Fig. 4

a DASH is used to selectively deplete one allele while keeping the other intact. An sgRNA in conjunction with Cas9 targets a wild-type (WT) KRAS sequence. However, since the G12D (c.35G>A) mutation disrupts the PAM site, Cas9 does not efficiently cleave the mutant KRAS sequence. Subsequent amplification of all alleles using flanking primers, as in the case of digital PCR, Sanger sequencing, or high-throughput sequencing, is only effective for non-cleaved and mutant sites. b Three human genomic DNA samples with varying ratios of wild-type to mutant (G12D) KRAS were treated either with KRAS-targeted DASH, a non-human control DASH, or no DASH. Counts of intact wild-type and G12D sequences were then measured by droplet digital PCR (ddPCR). c Same data as in (b), presented as percentage of mutant sequences detected. Inset shows fold enrichment of the percentage of mutant sequences with KRAS-targeted DASH versus no DASH. For both (b) and (c), values and error bars are the average and standard deviation, respectively, of three independent experiments