In vivo CRISPR screens identify a dual function of MEN1 in regulating tumor-microenvironment interactions [CRISPR screening]

In this study, we used a targeted CRISPR/Cas9 screen to identify genes that determine growth of A549 cells in vivo and in vitro respectively. Overall design: The CRISPR screen employed in our pooled Epidrug library consisted of ~12,500 sgRNAs targeting 317 epigenetic regulators, 657 FDA-approved drug targets based on Drugbank v4.3, and control genes, with 10 sgRNAs per gene on average. The sgRNAs were designed using the CRISPR-DO tool. Stable Cas9-expressing A549 cell line was generated. Cas9-expressing A549 cell line was infected with the library at an MOI of ~ 0.3 and coverage of 300x. 24-48h post-infection, cells were selected with puromycin for 72h and part of cells were sujected to DNA extraction as day 0. Other cells were inoculated in NOD/SCID mice or cultured in vitro, maintaining 300x coverage prior to our screening assay. Genomic DNA was extracted from replicate samples and sgRNA inserts were amplified by PCR. The input amount of genomic DNA was calculated to achieve 250x coverage of the library and resulting libraries were sequenced on an Illumina HiSeq2500.