Cas3-Derived Target DNA Degradation Fragments Fuel Primed CRISPR Adaptation

Abstract

Summary 14Prokaryotes use a mechanism called priming to update their CRISPR immunological memory to rapidly counter 15 revisiting, mutated viruses and plasmids. Here we have determined how new spacers are produced and 16 selected for integration into the CRISPR array during priming. We show that Cas3 couples CRISPR interference 17 to adaptation by producing DNA breakdown products that fuel the spacer integration process in a two-step,

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

“…We show that the activated nuclease initially degrades the displaced (i.e., noncomplementary) strand in the 3-to-5 prime directions (Fig. 4), and degradation products may serve as substrates for new sequence acquisition (52). Initial degradation of the noncomplementary strand is similar to Cas3-mediated degradation of DNA targets in the I-E system.…”

“…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.…”

“…target degradation (14). However, recent evidence suggests that some Cas3 proteins also participate (either directly or indirectly) in new sequence acquisition (26,51,52). In fact, early evidence supporting this functional overlap came from bioinformatic studies that identified a unique fusion in type I-F systems that links a Cas2-like adaptation protein to the amino terminus of the Cas3 interference protein (i.e., Cas2/3) (54).…”

Spacer capture and integration by a type I-F Cas1–Cas2-3 CRISPR adaptation complex

“…For type I-E systems, RecBCD may provide one route for the generation of precursor substrates for Cas1-Cas2 during naïve adaptation (16). Similarly, during priming, Cas3 has a role in substrate generation (45) and/or adaptation complex translocation (29). Therefore, we tested whether Cas1-Cas2-3 bound and processed (captured) substrates to generate protospacers proficient for integration.…”

“…However, during type I-E interference, Cas3 is recruited to Cascade-generated R-loops, unwinds DNA via its helicase activity, and feeds ssDNA to the HD nuclease for degradation (9,10). Interference can also stimulate priming in both type I-E and I-F systems (29,32) and, in type I-E systems, DNA degradation from Cas3 fuels priming by providing substrates to the adaptation complex (45). The Cas1-Cas2-3 structure reveals that the Cas3 HD nuclease active site is adjacent to the catalytic integrase lobe of Cas1.…”