CRISPR screen identifies genes that sensitize AML cells to double negative T cell therapy [guide RNA]

Here, we used a targeted CRISPR/Cas9 screen to identify genes that confer susceptibility of AML cells to DNT cell therapy. 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 cell lines (AML2 and AML3) were generated. Cas9-expressing AML cell lines were 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 then cultured for ~7 days, maintaining 300x coverage prior to our screening assay. To detect early apoptotic AML cells, we co-incubated the transduced AML3 cells with DNTs at a 1:1 E:T ratio for 2h to induce apoptosis in ~40-50% of the targets, depleted DNTs by magnetic separation, and performed FACS to separate Annexin V+ (live) and Annexin V- (dead or dying) target cells. 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.