摘要：SummaryOverwriting counterselectable markers is an efficient strategy for removing wild-type DNA or replacing it with payload DNA of interest. Currently, one bottleneck of efficient genome engineering in mammals is the shortage of counterselectable (negative selection) markers that work robustly without affecting organismal developmental potential. Here, we report a conditionalPigaknockout strategy that enables efficient proaerolysin-based counterselection in mouse embryonic stem cells. The conditionalPigaknockout cells show similar proaerolysin resistance as full (non-conditional)Pigadeletion cells, which enables the use of aPIGAtransgene as a counterselectable marker for genome engineering purposes. NativePigafunction is readily restored in conditionalPigaknockout cells to facilitate subsequent mouse development. We also demonstrate the generality of our strategy by engineering a conditional knockout of endogenousHprt. Taken together, our work provides a new tool for advanced mouse genome writing and mouse model establishment.Graphical abstractDisplay OmittedHighlights•Transcriptional inactivation ofPigarenders mESCs full resistance to proaerolysin•PIGA serves as counterselectable marker in endogenousPiga-inactivated mESCs•Pigafunction is readily restored by Cre-mediated STOP cassette excision•Conditional knockout and restoration strategy is also applicable toHprtBiotechnology; Genetic engineering; Bioengineering; Synthetic biology
