Fig 1.
Protospacer Adjacent Mutated Motif-mediated targeting.
(a) The gRNA-Cas9 complex targets DNA through complementarity with the gRNA sequence. Red color is the DNA binding domain. Green color is the PAM. (b) Example of the PAM constraint function for a random target sequence. (c) Schematic illustration of the fluorescence reporter plasmid. The variable regions, which correspond to either KRAS wild-type or p.G13A mutant alleles, were shown in the box and cloned after the mKate start codon. (d) Flow cytometry and fluorescence microcopy results for the normalized mKate expression. Both assays demonstrated that CRISPR specifically silenced the mKate carrying the KRAS p.G13A mutant sequence.
Fig 2.
CRISPR/Cas9-mediated generation of a heterozygous KRAS G13A/+ stable SW48 cell line.
Genomic DNA or mRNA were prepared from the KRAS wild type (WT) or G13A/+ SW48 (HT) cells and were used as PCR templates to isolate the cDNA fragments harboring the 13th codon of the KRAS gene. (a) The Sanger sequencing results of the genomic DNA or mRNA samples from SW48 WT cells. (b) The Sanger sequencing results of the genomic DNA or mRNA samples from SW48 HT cells.
Fig 3.
Protospacer Adjacent Mutated Motif-mediated targeting of the KRAS p.G13A mutation in colorectal cancer cells.
Genomic DNA or mRNA were prepared from the KRAS WT-Cas9 or HT-Cas9 cells and were used as PCR templates to isolate the cDNA fragments harboring the 13th codon of the KRAS gene. (a) The Sanger sequencing results of the genomic DNA or mRNA samples from SW48 WT-Cas9 cells. (b) The Sanger sequencing results of the genomic DNA or mRNA samples from SW48 HT-Cas9 cells. (c) The drug response curves of the KRAS HT, WT-Cas9 and HT-Cas9 SW48 cells to the MEK inhibitor AZD6244. The WT-Cas9 and HT-Cas9 cell lines show the same AZD6244 sensitivity.