Abstract

Various mechanisms of mitochondrial quality control maintain mitochondrial homeostasis, in order to regulate normal organelle and cellular functionality. In parallel to mitochondrial biogenesis and fission-fusion dynamics, mitochondria can be selectively degraded by the lysosome through a process known as mitophagy. PINK1/Parkin mediated mitophagy is the most well characterised mitophagy pathway in Parkinson’s disease (PD) pathogenesis, with monogenic mutations in PINK1 and PRKN being responsible for a form of autosomal recessive early-onset familial PD. However, knowledge of PINK1-independent mitophagy pathways has begun to emerge. To address the questions regarding the regulation of these various mitophagy pathways and their interplay with mitochondrial functionality, we conducted a multi-platform CRISPRi-based screen in SH-SY5Y.

CRISPR interference (CRISPRi) utilises a catalytically inactive Cas9 protein conjugated to a KRAB repressor domain, therefore when directed to the target genetic site according to the sgRNA guide it causes a steric block in transcription, leading to selective gene repression. The fluorescent probe mtKeima is monomeric protein targeted to the inner mitochondrial membrane which exhibits a pH sensitive stokes shift in excitation spectra. Therefore, the excitation shift of this fluorescent reporter can be used as a direct readout of the engulfment of mitochondria by the lysosome during mitophagy.

An SH-SY5Y line was engineered to constitutively express both CRISPRi machinery and also the doxycycline inducible expression of the mitophagy reporter mtKeima (CRISPRi-mtKeima SH-SY5Y). We assessed mitochondrial morphology and mitophagy in 16 independent CRISPRi-mtKeima stable KD lines generated for this screening panel, using sgRNA guides for 15 mitophagy-related genes and a non-targeting sgRNA control.