Cas9 specifies functional viral targets during CRISPR–Cas adaptation

Abstract

Clustered, regularly interspaced, short palindromic repeat (CRISPR) loci and their associated (Cas) proteins provide adaptive immunity against viral infection in prokaryotes. Upon infection, short phage sequences known as spacers integrate between CRISPR repeats and are transcribed into small RNA guides that identify the viral targets (protospacers) of the Cas9 nuclease. Streptococcus pyogenes Cas9 cleavage of the viral genome requires the presence of an NGG protospacer adjacent motif (PAM) sequence immediately downstream of the target. It is not known if and how viral sequences with the correct PAM are chosen as new spacers. Here we show that Cas9 specifies functional PAM sequences during spacer acquisition. The replacement of cas9 with alleles that lack the PAM recognition motif or recognize an NGGNG PAM eliminated or changed PAM specificity during spacer acquisition, respectively. Cas9 associates with other proteins of the acquisition machinery (Cas1, Cas2 and Csn2), presumably to provide PAM-specificity to this process. These results establish a new function for Cas9 in the genesis of the prokaryotic immunological memory.

CRISPR–Cas9 Structures and Mechanisms

“…It snips dsDNA 3 bp upstream of the PAM through its two distinct nuclease domains: an HNH-like nuclease domain that cleaves the DNA strand complementary to the guide RNA sequence (target strand), and an RuvC-like nuclease domain responsible for cleaving the DNA strand opposite the complementary strand (nontarget strand) (Figure 2) (13,27,48). In addition to its critical role in CRISPR interference, Cas9 also participates in crRNA maturation and spacer acquisition (32).…”

Evolutionary Ecology of Prokaryotic Immune Mechanisms

“…Class 2 systems can be subdivided into type II systems (44), which encode the Cas9 enzyme that is presently widely used for genome editing (80,81); type V systems, which encode the Cpf1, C2c1, or C2c3 effector enzyme (44); and type VI systems, which encode the C2c2 effector enzyme (45). Type II systems require Cas1 and Cas2 for spacer acquisition as well as Cas9 for PAM specificity (82). The expression stage requires a trans-encoded crRNA (tracrRNA) molecule that pairs with pre-CRISPR RNA repeat sequences, which, in the presence of Cas9, triggers RNase III-mediated cleavage in the resulting stretch of double-stranded RNA (83).…”

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.…”