Figure 1.

Guide RNAs to Target Mouse VEGF-A Gene

(A) A schematic diagram of CRISPR target sequences in exon 1 of mouse VEGF-A on chromosome 17. (B) CRISPR target sequences and protospacer adjacent motif (PAM) sequences with on-target and off-target scores indicating predicted Cas9 efficiency and off-target probability, respectively. (C) Schematic diagrams illustrating dual AAV vector system to express SpCas9 and gRNA with EGFP reporter or single AAV vector system to express both SaCas9 and corresponding gRNA. Both HA-tagged Cas9 orthologs and EGFP are driven by CMV promoters, while gRNAs are driven by U6 promoters. (D) T7EI mismatch assays demonstrating frequency of indel formation in mouse VEGF-A gene using NIH 3T3 cells transfected with constructs expressing SpCas9 or SaCas9 with different corresponding gRNAs. % indel frequencies are expressed in mean ± SEM (n = 3).