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Updated: Aug 10, 2025

One-step CRISPR-based Strategy for Endogenous Gene Tagging in Drosophila melanogaster
Published on: January 26, 2024
High-efficiency knock-in of degradable tags (dTAG) at endogenous loci in cell lines
Stuti Mehta1, Altantsetseg Buyanbat1, Stuart Orkin1
1Dana-Farber/Boston Children's Cancer and Blood Disorders Center, Boston, MA, United States.
Abstract:
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 (1-2kb) 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.
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