Loss-of-function studies are key for investigating gene function， and CRISPR technology has made genome editing widely accessible in model organisms and cells. However， conditional gene inactivation in diploid cells is still difficult to achieve. Here， we present CRISPR-FLIP， a strategy that provides an efficient， rapid and scalable method for biallelic conditional gene knockouts in diploid or aneuploid cells， such as pluripotent stem cells， 3D organoids and cell lines， by co-delivery of CRISPR-Cas9 and a universal conditional intronic cassette.