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

TL;DR

This study uses CRISPR-Cas9 to identify genes and miRNAs involved in human fibroblast migration, combining gene editing with functional assays to discover new candidates.

Contribution

The study introduces a method that integrates CRISPR-Cas9 knockout screening with transwell migration assays to identify non-essential genes and miRNAs involved in directional cell migration.

Findings

The screening confirmed known genes involved in cell migration.

The study identified new candidate genes and miRNAs associated with fibroblast migration.

The method effectively removes essential genes to focus on migration-specific targets.

Abstract

Directional cell migration plays a central role in a wide range of physiological and pathological conditions, such as embryonic development or tumor metastasis. 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. Overall, it is a complex phenomenon at the interface between mechanical forces and biochemical signaling, with cell‐specific and context‐specific molecular events acting in the process. Here, we focus on human fibroblast migration induced by a biochemical gradient with an approach that connects the identification of molecular players with the actual mechanical function. 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. Moreover, the screening has been performed after an expansion step aiming at the removal of all the essential genes and miRNAs, so as to identify targets related to the cell migratory ability without affecting other major cellular functions. The results confirm known genes involved in migration, but also highlight new candidates. This work establishes a methodological advancement in the use of CRISPR technology for functional screening and represents a resource for candidate genes and miRNAs playing a role in human fibroblast directional migration under biochemical gradient.

Figures