CRISPRa-Mediated Increase of OPA1 Expression in Dominant Optic Atrophy

Abstract

Dominant Optic Atrophy (DOA) is the most common inherited optic neuropathy and presents as gradual visual loss caused by the loss of retinal ganglion cells (RGCs). Over 60% of DOA cases are caused by pathogenic variants in the OPA1 gene, which encodes a mitochondrial GTPase essential in mitochondrial fusion. Currently, there are no treatments for DOA. Here, we tested the therapeutic potential of an approach to DOA using CRISPR activation (CRISPRa). Homology directed repair was used to introduce a common OPA1 pathogenic variant (c.2708_2711TTAGdel) into HEK293T cells as an in vitro model of DOA. Heterozygous c.2708_2711TTAGdel cells had reduced levels of OPA1 mRNA transcript, OPA1 protein, and mitochondrial network alterations. The effect of inactivated Cas9 fused to an activator (dCas9-VPR) was tested with a range of guide RNAs (gRNA) targeted to the promotor region of OPA1. gRNA3 and dCas9-VPR increased OPA1 expression at the RNA and protein level towards control levels. Importantly, the correct ratio of OPA1 isoform transcripts was maintained by CRISPRa. CRISPRa-treated cells showed an improvement in mitochondrial networks compared to untreated cells, indicating partial rescue of a disease-associated phenotype. Collectively, these data support the potential application of CRISPRa as a therapeutic intervention in DOA.

Keywords: CRISPR; CRISPR activation; OPA1; alternative splicing; gene editing; gene expression; mitochondria; mitochondrial fusion; optic atrophy; retinal ganglion cell.

Figure 1

Characterisation of c.2708_2711del HEK293 cell line. (A) Sanger sequencing results from the heterozygous and homozygous clones obtained after CRISPR edit, with reference WT sequence above. The bases targeted for deletion are highlighted in purple. Exon 27 is overlined in black. (B) Quantification of OPA1 transcript levels in edited cells compared to WT through qPCR. Bars indicate means ± SD. A total of 50 µg/mL of emetine was added to (+) cell samples for 4 h prior to RNA extraction. GAPDH and Actin were used as reference genes. p values were determined through two-way ANOVA. Values between untreated (blue bars) and emetine-treated samples (red bars) showed no significant difference in two-way ANOVA. (C) Western blots showing OPA1 protein, both l- and s-forms (upper and lower bands, respectively). (D) Quantification of total OPA1 levels (sum of OPA1 l- and s-forms) shows a relative decrease in edited cells. Actin stain used as a loading control. A total of 50 µg/mL of emetine was added to (+) cell samples for 4 h prior to cell lysis. A total of 8 µg of protein was loaded for each sample. * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; **** p ≤ 0.0001.

Figure 2

Mitochondrial network analysis of WT and c.2708_2711TTAGdel cells. (A) Representative images of the maximal intensity of WT and c.2708_2711del HEK293T cells stained with TOMM20. Scale bar represents 20 µm. Analysis was achieved using the Fiji MiNa plugin, which provided measures of (B) mitochondrial footprint; (C) mean branch lengths; (D) summed branch lengths; and (E) network branches. p values were determined through unpaired t test with Welch’s correction. A total of 45 cells were analysed per sample. * p ≤ 0.05; ns = not significant.

Figure 3

OPA1 transcript and protein upregulation mediated by dCas9–VPR. (A) Diagrammatic representation of the position of gRNAs in relation to OPA1’s transcription start site. (B) Relative OPA1 mRNA transcript upregulation measured through qPCR, with ACTIN and GAPDH used as reference genes. (C) OPA1 protein expression detected through Western blot and (D) quantification of the relative OPA1 protein level using actin immunoreactivity as a reference protein. A total of 8 µg of protein was loaded for each sample. n = 3. p values were determined through one-way ANOVA. * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; **** p ≤ 0.0001.

Figure 4

Maintenance of OPA1 isoform ratios in CRISPRa-treated cells. (A) Diagrammatic representation of OPA1 isoforms with alternatively spliced exons marked in red. Two sets of RT-PCR primers were used to separate isoforms containing and excluding exon 5b, with these primers marked by the arrows. The product size for each primer was marked accordingly. (B) RT-PCR isoform products separated on a 2% agarose gel. Black arrows indicate the bands detected (C,D) Each band was quantified as a percentage of the total transcripts for each amplicon. n = 3. Only isoform 5 showed any significant differences between the conditions and is marked in blue. p values were determined through two-way ANOVA. * p ≤ 0.05; ** p ≤ 0.01; **** p ≤ 0.0001.

Figure 5

Mitochondrial network analysis of CRISPRa-treated cells. (A) Representative images of the maximal intensity of c.2708_2711del HEK293T cells treated with different CRISPRa systems and stained with anti-TOMM20. Mitochondrial network analysis of transfected cells identified through intrinsic GFP fluorescence from the dCas9–VPR plasmid and mCherry fluorescence from the gRNA plasmids, achieved through 3D z-stack analysed by the Fiji MiNA plugin. Scale bar represents 10 µm. The analysis produced values for (B) mitochondrial footprint; (C) mean branch lengths; (D) summed branch lengths; and (E) network branches. p values were determined using the Kruskal–Wallis test. A total of 30 cells were analysed per sample from one experiment. * p ≤ 0.05; ** p ≤ 0.01; ns = not significant.