Figure 1.

Schematic outline of the screen. dCas9-expressing B16-F10 mouse melanoma cells were transduced with a library of lentiviral gRNAs targeting the TSS of 2195 “membrane protein” genes (five gRNAs/gene) and 250 nontargeting (‘NT’) controls. These cells (5.5 × 10⁵, which represents a 50-fold coverage of the library) were then subcutaneously injected into the flank of immunodeficient mice. The resulting masses were regularly measured and when they reached ∼2.5 cm² (sufficient time for metastasis and pulmonary colonization to have occurred; 14–21 days after administration of the tumor cells), the mice were humanely sacrificed. Their lungs were then collected for genomic DNA (gDNA) extraction and barcoded PCR was used to amplify the gRNAs present in the gDNA. The pooled PCRs were then sequenced using next-generation sequencing (NGS) and bioinformatic analysis was performed to identify the gRNAs that were enriched in each lung/mouse.