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Farhad Ghaseminejad, Beatrice M Tam, Orson L Moritz; Developing CRISPR-Based Treatment Strategies for Selective Editing of Mutant Rhodopsin in Xenopus laevis. Invest. Ophthalmol. Vis. Sci. 2021;62(8):1466.
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Given that mutations in the rhodopsin gene (RHO) are associated with autosomal dominant retinitis pigmentosa (adRP), highly specific gene-editing strategies in RHO may provide effective treatments for this blinding disease. The Moritz lab has previously described CRISPR-based genetically modified X. laevis that carry a 12-base pair (bp) deletion in the Rho.L gene, immediately downstream of the start codon. Phenotypically, we observed significantly lower levels of rhodopsin protein as well as substantial retinal degeneration (RD) in these animals. Utilizing this model, we aimed to prevent the detrimental effects of this 12bp deletion by inactivating the dysfunctional gene.
Methods: Our approaches involved removing the dysfunctional Rho.L start codon by inducing simultaneous double-strand breaks on both sides of the start codon, generating a loss-of-function allele. We designed a single-guide RNA (sgRNA) to target the defective Rho.L gene, but not the other X. laevis rhodopsin encoding genes (Rho.2.L and Rho.S) or the wildtype Rho.L allele. A second sgRNA targeted a relatively non-conserved region of the Rho.L promoter which was not specific for the mutant allele. Both sgRNAs were injected alone and in combination into X. laevis embryos. Tadpoles from treated and untreated groups were raised to 14 days. One eye from each sacrificed tadpole was solubilized for evaluating rhodopsin levels using a dot-blot assay. The contralateral eye was sectioned, labelled and imaged for assessing histology. Genetic changes were examined by PCR and Sanger sequencing.
Results: Compared to the untreated animals carrying the 12bp deletion, significantly higher levels of rhodopsin were detected in the CRISPR-edited animals. Histologically, considerable RD in the untreated groups was also prevented in animals receiving the sgRNA edit. Treatment with even the single mutation-specific sgRNA dramatically prevented RD, likely due to frameshift mutations similarly causing loss of function. Treatment of wildtype animals with the sgRNAs did not induce any detrimental effects.
Conclusion: We have demonstrated prevention of RD in animals treated with mutation-specific sgRNAs, compared to untreated animals. Both frameshifts and generation of large inactivating deletions dramatically prevented RD, showing that complex approaches may not be necessary for developing effective treatments.
This is a 2021 ARVO Annual Meeting abstract.
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