Correction of NR2E3 Associated Enhanced S-cone Syndrome Patient-specific iPSCs using CRISPR-Cas9

Abstract

Enhanced S-cone syndrome (ESCS) is caused by recessive mutations in the photoreceptor cell transcription factor NR2E3. Loss of NR2E3 is characterized by repression of rod photoreceptor cell gene expression, over-expansion of the S-cone photoreceptor cell population, and varying degrees of M- and L-cone photoreceptor cell development. In this study, we developed a CRISPR-based homology-directed repair strategy and corrected two different disease-causing NR2E3 mutations in patient-derived induced pluripotent stem cells (iPSCs) generated from two affected individuals. In addition, one patient’s iPSCs were differentiated into retinal cells and NR2E3 transcription was evaluated in CRISPR corrected and uncorrected clones. The patient’s c.119-2A>C mutation caused the inclusion of a portion of intron 1, the creation of a frame shift, and generation of a premature stop codon. In summary, we used a single set of CRISPR reagents to correct different mutations in iPSCs generated from two individuals with ESCS. In doing so we demonstrate the advantage of using retinal cells derived from affected patients over artificial in vitro model systems when attempting to demonstrate pathophysiologic mechanisms of specific mutations.

Novel Pathogenic Sequence Variants in NR2E3 and Clinical Findings in Three Patients

“…Most previous studies have suggested that the c.119-2A > C variant causes the skipping of exon 2 and a premature termination codon in exon 3, leading to nonsense-mediated decay of the aberrant protein [44,45]. However, in a study by Bohrer et al using patient induced Pluripotent Stem Cell (iPSCs), the authors suggest that a cryptic acceptor site in intron 1 is activated by the variant and a region of intron one is incorporated into the transcript as well as exon two, which would also result in a termination codon in exon 3 [46]. It has been suggested by Bandah et al that, due to the mutation occurring in the second intronic base of the donor splice site, a variable amount of wild type protein can still be generated in patients with the c.119-2A > C variant [44].…”

Patient derived stem cells for discovery and validation of novel pathogenic variants in inherited retinal disease

“…9B). A hallmark example is NR2E3, which encodes a retinal nuclear receptor transcription factor that suppresses the development of cone photoreceptors (Bohrer et al, 2019;Cheng et al, 2004). In a recent scRNA-seq investigation of retinal organoid differentiation, NR2E3 was only expressed in progenitor photoreceptor populations of day 90-110 organoids and remained absent from both day 60 and day 125 organoids (Sridhar et al, 2020).…”

Retinal Organoid Technology: Where Are We Now?

“…Bohrer et al developed a CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-based homology-directed repair strategy and corrected the NR2E3 premature mutation (c.119-2-A>C) in patient-derived iPSCs. The organoids derived from the gene-corrected iPSCs regained the ability to express wild-type NR2E3 [56]. NRL (Neural Retina Leucine Zipper) mutations also cause enhanced S-cone syndrome and retinitis pigmentosa, as it is critical for rod fate determination and maintenance [57,58].…”