CRISPR-Cas targeting in Haloferax volcanii promotes within-species gene exchange by triggering homologous recombination

Abstract CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-Cas (CRISPR-associated genes) systems provide adaptive immunity in bacteria and archaea against mobile genetic elements, but the role they play in gene exchange and speciation remains unclear. Here, we investigated how CRISPR-Cas targeting affects mating and gene exchange in the halophilic archaeon Haloferax volcanii. Surprisingly, we found that CRISPR-Cas targeting significantly increased mating efficiency between members of the same species, in contrast to its previously documented role in reducing interspecies mating. This enhanced mating efficiency was dependent on the Cas3 nuclease/helicase and extended beyond the targeted genomic regions. Further analysis revealed that CRISPR-Cas targeting promoted biased recombination in favor of the targeting strain (the strain containing the CRISPR-Cas system) during mating, resulting in an increased proportion of recombinant progeny that are positive for CRISPR-Cas. To test whether an increase in recombination is sufficient to increase mating efficiency, we tested whether strains lacking the Mre11–Rad50 complex, which are known to have elevated recombination activity, also exhibited higher mating success. Indeed, these strains showed higher mating, as did cells that were exposed to DNA damage using methyl methanesulfonate. These findings suggest that CRISPR-Cas systems in archaea play roles beyond their canonical immune function. They may contribute to speciation by facilitating within-species gene exchange while limiting between-species genetic transfer, thereby maintaining species boundaries.