Zebrafish as a Model to Evaluate a CRISPR/Cas9-Based Exon Excision Approach as a Future Treatment Option for EYS-Associated Retinitis Pigmentosa

Abstract

Retinitis pigmentosa (RP) is an inherited retinal disease (IRD) with an overall prevalence of 1 in 4000 individuals. Mutations in EYS (Eyes shut homolog) are among the most frequent causes of non-syndromic autosomal recessively inherited RP and act via a loss-of-function mechanism. In light of the recent successes for other IRDs, we investigated the therapeutic potential of exon skipping for EYS-associated RP. CRISPR/Cas9 was employed to generate zebrafish from which the region encompassing the orthologous exons 37-41 of human EYS (eys exons 40-44) was excised from the genome. The excision of these exons was predicted to maintain the open reading frame and to result in the removal of exactly one Laminin G and two EGF domains. Although the eysΔexon40-44 transcript was found at levels comparable to wild-type eys, and no unwanted off-target modifications were identified within the eys coding sequence after single-molecule sequencing, EysΔexon40-44 protein expression could not be detected. Visual motor response experiments revealed that eysΔexon40-44 larvae were visually impaired and histological analysis revealed a progressive degeneration of the retinal outer nuclear layer in these zebrafish. Altogether, the data obtained in our zebrafish model currently provide no indications for the skipping of EYS exons 37-41 as an effective future treatment strategy for EYS-associated RP.

Disruption of the foxe1 gene in zebrafish reveals conserved functions in development of the craniofacial skeleton and the thyroid

“…Using CHOPCHOP ( https://chopchop.cbu.uib.no ) an efficient single guide (sg) RNA was selected at the 5′ end of the target exon. sgRNAs were generated using templates for in vitro transcription as previously described ( Schellens et al, 2021 ). Briefly, a 20 nt (GGC​CGC​AAA​GAG​GCC​GTC​GG) target specific oligonucleotide with a T7 promotor sequence (5′-TAA​TAC​GAC​TCA​CTA​TA-3′) and a complementary region (5′-GTT​TTA​GAG​CTA​GAA​ATA​GCA​AG-3′) to a constant oligonucleotide encoding the reverse complement of the tracrRNA tail were annealed.…”

“…Immunohistochemistry was performed as described earlier ( Schellens et al, 2021 ). Larvae were euthanized and fixated in 4% PFA overnight and embedded in Optimal Cutting Temperature (Agar Scientific) for cryo-sectioning.…”

Genotypic and phenotypic profiles of EYS gene-related retinitis pigmentosa: a retrospective study

“…Unfortunately, a large gene such as EYS spans with a coding size of more than twice of the packing capacity of the AAV vectors, making it difficult to utilise this technique. As such, the options were narrowed down to the genome editing technology, including the use of the clustered regularly interspaced short palindromic repeats (CRISPR) system that had been attempted in the zebrafish model 29 . The induced pluripotent stem cell (iPSC) replacement therapy could also be considered albeit some challenges on cell differentiation and neural connection 30 .…”

Efficient Generation of Knock-In Zebrafish Models for Inherited Disorders Using CRISPR-Cas9 Ribonucleoprotein Complexes

“…Likely, our approach will also be successful to introduce shorter and slightly larger genomic deletions. We recently also published as strategy using 2 sgRNA-Cas9 RNP complexes to introduce larger deletions in the zebrafish genome, such as entire exons [31]. Our approach for the introduction of nucleotide substitutions and small, in-frame deletions is summarized in Figure 4, where we provide our recommended criteria to assess RNP efficiency and knock-in efficiency to maximize the chances of identifying founder fish with minimal screening efforts.…”