Aug 26, 2023
CRISPR/Cas9 genome editing
- 1Department of Molecular and Cell Biology, Howard Hughes Medical Institute, University of California, Berkeley, Berkeley, United States
Protocol Citation: wusj, schekman 2023. CRISPR/Cas9 genome editing. protocols.io https://dx.doi.org/10.17504/protocols.io.rm7vzxb38gx1/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: August 03, 2023
Last Modified: August 26, 2023
Protocol Integer ID: 85898
Funders Acknowledgement:
Randy Schekman
Grant ID: Howard Hughes Medical Institute
Abstract
This protocol describes the generation of DNAJC5 KO using CRISPR/Cas9 edition
CRISPR/Cas9 genome editing
CRISPR/Cas9 genome editing
gRNA targeting exon 4 of DNAJC5 ( CACC GGAG GCCG CAGA AGAC AAAC A) was inserted into a
pX330-based plasmid expressing Venus fluorescent protein
HEK293T cells were transfected with pX330-pX330-Venus-DNAJC5-Exon4-gRNA by Lipofectamine 2000 followed the commercial protocol by Thermo Fisher.
After 48 hr, we diluted the cells and single colonies were isolated from 96-well plate, expanded, and determined for DNAJC5 KO by immunoblot.