1. CRISPR/Cas targeting of RFP in C. albicans.
CRISPR/Cas targeting of RFP in C. albicans. (A) Yeast colonies that express functional RFP are pink and are fluorescent and can be easily distinguished from colonies that arise through CRISPR-mediated deletion of RFP, which are white and nonfluorescent. Panel B depicts the strategy for quantitating Cas9- and sgRNA-dependent cleavage of RFP. RFP (EPC1) or RFP CaCAS9 (EPC2) hemizygous strains were constructed as described in Materials and Methods. These strains were transformed with or without a donor healing fragment and a series of URA3-marked plasmids that differ in expression of an RFP sgRNA (Fig. 3). The gRNA targets a 20-bp DNA sequence proximal to the RFP PAM site at position 132. The number of white and red colonies in each transformation was counted, and cleavage efficiency was calculated as the percentage of white colonies in the population.
Henry Ng, and Neta Dean mSphere 2017; doi: /mSphere