| 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
|
| Resource |
| Human and Animal Cells |
HUDEP-2(RCB4557) |
