CRISPRi inDeinococcus radiodurans

Abstract

AbstractThe extremely radiation resistant bacterium,Deinococcus radiodurans, is a microbe of importance, both, for studying stress tolerance mechanisms and as a chassis for industrial biotechnology. However, the molecular tools available for use in this organism continue to be limiting. In view of this, the CRISPR-Cas tools provide a large repertoire of applications for gene manipulation. We show the utility of the type I-E Cascade system for knocking down gene expression in this organism. A single-vector system was designed for expression of the Cascade components as well as the crRNA. The type I-E Cascade system was better tolerated than the type II-A Cas9 system inD. radiodurans. An assayable acid phosphatase gene,phoNintegrated into the genome of this organism could be knocked down to 10% of its activity using the Cascade system. Cascade-based knockdown ofssb, a gene important for radiation resistance resulted in poor recovery post irradiation. Targeting the Radiation and Desiccation Resistance Motif (RDRM), upstream of thessb, prevented de-repression of its expression upon radiation exposure. In addition to this, multi-locus targeting was demonstrated on the deinococcal genome, by knocking down bothphoNandssbexpression simultaneously. The programmable CRISPRi tool developed in this study will facilitate study of essential genes, hypothetical genes, cis-elements involved in radiation response as well as enable metabolic engineering in this organism. Further the tool is amenable for implementing high-throughput approaches for such studies.