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

Laura Bohrer,Edwin Stone,Budd Tucker,Joseph Giacalone,Kristin Anfinson,Robert Mullins,Jessica Cooke,Erin Burnight,Jeaneen Andorf,Luke Wiley

doi:10.3390/genes10040278

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.

Highlights

Enhanced S-cone syndrome (ESCS) is an autosomal recessive retinopathy that results from mutations in the photoreceptor cell transcription factor, NR2E3 (Nuclear Receptor Subfamily 2, GroupE, Member 3)
The goal of this study was to develop a CRISPR-Cas9 homology-directed repair strategy suitable for the correction of disease-causing mutations in induced pluripotent stem cells (iPSCs) generated from two independent patients with molecularly confirmed NR2E3-associated enhanced S-cone syndrome (Figure 1A,B)
S-cone syndrome has a recessive mode of inheritance and the p.(Arg73Ser) mutation on the paternal allele of Patient 2 is within 100 bps of the c.119-2A>C mutations in Patient 1, we hypothesized that a single CRISPR-Cas9 homology-directed repair (HDR) cassette would be sufficient for the correction of iPSCs generated from both individuals

Summary

Introduction

Enhanced S-cone syndrome (ESCS) is an autosomal recessive retinopathy that results from mutations in the photoreceptor cell transcription factor, NR2E3 (Nuclear Receptor Subfamily 2, GroupE, Member 3). Enhanced S-cone syndrome (ESCS) is an autosomal recessive retinopathy that results from mutations in the photoreceptor cell transcription factor, NR2E3 NR2E3, which is expressed in the outer nuclear layer of the human retina [1], is a direct transcriptional target of NRL, a key regulator of photoreceptor cell genesis. Loss of NR2E3 function hinders rod photoreceptor cell development and drives over-expansion of the S-opsin-positive cone photoreceptor cell population (i.e., blue cones) [2,3,4,5,6], which is normally the least prevalent of the photoreceptor cell subtypes. The disease is progressive in nature, clinically evident retinal degeneration is often

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