Simple and reliable in situ CRISPR-Cas9 nuclease visualization tool is ensuring efficient editing in Streptomyces species

Abstract

CRISPR-Cas9 technology has emerged as a promising tool for genetic engineering of Streptomyces strains. However, in practice, numerous technical hurdles have yet to be overcome when developing robust editing procedures. Here, we developed an extension of the CRISPR-Cas toolbox, a simple and reliable cas9 monitoring tool with transcriptional fusion of cas9 nuclease to a beta glucuronidase (gusA) visual reporter gene. The Cas9-SD-GusA tool enables in situ identification of cells expressing Cas9 nuclease following the introduction of the plasmid carrying the CRISPR-Cas9 machinery. Remarkably, when the Cas9-SD-GusA system was applied under optimal conditions, 100% of the colonies displaying GusA activity carried the target genotype. In contrast, it was shown that the cas9 sequence had undergone major recombination events in the colonies that did not exhibit GusA activity, giving rise to "escaper colonies" carrying unedited genotype. Our approach allows a simple detection of "escaper" phenotype and serves as an efficient CRISPR-Cas9 optimisation tool.