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

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

Research output: Contribution to journalArticlepeer-review


Haloferax volcanii is, 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 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 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 naïve spacer acquisition at a low level in WT 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.

Original languageEnglish
Pages (from-to)13502-13515
Number of pages14
JournalThe Journal of biological chemistry
Issue number39
StatePublished - 25 Sep 2020


  • adaptation
  • archaea
  • CRISPR-Cas
  • crRNA
  • Haloferax volcanii
  • homologous recombination
  • naive adaptation
  • self-targeting
  • type I-B


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