One-step generation of conditional and reversible gene knockouts

Abstract

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.

Figure 1. FLIP cassette strategy for bi-allelic conditional gene modification

(a) Schematic drawing of the FLIP cassette strategy for bi-allelic conditional gene modification. (b) The design of the FLIP cassette. SD – splice donor, SA1, SA2 – splice acceptor, Purple triangles - LoxP1 sites Pink triangles – Lox5171 sites BP1, BP2 (blue circles) – branching point, pA - polyadenylation signal. (c) Schematic of the FLIP cassette containing a DsRed reporter gene (d) Images of HEK 293 cells transfected with the FLIP cassette. Both eGFP and DsRed proteins are expressed (top row). After Cre recombination the eGFP expression is disrupted, and only DsRed expression is maintained (bottom row). Scale bar 400 µm.

Figure 2. Insertion of the FLIP cassette in the endogenous Ctnnb1 gene of mouse embryonic stem cells.

(a) Immunofluorescence of β-catenin before and after Cre transfection. (b) Representative bright field images of the ESC clones before (top) and after (bottom) Cre transfection. Scale bar 400μm.