RFP CRISPR mutagenesis as a function of sgRNA delivery. RFP CRISPR mutagenesis as a function of sgRNA delivery. RFP Cas9 strain EPC2 was transformed with each of the plasmids depicted in Fig. 3. Shown are the percentages of white mutant (rfpΔ), red (RFP), and sectored colonies that arose after 2 days at 30°C. Transformations were performed in the presence of a donor homologous repair fragment (A) or in its absence (B). The data represent an average from three separate experiments in which all plasmids (plus or minus the repair fragment) were transformed side by side. Approximately 200 colonies per plate were counted and scored as red, white, or sectored. For each experiment, controls were included in which all plasmids were transformed in the isogenic strain that lacks Cas9 (not shown). (C) Light and red fluorescent images of colony phenotype as a function of different sgRNA delivery plasmids depicted in Fig. 3. Note the near absence of sectored colonies when sgRNA is efficiently delivered (PADH-tRNA-gRFP-HDV) compared to when poorly delivered (PtRNA or PSNR52). Henry Ng, and Neta Dean mSphere 2017; doi:10.1128/mSphere.00385-16