RESUMO
Somatic mutations are desirable targets for selective elimination of cancer, yet most are found within noncoding regions. We have adapted the CRISPR-Cas9 gene editing tool as a novel, cancer-specific killing strategy by targeting the subset of somatic mutations that create protospacer adjacent motifs (PAMs), which have evolutionally allowed bacterial cells to distinguish between self and non-self DNA for Cas9-induced double strand breaks. Whole genome sequencing (WGS) of paired tumor minus normal (T-N) samples from three pancreatic cancer patients (Panc480, Panc504, and Panc1002) showed an average of 417 somatic PAMs per tumor produced from single base substitutions. Further analyses of 591 paired T-N samples from The International Cancer Genome Consortium found medians of â¼455 somatic PAMs per tumor in pancreatic, â¼2800 in lung, and â¼3200 in esophageal cancer cohorts. Finally, we demonstrated 69-99% selective cell death of three targeted pancreatic cancer cell lines using 4-9 sgRNAs designed using the somatic PAM discovery approach. We also showed no off-target activity from these tumor-specific sgRNAs in either the patient's normal cells or an irrelevant cancer using WGS. This study demonstrates the potential of CRISPR-Cas9 as a novel and selective anti-cancer strategy, and supports the genetic targeting of adult cancers.
RESUMO
When we transduced pancreatic cancers with sgRNAs that targeted 2-16 target sites in the human genome, we found that increasing the number of CRISPR-Cas9 target sites produced greater cytotoxicity, with >99% growth inhibition observed by targeting only 12 sites. However, cell death was delayed by 2-3 weeks after sgRNA transduction, in contrast to the repair of double strand DNA breaks (DSBs) that happened within 3 days after transduction. To explain this discrepancy, we used both cytogenetics and whole genome sequencing to interrogate the genome. We first detected chromatid and chromosome breaks, followed by radial formations, dicentric, ring chromosomes, and other chromosomal aberrations that peaked at 14 days after transduction. Structural variants (SVs) were detected at sites that were directly targeted by CRISPR-Cas9, including SVs generated from two sites that were targeted, but the vast majority of SVs (89.4%) were detected elsewhere in the genome that arose later than those directly targeted. Cells also underwent polyploidization that peaked at day 10 as detected by XY FISH assay, and ultimately died via apoptosis. Overall, we found that the simultaneous DSBs induced by CRISPR-Cas9 in pancreatic cancers caused chromosomal instability and polyploidization that ultimately led to delayed cell death.
RESUMO
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.