Abstract
Purpose :
Autosomal dominant optic atrophy (DOA) is the most common inherited optic neuropathy. The majority of over 250 OPA1 mutations identified in DOA patients cause reduced OPA1 protein expression. To model DOA, we developed an inducible CRISPR inference (CRISPRi) knockdown system in human pluripotent stem cells (hPSCs) that reduces OPA1 expression. We then characterized the effects reduced OPA1 expression has on DOA-associated mitochondria-related phenotypes and tested the genetic interaction between OPA1 knockdown and knockdown of DRP1, a gene which encodes a mitochondrial fission protein.
Methods :
An inducible CRISPRi system was generated by integration of doxycycline-inducible catalytically-dead Cas9 fused with the transcriptional inhibitory KRAB domain (dCAS9-KRAB) into the AAVS1 safe-harbor site of an hPSC line. Clones were selected and infected with lentivirus containing constitutively expressed sgRNAs complementary to regions proximal to the transcription start site of OPA1 and/or DRP1 genes to target transcriptional inhibition to the endogenous genes.
Results :
Addition of doxycycline to the inducible CRISPRi hPSCs expressing OPA1-targeting gRNA caused rapid induction of dCas9-KRAB expression and concurrent inhibition of OPA1 mRNA and protein expression. Examination of mitochondria morphology by confocal laser- scanning and transmission-electron microscopy revealed fragmented mitochondria, consistent with the role of OPA1 in mitochondrial fusion. Inhibition of OPA1 also led to reduced oxidative phosphorylation (OXPHOS), as measured by the Seahorse mitochondrial assay, and resulted in lower cell viability. Interestingly, CRISPRi of DRP1, the gene encoding a mitochondrial fission regulator, suppressed the phenotypes of OPA1 knockdown; compared to the OPA1 knockdown line, the OPA1/DRP1 knockdown dramatically reduced mitochondrial fragmentation and restored OXPHOS activity and cell viability back to wild-type levels.
Conclusions :
These data suggest that together OPA1 and DRP1 play critical roles in mitochondrial homeostasis in hPSCs. We are currently differentiating these CRISPRi hPSC lines into retinal ganglion cells (RGCs) to probe the role of OPA1 in RGCs as well as test whether there is a similar OPA1/DRP1 genetic interaction that modulates mitochondrial function in RGCs.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.