The technology includes USP30 knockout HeLa cells that were generated using CRISPR technology. Parkin and Pink1 are key master genes for triggering the degradation of mitochondria (mitophagy). In contrast, USP30 has been found to be localized in the mitochondria and have a role in antagonizing Parkin function, thereby impeding mitophagy.
To create the HeLa cells, one CRISPR gRNAs targeting exon 5 of USP30 genome was used for transfection with Cas9 and a GFP-C1 reporter. Cells were sorted 2 days after transfection and plated out in 96-well plates. 10 days later, single colonies were transferred to 24-well plates and then genomic DNA of each colony was isolated for PCR genotyping. The positive clones were then confirmed by sequencing.
USP30 is constantly degraded in normal conditions, and it has been difficult to raise a good and specific USP30 antibody. The USP30 knockout cell line can be the gold standard to verify if an antibody is functional and specific.