Fig. 1: Reporter strain for type II-A CRISPR-Cas spacer acquisition.
a Schematic representation of the reporter strain. Spacer acquisition from a prespacer (PS) containing a ribosome binding site (RBS) and a start codon (ATG) results in the installation of a translation start site, leading to downstream reporter expression. Pcr, CRISPR array promoter; Pcas, cas operon promoter; R, repeat. b Fluorescence microscopy of S. aureus cells carrying a reporter plasmid expressing mNG (green fluorescence) with a single CRISPR repeat, a spacer with a translation start site (spc1(ATG)) or a spacer with a defective start site in which the ATG start codon was replaced by a stop codon (spc1(TAG)). Each spacer construct was combined with different tracrRNAs or transformed into staphylococci lacking RNase III (Δrnc). Representative images are shown (n = 2 biological replicates). c Spacer acquisition rate as determined by the ratio of CFUs on erythromycin and chloramphenicol (ErmR/CmR) after electroporation of reporter strains with the indicated prespacers (or no prespacer) in the presence or absence of csn2. Individual data points are shown with error bars representing the mean ± s.d. of four technical replicates.
