Research Article
Aurélie Van Tongelen
Abstract
Studying the cellular function of microRNAs requires genetic strategies to generate their loss-of-function. Recently, a novel approach of targeted genomic deletion was proposed, which is based on induction of site-specific DNA cuts with Cas9/gRNA ribonucleoprotein complexes, combined with homologous recombination-dependent insertion of cassettes that contain sequences for Cre-lox or FLP-FRT systems. Here, we provide a technical report describing application of this CRISPR/Cas9-directed homologous recombination procedure to the generation of human tumor cells in which conditional knockout of an X-linked cluster of microRNAs (miR-105/miR-767) can be induced. We describe the successive steps of genetic engineering and cell clone selection that allowed us to generate cells with the expected genome editing.