CRISPR screening in head and neck cancer with mTOR inhibitor

To elucidate the mechanism by which head and neck squamous cell carcinoma (HNSCC) acquires resistance to mTOR inhibitors and to select effective synergistic drugs, a wide-genome CRISPR screen was performed using HNSCC cell lines.LentiCas9 plasmid (#52962, Addgene) was a gift from Feng Zhang and was used to generate Cal27-Cas9 expressing cells. Cal27-Cas9 cells were infected with Human genome-wide CRISPR pooled library (Brunello) that was a gift from John Doench & David Root (#73178-LV, Addgene). And cells were amplified and seeded into 500cm2 square cell culture plate (2,500,000 cells/plate), and were divided into two different groups; 3 replicate plates for either vehicle/DMSO or INK128 (10nM). The treatment was continued until the population doubling reached 20. The genomic DNA was extracted. The entirety of isolated genomic DNA was used for subsequent PCR to ensure capturing the full representation of the libraries. PCR products were purified and sequenced on a NovaSeq6000.