Harnessing the native type I-B CRISPR-Cas for genome editing in a polyploid archaeon

Abstract Research on CRISPR-Cas (clustered regularly interspaced short palindromicrepeats- CRISPRassociated protein) systems has led to the revolutionary CRISPR/ Cas9 genome editingtechnique. However, for most archaeaand half of bacteria, exploitation of their native CRISPR-Cas machineries may be more straightforward and convenient. In this study, we harnessed the native type I-B CRISPR-Cas system for precise genome editingin the polyploidhaloarchaeon Haloarcula hispanica. After testing different designs, the editing tool was optimized to be a single plasmid that carries both the self-targeting mini- CRISPRand a 600–800 bp donor. Significantly, chromosomal modifications, such as gene deletion, gene tagging or single nucleotide substitution, were precisely introduced into the vast majority of the transformants. Moreover, we showed that simultaneous editingof two genomic loci could also be readily achieved by one step. In summary, our data demonstrate that the haloarchaeal CRISPR-Cas system can be harnessed for genome editingin this polyploidarchaeon, and highlight the convenience and efficiency of the native CRISPR-based genome editingstrategy.