Foreign DNA acquisition by the I-F CRISPR–Cas system requires all components of the interference machinery

Abstract

CRISPR immunity depends on acquisition of fragments of foreign DNA into CRISPR arrays. For type I-E CRISPR–Cas systems two modes of spacer acquisition, naïve and primed adaptation, were described. Naïve adaptation requires just two most conserved Cas1 and Cas2 proteins; it leads to spacer acquisition from both foreign and bacterial DNA and results in multiple spacers incapable of immune response. Primed adaptation requires all Cas proteins and a CRISPR RNA recognizing a partially matching target. It leads to selective acquisition of spacers from DNA molecules recognized by priming CRISPR RNA, with most spacers capable of protecting the host. Here, we studied spacer acquisition by a type I-F CRISPR–Cas system. We observe both naïve and primed adaptation. Both processes require not just Cas1 and Cas2, but also intact Csy complex and CRISPR RNA. Primed adaptation shows a gradient of acquisition efficiency as a function of distance from the priming site and a strand bias that is consistent with existence of single-stranded adaption intermediates. The results provide new insights into the mechanism of spacer acquisition and illustrate surprising mechanistic diversity of related CRISPR–Cas systems.

Cas1 and the Csy complex are opposing regulators of Cas2/3 nuclease activity

“…S2E). This model may explain how AcrF3 blocks both target interference and new sequence adaptation (26).…”

“…However, strict sequence requirements present a potential weakness in the immune system because mutations in either the PAM or specific positions in the protospacer allow viruses to escape CRISPR-Cas immunity (45)(46)(47). Bacteria with type I immune systems can restore immunity against "escape" mutants by using a positive feedback loop that rapidly updates the CRISPR locus with new spacers derived from the same region of the foreign DNA that contains the mutated target (26,46,(48)(49)(50). This process of rapid acquisition, called "primed adaptation," requires not only the adaptation proteins (i.e., Cas1 and Cas2) but also Cas3 and the crRNA-guided surveillance complex (26,46,51,52).…”

“…Bacteria with type I immune systems can restore immunity against "escape" mutants by using a positive feedback loop that rapidly updates the CRISPR locus with new spacers derived from the same region of the foreign DNA that contains the mutated target (26,46,(48)(49)(50). This process of rapid acquisition, called "primed adaptation," requires not only the adaptation proteins (i.e., Cas1 and Cas2) but also Cas3 and the crRNA-guided surveillance complex (26,46,51,52). Priming in type I-F and type I-E CRISPR systems is similar, but there are important differences.…”

“…Cas2 proteins play a critical role in CRISPR adaptation (i.e., the integration of foreign DNA into CRISPR loci) (21)(22)(23)(24)(25), and fusion of the Cas2 adaptation protein to the Cas3 interference protein suggests a functional connection between the two stages of CRISPR immunity. In fact, recent work by Vorontsova et al has shown that new sequence acquisition (both naïve and primed) in type I-F systems requires all of the Cas proteins, including those that were previously thought to be involved exclusively in interference (26).…”

Recruitment of CRISPR-Cas systems by Tn7-like transposons

“…At the adaptation stage, the Cas1-Cas2 protein complex, in some instances with additional involvement of accessory adaptation proteins and/or effector module proteins, captures a segment of the target DNA (known as the "protospacer") and inserts it at the 5′ end of a CRISPR array (19)(20)(21)(22)(23). In the second processing stage, a CRISPR array is transcribed into a long transcript known as "precrRNA" that is bound by Cas proteins and processed into mature, small crRNAs.…”