CRISPR-Cas9 for selective targeting of somatic mutations in pancreatic cancers.

Somatic mutations are desirable targets for selective elimination of cancer, yet most are found within the noncoding regions. We propose a novel, cancer-specific killing approach using CRISPR-Cas9 which exploits the requirement of a protospacer adjacent motif (PAM) for Cas9 activity. Through whole genome sequencing (WGS) of paired tumor minus normal (T-N) samples from three pancreatic cancer patients (Panc480, Panc504, and Panc1002), we identified an average of 417 somatic PAMs per tumor produced from single base substitutions. We analyzed 591 paired T-N samples from The International Cancer Genome Consortium and discovered medians of ~455 somatic PAMs per tumor in pancreatic, ~2800 in lung, and ~3200 in esophageal cancer cohorts. Finally, we demonstrated >80% selective cell death of two targeted pancreatic cancer cell lines in co-cultures using 4-9 sgRNAs, targeting noncoding regions, designed from the somatic PAM discovery approach. We also showed no off-target activity from these tumor-specific sgRNAs through WGS.