We used CRISPR to inactivate mutant KRAS in CFPAC1 (KRASG12V) pancreatic cancer cell lines. Gene expression analysis of KRAS intact vs. knockout cells identified sets of genes involved in protein synthesis, cell differentiation, and metabolic processes, while the expression of MAPK/ERK target genes remained unperturbed.

For CRISPR/Cas9-mediated knockouts, we used sgRNAs specific for KRAS. Knockout efficiency was measured by Western blotting. Multiple technical replicates were obtained for each CRISPR targeted gene. Genomic DNA from clonal cell lines was collected using QiaAmp DNA Mini Kit (Qiagen). PCR products for sequencing were amplified using Taq DNA polymerase (Invitrogen) and cloned into a pBluescript vector. At least 10-15 bacterial colonies were sequenced per cell line. For RNA isolation, cells were harvested with TRIzol reagent (Invitrogen). Cells were maintained in DME medium supplemented with 5% fetal bovine serum (FBS). RNA sequencing of tumor cells with basic bioinformatics and statistical analyses were performed by Novogene Corp. The FPKM counts represent fragments per kilobase of transcript per million mapped reads.