Functional CRISPR-Cas9 knockout screening of the genetic determinants of human fibroblast migration propensity

Directional cell migration plays a central role in a wide range of physiological and pathological conditions, such as inflammation and cancer. Steps involved in cell migration include cell polarization, formation of membrane protrusions at the cell front side and adhesion disassembly at the rear side, and a general cytoskeletal rearrangement. However, there are cell-specific and context-specific molecular events acting in the process. Here, we show how to screen for genes and miRNAs involved in migration by the direct integration of a high-throughput gene editing method, the CRISPR-Cas9 knockout pool screening, and a well-established functional assay, the transwell migration assay. We applied this methodology to human fibroblasts migrating in the presence of a biochemical gradient. The results confirm known genes involved in migration, but also highlight new candidates, like the ectopically expressed family of olfactory receptors. This work establishes a methodological advancement in the use of CRISPR technology for functional screening, and represents a resource for candidate genes and miRNA playing a role in human fibroblast directional migration. Overall design: We sequenced the plasmid library amplified from Human CRISPR Knockout Pooled Library (GeCKO v2 library). Then, human fibroblasts were transduced with this library, cells were expanded for 14 days under puromycin selection conditions, before starting the migration selective pressure in tranwells. Three rounds of selection were performed collecting separately the cells attached to the top and the bottom of the transwell membrane. Control culture was performed in parallel in conventional flasks.