RRC ID 74622
Author Mehta S, Buyanbat A, Orkin S, Nabet B.
Title High-efficiency knock-in of degradable tags (dTAG) at endogenous loci in cell lines.
Journal Methods Enzymol
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.
Volume 681
Pages 1-22
Published 2023-1-1
DOI 10.1016/bs.mie.2022.08.045
PII S0076-6879(22)00370-6
PMID 36764753
MeSH Animals CRISPR-Cas Systems* DNA DNA, Single-Stranded Gene Editing* Humans Mice Recombinational DNA Repair
Human and Animal Cells HUDEP-2(RCB4557)