CRISPR/Cas9 genome editing
schekman, wusj
Published: 2023-08-26 DOI: 10.17504/protocols.io.rm7vzxb38gx1/v1
Abstract
This protocol describes the generation of DNAJC5 KO using CRISPR/Cas9 edition
Steps
CRISPR/Cas9 genome editing
1.
gRNA targeting exon 4 of DNAJC5 ( CACC GGAG GCCG CAGA AGAC AAAC A) was inserted into a
pX330-based plasmid expressing Venus fluorescent protein
2.
HEK293T cells were transfected with pX330-pX330-Venus-DNAJC5-Exon4-gRNA by Lipofectamine 2000 followed the commercial protocol by Thermo Fisher.
3.
After 48 hr, we diluted the cells and single colonies were isolated from 96-well plate, expanded, and determined for DNAJC5 KO by immunoblot.
