A Simple Combined Use of CRISPR-Cas9 and Cre-LoxP Technologies for Generating Conditional Gene Knockouts in Mammalian Cells.

Gene knockout technologies have contributed fundamentally to our understanding of the cellular functions of various genes. Two prevalent systems used for the efficient elimination of the expression of specific genes are the Cre-LoxP system and the CRISPR-Cas9 system. Here, we present a simple method that combines the use of CRISPR-Cas9 and Cre-LoxP for the conditional deletion of essential genes in mammalian cells. First, an inducible Cre recombinase is stably expressed in the cells. Next, CRISPR-Cas9 is used to knock out an essential gene, whose function is complemented by stable expression of a FLAG-tagged version of the same protein encoded from a floxed transcription unit containing silent mutations, making it refractory to the CRISPR-Cas9 guide. This FLAG-tagged protein can be deleted by activating the expressed Cre protein, enabling evaluation of the cellular consequences of its deletion. We have further used this system to evaluate the ability of phylogenic homologues and of potential mutants to cover functionally for the deleted gene.