期刊
JOURNAL OF BIOLOGICAL CHEMISTRY
卷 295, 期 39, 页码 13502-13515出版社
ELSEVIER
DOI: 10.1074/jbc.RA120.014030
关键词
CRISPR-Cas; type I-B; archaea; Haloferax volcanii; adaptation; self-targeting; crRNA; homologous recombination; naive adaptation
资金
- Deutsche Forschungsgemeinschaft (DFG) [Ma1538/27-1]
- BBSRC [BB/R007543/1] Funding Source: UKRI
Haloferax volcaniiis, to our knowledge, the only prokaryote known to tolerate CRISPR-Cas-mediated damage to its genome in the WT background; the resulting cleavage of the genome is repaired by homologous recombination restoring the WT version. In mutantHaloferaxstrains 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 inH. volcaniiCRISPR-Cas type I-B. We show that self-targeting and genome breakage events that are induced by self-targeting, such as those catalyzed 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 centered on the targeting site. Furthermore, we observed naive spacer acquisition at a low level in WTHaloferaxcells and with higher efficiency upon overexpression of the Cas proteins Cas1, Cas2, and Cas4. Taken together, these findings indicate that naive adaptation is a regulated process inH. volcaniithat operates at low basal levels and is induced by DNA breaks.
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