Generation of Flag-Rad knock-in mice. (A) Graphical representation of the RRAD gene engineering strategy. Two transgenic mouse models were generated with CRISPR/Cas9 targeting the endogenous RRAD gene in this study. First, a 3xFlag epitope was inserted at the N-terminus of RRAD, generating 3xFlag-Rad mice. Subsequent CRISPR/Cas9 engineering of 3xFlag-Rad embryo introduced a stop codon at amino acid position 277, removing a large portion of the polybasic C-terminus of Rad important for membrane anchoring in cultured cells (Flag-RadΔCT). Created with BioRender.com. (B) Validation of stop codon knock-in showing the location of base changes (upper) and stop codon insertion in place of Ala277 (lower). (C) Western blot for Rad and Flag-Rad expression in protein lysates from wildtype (WT), Flag-Rad, and Flag-RadΔCT whole heart lysates. Note that total Rad protein levels are not significantly changed in either transgenic model. Source data are available for this figure: SourceData F1.