Figure 3. Multi-gene disruption using the mono-promoter-driven CRISPR/Cas9 system.

(A) Schematics of the gRNA and Cas9 expression vectors tested in (B,C). All gRNAs targeted HPRT1 and/or FAN1. (B) Restriction fragment-length polymorphism analysis. HEK 293 cells (1 × 10⁵) were transfected with one of the vectors (1 μg) shown in (A), and the genomic DNAs of the cells were extracted 72 h after transfection. The DNA was subjected to PCR, and 300 ng of the PCR products were subjected to agarose gel electrophoresis after incubation with or without XcmI for HPRT1 and BamHI for FAN1. Cleaved and uncleaved fragments indicate unmodified and modified genomes, respectively. (C) The indel rates of the human HPRT1 and FAN1 loci in HEK 293 cells were calculated from RFLP analysis, as shown in (B). The band intensity was analyzed by ImageJ software. All values represent means ± SEM of three separate experiments. Different letters indicate significant differences (P < 0.05), as determined by ANOVA followed by Tukey’s multiple comparison test (n = 3).