Adaptation induced by self-targeting in a type I-B CRISPR-Cas system.

Stachler, Aris-Edda; Wörtz, Julia; Alkhnbashi, Omer S; Turgeman-Grott, Israela; Smith, Rachel; Allers, Thorsten; Backofen, Rolf; Gophna, Uri; Marchfelder, Anita

Haloferax volcanii is to our knowledge the only prokaryote known to tolerate CRISPR-Cas mediated damage to its genome in the wild type background; the resulting cleavage of the genome is repaired by homologous recombination restoring the wild type version. In mutant Haloferax strains with enhanced self-targeting, cell fitness decreases and microhomology-mediated end joining becomes active, generating deletions in the targeted gene. Here we use self-targeting to investigate adaptation in H. volcanii CRISPR-Cas type I-B. We show that self-targeting and genome breakage events that are induced by self-targeting, such as those catalysed by active transposases, can generate DNA fragments that are used by the CRISPR-Cas adaptation machinery for integration into the CRISPR loci. Low cellular concentrations of self-targeting crRNAs resulted in acquisition of large numbers of spacers originating from the entire genomic DNA. In contrast, high concentrations of self-targeting crRNAs resulted in lower acquisition that was mostly centred around the targeting site. Furthermore, we observed naïve spacer acquisition at a low level in wild type Haloferax cells and with higher efficiency upon overexpression of the Cas proteins Cas1, Cas2 and Cas4. Taken together these findings indicate that naïve adaptation is a regulated process in H. volcanii that operates at low basal levels and is induced by DNA breaks.