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

Abstract

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.

Protospacer-Adjacent Motif Specificity during Clostridioides difficile Type I-B CRISPR-Cas Interference and Adaptation

“…7 ). This result is consistent with preferential spacer acquisition from regions prone to the generation of free DNA termini, as was also reported for other CRISPR-Cas systems ( 44 – 46 ).…”

“…The PAM requirements and proteins involved in motif recognition differ during interference and adaptation ( 47 ). In H. volcanii type I-B CRISPR-Cas, only the TAC PAM was associated with spacer acquisition, suggesting a PAM recognition mechanism that is stricter for adaptation than for interference ( 45 ). The PAM requirements for spacer acquisition remained unexplored in bacterial I-B CRISPR-Cas systems.…”

“…In general, the spacer acquisition process in various CRISPR-Cas systems has been extensively studied, including recent reports on naive, self-targeting-induced, and primed adaptation for archaeal type I-B CRISPR-Cas ( 45 , 46 , 51 – 53 ). However, no data on the bacterial type I-B CRISPR-Cas spacer acquisition have been reported so far.…”

“…This protein participates in the selection and processing of prespacers, defines the correct PAM, and provides the correct orientation for new spacers during their integration into the CRISPR array ( 27 – 31 ). Recent studies in Pyrococcus furiosus and H. volcanii showed that the overexpression of Cas1, Cas2, and Cas4 elevates basal adaptation levels ( 45 , 46 ). However, overexpression of the adaptation proteins resulted in PAM-independent acquisition in H. volcanii ( 45 ), which is clearly distinct from our observations in C. difficile .…”

Heavily Armed Ancestors: CRISPR Immunity and Applications in Archaea with a Comparative Analysis of CRISPR Types in Sulfolobales

“…By expressing a chromosome-targeting crRNA, it was shown that H. volcanii is one of the few prokaryotes that tolerates CRISPR-Cas self-targeting potentially because of a potent microhomology-repair pathway [168], marking it as a distinguished model organism to study CRISPR autoimmunity. For instance, a very recent study demonstrated that overexpression of a self-targeting spacer triggered adaptation of novel spacers collected from the vicinity of the originally targeted chromosomal locus [169]. Apart from the own chromosome, H. volcanii and H. mediterranei were recently shown to acquire spacers from each other s chromosomes in mating assays [170], where cells of both species fuse by forming cytoplasmatic bridges [171].…”