Determination of Cas9/dCas9 associated toxicity in microbes

Abstract

The CRISPR-Cas9 system has been used extensively in eukaryotic and prokaryotic systems for various applications. In case of the latter, a couple of previous studies had shown Cas9 protein expression associated toxicity. We studied the same in five microbes, viz Escherichia coli, Salmonella typhimurium, Mycobacterium smegmatis, Xanthomonas campestris and Deinococcus radiodurans. Transformation efficiency of plasmids carrying genes coding for Cas9 or dCas9 was used to gauge toxicity associated with Cas9 protein expression. Results showed differential levels of Cas9 toxicity among the bacteria and lower transformation efficiency for cas9/dcas9 bearing plasmids compared to controls in general. This indicated lethal effect of Cas9/dCas9 expression. While E. coli and S. typhimurium seemed to tolerate Cas9/dCas9 fairly well, in GC rich microbes, M. smegmatis, X. campestris and D. radiodurans, Cas9/dCas9 associated toxicity was acute.