Dumais, B.; Bossaert, M.; Seigneur, P.; Rozie, A.; Gasmi, S.; Caroff, M.; Maraval, V.; Bernardes-Genisson, V.; Gomez, D.; Frit, P.; Ballereau, S.; Genisson, Y.; Britton, S.

Several approaches are available to increase the efficiency of CRISPR/Cas9-based genome editing, including the co-inactivation of a gene that mediates the cytotoxic activity of a compound which can be used to enrich the population in edited cells. Here we show in multiple cell lines how inactivating HSD17B11, a non-essential Short-chain Dehydrogenase/Reductase, confers a strong resistance (29- to 130-fold resistance) in both human and mouse cells to a Phenyl diAlkynylCarbinol compound (PAC) without impacting cell viability and proliferation. We show how co-inactivating HSD71B11 along with selection with PAC is usable to quickly identify efficient guide(s) against a gene of interest and to readily isolate fully inactivated clones. Altogether, this work provides an experimental framework for the facile generation of knockouts using PAC for selecting successfully inactivated cells.