CRISPR/Cas9 knock-in of PINK1-GFP at the endogenous PINK1 locus in U-2 OS cells v1

A CRISPR/Cas9-based strategy is presented for generating U-2 OS cells with a PINK1-GFP knock-in at the N-terminus of the endogenous PINK1 locus (replacing the endogenous PINK1 gene with PINK1-GFP cDNA). The method uses Cas9 nickase and paired sgRNAs to introduce a double-stranded break in exon 1 of PINK1, enabling homology-directed repair using a donor plasmid encoding PINK1-GFP cDNA flanked by approximately 500 bp homology arms as the template. Following plasmid transfection, cells undergo a 10 day recovery period to allow depletion of transiently expressed PINK1-GFP. Mitochondrial depolarisation is then induced using antimycin and oligomycin to increase PINK1-GFP expression levels and therefore enhance GFP signal detection. GFP positive cells were isolated by fluorescence-activated cell sorting into 96-well plates using a low threshold gate to capture clones with physiologically relevant expression and were subsequently expanded and validated by immunoblotting.