High-efficiency knock-in of degradable tags (dTAG) at endogenous loci in cell lines

The dTAG system is a versatile strategy for tunable control of protein abundance and facilitates the time-resolved assessment of disease-associated protein function. A "co-opted" fusion-based degron peptide, the "dTAG" facilitates the study of endogenous protein function when knocked-in at the endogenous genetic loci of proteins of interest. We combine CRISPR/Cas9 mediated induction of double-strand breaks (DSB) with the delivery of a single-stranded DNA HDR-donor-template via crude preparations of recombinant adeno-associated virus (rAAV). Our approach to knock-in of large (1–2 kb) DNA fragments via crude-rAAV mediated HDR donor delivery is rapid and inexpensive. It facilitates genetic modification of a variety of human as well as mouse cell lines at high efficiency and precision.