Fig. 1.

Identification of self-targeting Staphylococcus strains that contain active type II CRISPR-Cas systems. (A) Acquisition of protospacers from MGEs can result in self-targeting if the MGE is capable of stably integrating (e.g., prophages) or associating (e.g., plasmid) with the genome. (B) Overview of TXTL assay to test for Acr activity. Cas9 template DNA is combined with an sfGFP reporter plasmid and a plasmid expressing a sgRNA targeting the reporter. In the absence of Acr activity, expression of the Cas9 RNP suppresses sfGFP expression. In the presence of Acrs, the Cas9 RNP is inhibited, resulting in increased fluorescence. (C) Genomic amplicons from S. schleiferi and S. haemolyticus containing Cas9 lower sfGFP expression with an sfGFP-targeting sgRNA, demonstrating that the natural CRISPR loci are active.