CRISPR–Cas system epitomizes prokaryote-specific quintessential adaptive defense machinery that limits the genome invasion of mobile genetic elements. It confers adaptive immunity to bacteria by capturing a protospacer fragment from invading foreign DNA, which is later inserted into the leader proximal end of CRIPSR array and serves as immunological memory to recognize recurrent invasions. The universally conserved Cas1 and Cas2 form an integration complex that is known to mediate the protospacer invasion into the CRISPR array. However, the mechanism by which this protospacer fragment gets integrated in a directional fashion into the leader proximal end is elusive. Here, we employ CRISPR/dCas9 mediated immunoprecipitation and genetic analysis to identify Integration Host Factor (IHF) as an indispensable accessory factor for spacer acquisition in Escherichia coli. Further, we show that the leader region abutting the first CRISPR repeat localizes IHF and Cas1–2 complex. IHF binding to the leader region induces bending by about 120° that in turn engenders the regeneration of the cognate binding site for protospacer bound Cas1–2 complex and brings it in proximity with the first CRISPR repeat. This appears to guide Cas1–2 complex to orient the protospacer invasion towards the leader-repeat junction thus driving the integration in a polarized fashion.

In type I CRISPR-Cas system, Cas3—a nuclease cum helicase—in cooperation with Cascade surveillance complex cleaves the target DNA. Unlike the Cascade/I-E, which is composed of five subunits, the Cascade/I-C is made of only three subunits lacking the CRISPR RNA processing enzyme Cas6, whose role is assumed by Cas5. How these differences in the composition and organization of Cascade subunits in type I-C influence the Cas3/I-C binding and its target cleavage mechanism is poorly understood. Here, we show that Cas3/I-C is intrinsically a single-strand specific promiscuous nuclease. Apart from the helicase domain, a constellation of highly conserved residues—which are unique to type I-C—located in the uncharacterized C-terminal domain appears to influence the nuclease activity. Recruited by Cascade/I-C, the HD nuclease of Cas3/I-C nicks the single-stranded region of the non-target strand and positions the helicase motor. Powered by ATP, the helicase motor reels in the target DNA, until it encounters the roadblock en route, which stimulates the HD nuclease. Remarkably, we show that Cas3/I-C supplants Cas3/I-E for CRISPR interference in type I-E in vivo, suggesting that the target cleavage mechanism is evolutionarily conserved between type I-C and type I-E despite the architectural difference exhibited by Cascade/I-C and Cascade/I-E.

Edited by Joel M. Gottesfeld 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-Especific 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.Prokaryotes utilize an adaptive immune response mediated by CRISPR and CRISPR-associated proteins (Cas) 2 to respond to infections by mobile genetic elements (MGE) (viz. phages and plasmids) (1-4). CRISPR encompasses a typical architec-This work was supported by Department of Biotechnology (DBT) Grants BT

The existence of adaptive immunity in prokaryotes came to light with the discovery of the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) in association with CRISPR-associated (Cas) proteins. This RNA mediated defence system confers resistance against the invading mobile genetic elements such as phages and plasmids. The CRISPR-Cas system operates by forming a ribonucleoprotein complex that comprises of an invader derived small RNA and Cas protein(s). Herein the small RNA acts as a guide to recognize the nucleic acid target whereas the Cas proteins facilitate target annihilation. Given the cardinal role adopted by this small RNA, its maturation from the pre-CRISPR transcript forms a pivot for successful adaptive immunity. The mandate to generate the guide CRISPR RNA (crRNA) is fulfilled by specific endoribonuclease, which processes the pre-crRNA transcript in between the repeats to liberate the individual interfering units. Intriguingly, while some endoRNases of the CRISPR system are able to process the pre-crRNA independently, others require participation of additional Cas proteins, which form a multi-protein complex for processing the pre-crRNA. Additionally, some CRISPR variants require non-Cas auxiliary factors to process the pre-crRNA. The mode of crRNA maturation further diversifies as the endoRNases in CRISPR variants coevolve with repeat clusters that exhibit high diversity in sequence and folding. Therefore, the maturation of a specific crRNA requires a distinct mechanistic solution for substrate discrimination by these endoRNases, the understanding of which is essential for appreciating the CRISPR biology. This review highlights the vivid modes adopted by the diverse CRISPR-Cas systems to generate the mature crRNA. Keywords: CRISPR RNA; CRISPR-Cas IntroductionIn order to survive, all organisms must overcome their predators. The prokaryotes and their viral predators coexist in natural and man-made environment and therefore the prokaryotes face a constant threat of getting infected by phages. This results in acute pressure on the microbial community to coevolve with their predators causing an evolutionary arms race between prey and predator. Pitted against a hostile environment, prokaryotes have developed multilayered antiviral defense systems, which act at various stages of the infection cycle of the invader. These include various innate defense systems like surface exclusion (receptor downregulation or masking), super infection exclusion (Sie systems), restrictionmodification systems (R-M and R-M like systems), and abortive infection systems (Abi) (Hyman and Abedon, 2010; Labrie et al., 2010;Westra et al., 2012a). These innate defense mechanisms are diffusive in nature and do not rely on the identity of the predator to elicit a response (Fig 1). Added to this repertoire of arsenals, the recently discovered Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) in association with CRISPRassociated (Cas) proteins endows the bacteria and archaea with an adaptive immunity (Ja...

In type I CRISPR-Cas system, Cas3 –a nuclease cum helicase– in cooperation with Cascade surveillance complex cleaves the target DNA. Unlike the Cascade/I-E, which is composed of five subunits, the Cascade/I-C is made of only three subunits lacking the CRISPR RNA processing enzyme Cas6, whose role is assumed by Cas5. How these differences in the composition and organisation of Cascade subunits in type I-C influences the Cas3/I-C binding and its target cleavage mechanism is poorly understood. Here, we show that Cas3/I-C is intrinsically a single-strand specific promiscuous nuclease. Apart from the helicase domain, a constellation of highly conserved residues –that are unique to type I-C– located in the uncharacterised C-terminal domain appears to influence the nuclease activity. Recruited by Cascade/I-C, the HD nuclease of Cas3/I-C nicks the single-stranded region of the nontarget strand and positions the helicase motor. Powered by ATP, the helicase motor reels in the target DNA, until it encounters the roadblock en route, which stimulates the HD nuclease. Remarkably, we show that Cas3/I-C supplants Cas3/I-E for CRISPR interference in type I-E in vivo, suggesting that the target cleavage mechanism is evolutionarily conserved between type I-C and type I-E despite the architectural difference exhibited by Cascade/I-C and Cascade/I-E.