Figure 3. Multiplex genome editing with Cas9 nickase and six gRNAs.

(A) Schematic illustration of the all-in-one vector expressing six gRNAs targeting three different genomic loci and Cas9 nuclease. The black box indicates exon 14 of the human APC gene. The numbers from 2118 to 10804 represent the base positions in the APC gene transcript (NCBI reference sequence: NM_001127511.2). (B) The sizes of PCR products and cut images in each locus. (C) Genomic cleavage analysis of the three genomic loci targeted with the all-in-one CRISPR/Cas9-nickase vector. The products from untransfected control cells (C) and cells transfected with the CRISPR/Cas9-nickase vector expressing six gRNAs (6) were analyzed by agarose gel electrophoresis. The arrowheads indicate the approximate positions of the cleaved fragments. % NHEJ was estimated using ImageJ software as previously described³⁰. W, Wide-Range DNA Ladder (100–2,000 bp) (Takara Bio, Shiga, Japan).