Figure 1. Improved editing of the Ai9 locus in vitro after optimization of gRNAs and modification of the gRNA scaffold. A. The schematic shows the Ai9 lox-stop-lox-tdTomato cassette and the positions of tested gRNA target sequences. B. gRNA sequences, PAMs and their predicted efficiency and on- and off-target editing ranks as predicted by the Broad Genetic Perturbation Platform https://portals.broadinstitute.org/gpp/public/analysis-tools/sgrna-design. Sequences that deviate from the NNGRRT consensus SaCas9 PAM are highlighted in red. C. A schematic of the Cas9-gRNA construct design. Modifications to the gRNA scaffold are highlighted in red. The position of the spacer is shown in blue. D. In vitro editing efficiency by SaCas9 with single or paired gRNA combinations. Ai9 fibroblasts were transfected with a single or pair of CMV-SaCas9 vectors with a U6-driven gRNA as indicated. The cells were then assessed for tdTomato expression as a marker of editing by flow cytometry. Plasmids harboring modifications to the gRNA scaffold enhance the editing of the Ai9 loxstop-lox cassette. E. Editing efficiency normalized to the recombination efficiency achieved by EF1a-Cre expression, which served as a positive control.