Allele-Specific Knockout by CRISPR/Cas to Treat Autosomal Dominant Retinitis Pigmentosa Caused by the G56R Mutation in NR2E3

Abstract

Retinitis pigmentosa (RP) is an inherited retinal dystrophy that causes progressive vision loss. The G56R mutation in NR2E3 is the second most common mutation causing autosomal dominant (ad) RP, a transcription factor that is essential for photoreceptor development and maintenance. The G56R variant is exclusively responsible for all cases of NR2E3-associated adRP. Currently, there is no treatment for NR2E3-related or, other, adRP, but genome editing holds promise. A pertinent approach would be to specifically knockout the dominant mutant allele, so that the wild type allele can perform unhindered. In this study, we developed a CRISPR/Cas strategy to specifically knockout the mutant G56R allele of NR2E3 and performed a proof-of-concept study in induced pluripotent stem cells (iPSCs) of an adRP patient. We demonstrate allele-specific knockout of the mutant G56R allele in the absence of off-target events. Furthermore, we validated this knockout strategy in an exogenous overexpression system. Accordingly, the mutant G56R-CRISPR protein was truncated and mis-localized to the cytosol in contrast to the (peri)nuclear localizations of wild type or G56R NR2E3 proteins. Finally, we show, for the first time, that G56R iPSCs, as well as G56R-CRISPR iPSCs, can differentiate into NR2E3-expressing retinal organoids. Overall, we demonstrate that G56R allele-specific knockout by CRISPR/Cas could be a clinically relevant approach to treat NR2E3-associated adRP.

Closely related type II-C Cas9 orthologs recognize diverse PAMs

“…Autosomal dominant diseases are mainly caused by single-nucleotide missense mutations (23). If missense mutations form novel PAMs, CRISPR/Cas9 nucleases can disrupt mutant alleles by a PAM-specific approach (24, 25). For clinical applications, it is crucial to develop a CRISPR toolbox capable of recognizing multiple PAMs.…”

“…Our study enhanced ability to perform allele-specific genome editing. Allele-specific gene disruption through non-homologous end joining (NHEJ) is a potential strategy to treat autosomal dominant diseases (24, 34), where the causative gene is haplosufficient. Autosomal dominant diseases are mainly caused by single-nucleotide missense mutations (23).…”

Human organoids in basic research and clinical applications

“… 190 Retinal genetic diseases have also been modeled with retinal organoids. 191 , 192 Similarly, NKX2-5- and HAND1-knockout (KO) cardiac organoids have been applied to model hypoplastic left heart syndrome, the most severe congenital defect in humans. 169 In particular, brain organoids have been widely used for research on various neurodevelopmental genetic disorders.…”

Retinal Organoid Technology: Where Are We Now?

“…For instance, the G56R mutation in NR2E3 is one of the most common mutations causing autosomal dominant RP. Diakatou et al generated by CRISPR/Cas9 editing allele-specific knockout of the mutant G56R allele in patient iPSCs and found that this knockout did not affect the differentiation potential of retinal organoids or NR2E3 expression [65].…”