Efficient targeting of Bmal1 and Per2 genes in U2OS cells by the all-in-one CRISPR-Cas9 vector. ( A ) Four exons were targeted in each gene based on their score in a predictive software tool. The PAM sequences are indicated in red. Note that target sites for Bmal1 exon 6, 7 and 9 are close to splicing sites indicated by dashes within the sequence. ( B ) mCherry-expressing cells (Pos) were selected by FACS and subjected to T7E1 assay to assess the efficiency of indels. Control cells (Neg) were from the same FACS sorting. Cells with low mCherry signal (middle) were discarded. All of the eight samples showed similarly efficient indels, proportional to transfection efficiency. Two representative samples are shown. ( C ) Targeting of the clock genes by the all-in-one vectors produces diverse indels including frame-shifting mutations. PCR amplicons from the genomic targets of Bmal1 exon 7 and Per2 exon 15 were sequenced (see Supplementary Fig. 1 for Per2 exon 15). 15–30% of clones were wt. Deletions and insertions are indicated by lines and green characters, respectively.