Cas9 Cleavage of Viral Genomes Primes the Acquisition of New Immunological Memories

Abstract

Highlights d Immune cells acquire additional spacers as Cas9 destroys the infecting virus d New spacers come from the immediate vicinity of Cas9 target site in the viral genome d Cleavage of the target DNA by Cas9 is required for additional spacer acquisition d Cleavage-mediated spacer acquisition anticipates the rise of target escaper viruses

Diversity of molecular mechanisms used by anti-CRISPR proteins: the tip of an iceberg?

“…CRISPR-Cas immunity divides into (1) the adaptation step, to build up the immunological memory through the acquisition of foreign DNA sequences, (2) the expression and assembly step, to produce functional effector complexes and (3) the interference step, to detect and cleave foreign nucleic acids, provided they are flanked by a protospacer-adjacent motif (PAM), with the exception of type III and type VI CRISPR-Cas systems, that allows self and non-self discrimination by the host [5,18] (Figure 1). In type I and type II CRISPR-Cas systems, the PAM is also important for appropriate spacers selection during adaptation [19,20]. Anti-CRISPR proteins have, therefore, the possibility to inactivate CRISPR-Cas immunity by interfering with one, or several, of these steps.…”

“…In addition, components of the interference machinery are also involved in the adaptation step. The type I helicase/nuclease Cas3 and effector Cascade-crRNA complex (glossary), and the type II effector Cas9-sgRNA complex (glossary), associate with the Cas1-Cas2 complex for naïve, primed and interference-driven spacer acquisition (glossary) [20,[22][23][24][25]. Noteworthy, Cas2 and Cas3 are fused into a single protein (Cas2/3) in the type I-F CRISPR-Cas system [3], pointing on the functional link between the adaptation and interference steps.…”

“…In type II-A CRISPR-Cas systems, the Cas9-tracrRNA complex (glossary) associates with the other components of the spacer acquisition machinery, Cas1, Cas2 and Csn2, to ensure that new spacers are flanked by the correct PAM [25]. Moreover, Cas9-mediated interference activity and protospacer cleavage have recently been shown to prime the acquisition of new spacers [20]. Therefore, type II-A anti-CRISPR proteins that inhibit Cas9 interference activity could also perturb the adaptation step.…”

How important is CRISPR-Cas for protecting natural populations of bacteria against infections by mobile genetic elements?

“…Mild or strong overexpression of the Type I-E CRISPR-Cas system of E. coli targeting the non-lysogenic mutant lvir provides full immunity, with efficiency of plaquing around 10 -6 (125, 129). Similarly, the Type II-A CRISPR-Cas immune system of Streptococcus pyogenes SF370 provides high levels of immunity when expressed in S. aureus RN4220 against the staphylococcal phage ϕNM4γ4, a lytic mutant of ϕNM4 (44)(45)(46) and the Type III-A from S. epidermidis RP62a provides high levels of immunity when expressed in S. aureus RN4220 against phage ϕNM1g6, a lytic mutant of the temperate phage ϕNM1 (101). Mapping the variability in the levels of protection conferred by CRISPR-Cas immunity using a wider range of CRISPR immune systems and phages will be critical to understand when and where these systems matter.…”

“…Since these pioneering studies, spacer uptake from phages and other mobile genetic elements in bacteria and archaea from natural and human-associated environments has been inferred from variation in spacer sequences within and between populations of the same species and from their homology to mobile genetic element (MGE) genomes (31)(32)(33)(34)(35)(36)(37)(38)(39)(40). Experimental observations of spacer uptake in the lab in response to plasmid and phage infection have been made amongst others in engineered E. coli strains (41)(42)(43) and Staphylococcus aureus (44)(45)(46)(47) and in wild type Pectobacterium atrosepticum (48), Pseudomonas aeruginosa (49,50), Roseburia intestinalis (51), Sulfolobus solfataricus (52), Streptococcus mutans (37) and other species (reviewed in (53)). Consistent with the hypothesis that CRISPR-Cas protects bacteria from infections, some mobile genetic elements encode so-called anti-CRISPR genes (reviewed in (54)).…”