA interference in type I-F1 systems is governed through a checkpoint provided by the Cascade complex, which verifies the appropriate length for the R-loop.

中文翻译：

---

DNA干扰由I-F1型CRISPR-Cas系统中的R环长度控制。

CRISPR-Cas系统可提供对原核生物中外源核酸的适应性免疫力，可作为基因组工程中多种应用的有用分子工具。源自不同原核生物的多样CRISPR-Cas系统通过共同的机制起作用，该机制涉及将小的crRNA分子和Cas蛋白组装成核糖核蛋白（RNP）效应物复合体，并在与靶DNA结合后形成R环结构。对IE亚型的广泛研究建立了I型系统中DNA干扰的原型机制，其中核糖核蛋白Cascade复合体和Cas3蛋白的协同作用破坏了外源DNA。然而，I型亚型之间不同的蛋白质组成表明DNA干扰机制的差异，可以将其用于新颖的实际应用中，从而需要进一步探索这些系统。在这里，我们研究了由集食放线菌D7S-1（Aa）的I-F1系统提供的DNA干扰机制。我们表明，功能性Aa-Cascade复合物不仅可以用32 nt的WT间隔子组装，而且可以用较短或更长（14–176 nt）的间隔子组装。当C或CT的原间隔子相邻基序（PAM）分别位于原间隔子的上游时（分别位于-1或-2，-1位置），由间隔子引导的所有复合物均与目标DNA序列（原间隔子）结合，形成一个R环。 ）。间隔子和原间隔子的互补范围决定了R环的长度；然而，只有WT长或更长的R环触发核酸酶/解旋酶Cas2 / 3，这会在WT R环的PAM远端引发ATP依赖的单向降解。同时，在PAM远端的WT R-环的截短消除了Cas2 / 3裂解。我们对I-F1 CRISPR-Cas系统中的DNA干扰机制进行了全面的表征，在某些方面与IE类型不同。首先，通常在IE型的PAM近端发生的DNA切割起始转移到I-F1型的WT R环的PAM远端。其次，R环长度控制I-F1型DNA干扰的开/关，而IE类型的裂解起始受限制。