Somatic mutations in malignant cells genetically distinguish malignant cells from normal cells. We attempted to determine the type of somatic mutation in cancers that would create the largest number of novel CRISPR-Cas9 target sites. Whole genome sequencing (WGS) of three pancreatic cancers revealed that single base substitutions, mostly in noncoding regions, produced the highest number of novel NGG protospacer adjacent motifs (PAMs; median=494) compared to structural variants (median=37) and single base substitutions in exons (median=4). Using our optimized PAM discovery pipeline, we detected large numbers of somatic PAMs (median=1127/tumor) in 587 individual tumors from ICGC via WGS analyses of different tumor types. Finally, we showed that these PAMs, which were absent in patient-matched normal cells, could be exploited for cancer-specific targeting, with >75% selective cell killing in mixed cultures of human cancer cell lines using CRISPR-Cas9.Statement of significanceWe developed a highly efficient somatic PAM discovery approach and found that a large number of somatic PAMs exist in individual tumors. These PAMs could serve as novel targets to selectively kill cancer cells.