High tolerance to self-targeting of the genome by the endogenous CRISPR-Cas system in an archaeon

CRISPR-Cas systems allow bacteria and archaea to acquire sequence-specific immunity against selfish genetic elements such as viruses and plasmids, by specific degradation of invader DNA or RNA. However, this involves the risk of autoimmunity if immune memory against host DNA is mistakenly acquired. Such autoimmunity has been shown to be highly toxic in several bacteria and is believed to be one of the major costs of maintaining these defense systems. Here we generated an experimental system in which a non-essential gene, required for pigment production and the reddish colony color, is targeted by the CRISPR-Cas I-B system of the halophilic archaeonHaloferax volcanii.We showthat under native conditions, where both the self-targeting and native crRNAs are expressed, self-targeting by CRISPR-Cas causes no reduction in transformation efficiency of the plasmid encoding the self-targeting crRNA. Furthermore, under such conditions, no effect on organismal growth rate or loss of the reddish colony phenotype due to mutations in the targeted region could be observed. In contrast, in cells deleted for the pre-crRNA processing gene cas6, where only the self-targeting crRNA exists as mature crRNA, selftargeting leads to moderate toxicity and the emergence of deletion mutants. Sequencing of the deletions caused by CRISPR-Cas self targeting indicated DNA repair viamicrohomology-mediated end joining.