Engineering the genome with a nested, self-excising selection cassette.

is a powerful model for dissecting biological processes . In particular, the ease of generating targeted knock-in alleles makes it possible to visualize and functionally modify endogenous proteins to gain fundamental insights into biological mechanisms. Methods for genome engineering typically utilize selectable markers, visual screening for fluorescence, or PCR genotyping to identify successfully edited animals. A common genetic tool known as the Self-Excising Cassette (SEC) combines drug and phenotypic selection, which makes it possible to screen large numbers of progeny rapidly and with minimal hands-on effort. However, N-terminal and internal knock-ins using the SEC cause loss of function until the selectable marker cassette is excised, which makes it impossible to isolate homozygous lines for essential genes prior to SEC excision. To simplify generating knock-ins for essential genes, we developed a Nested, Self-Excising selection Cassette (NSEC) that is located entirely within a synthetic intron and does not interfere with the expression of endogenous, N-terminally-tagged NSEC-fusion proteins. This innovation makes it possible to isolate homozygous lines for N-terminally tagged genes prior to selectable marker excision and allows for a standardized workflow to generate N-terminal and internal tags in any background and without the need for genetic balancers. We designed versions of NSEC that include an optional auxin-inducible degron tag and mTurquoise2, GFP, mStayGold, mNeonGreen, or mScarlet-I fluorescent proteins for experimental flexibility. The NSEC expands our molecular toolbox and enhances the scalability, efficiency, and versatility of genome engineering.