Generation of FLIP and FLIP-FlpE Targeting Vectors for Biallelic Conditional and Reversible Gene Knockouts in Mouse and Human Cells

Abstract

Rapid generation of conditional knockout models in a diploid system is challenging. Recently, CRISPR-FLIP strategy has been introduced, which facilitates the generation of biallelic conditional or reversible gene knockouts in various mammalian cell lines including mouse and human pluripotent stem cells by codelivery of the CRISPR/Cas9 system and a universal intronic cassette—FLIP and FLIP-FlpE. Here, I describe the design and cloning method of FLIP and FLIP-FlpE targeting vectors for conditional and reversible gene knockouts. This method is applicable to mouse embryonic stem cells, human induced pluripotent stem cells, and adult stem cell-derived organoids.