Figure 1.

CRISPR/Cas9 engineering of isogenic A375 models of drug-resistant melanoma. (A) Schematic diagram of the CRISPR/Cas9 editing strategy used for the introduction of point mutations associated with BRAF and MEK inhibitor resistance into A375 melanoma cells. Two guide RNAs in complex with Cas9 (sg1 and sg2, scissors) were used to create double-stranded breaks in the intronic regions (black lines) to either side of the target exon (black box). A donor plasmid containing a copy of the target exon with the desired point mutation (star) and flanking intronic sequences was used as a repair template. This strategy ensures that any indels resulting from imperfect sequence repair at the Cas9 cut sites (red lines) are spliced out during mRNA processing and do not affect the resulting cellular protein; (B) Sanger sequencing of genomic DNA from the resulting KRAS G13D heterozygous (left), NRAS Q61K heterozygous (middle), and MEK1 Q56P homozygous (right) A375 isogenic lines.