High-Content Imaging-Based Pooled CRISPR Screens in Mammalian Cells

We developed a microscopy-based approach, which we name optical enrichment, to select cells displaying a particular CRISPR-induced phenotype by automated imaging-based computation, mark them by photo-activation of an expressed photo-activatable fluorescent protein, and then isolate the fluorescent cells using fluorescence-activated cell sorting (FACS). One proof-of-principle screen, a nuclear size screen, a FSC screen and a H2B-mGFP screen were performed to validate the method. For mIFP proof-of-principle screen, sorted mIFP positive sample, sorted mIFP negative sample and unanalyzed sample identified from microscopy were separately collected by FACS and sequenced with 2 replicates. For nuclear size screen, cells with nuclear size larger than 1,000 μm² were identified from microscopy and collected together with unanalyzed sample by FACS and prepared for sequencing with 2 replicates (4 runs per replicate). For H2B-mGFP screen and FSC screen, the top 10 percentile of cells based on either GFP fluorescence or FSC signal were separately sorted and prepared for high throughput sequencing with 2 replicates.