| Summary: | Background: Dominant mutations account for a substantial proportion of inherited retinal degenerations (IRDs) through haploinsufficiency or gain-of-function (GOF) effects. This work explores a potential strategy to treat diseases caused by dominant GOF mutations by specifically suppressing the function of the mutant allele at the genetic level using the CRISPR/Cas9 system. After taking into account the likelihood of allele pathogenicity, we identified six targets suitable for this form of genome editing therapy, including mutations in the RHO, BEST1, IMPDH1, SNRNP200, PRPH2, and PRPF8 genes.
Methods: Cas9 single-guide RNAs (sgRNAs) for target sequences were tested in COS-7 cells with the pEGxxFP assay. Cas9-sgRNA plasmids were co-transfected with reporter plasmids containing either the mutated allele target sequence or the wildtype sequence of the identified genes and the efficiency of Cas9 cleavage was quantified by FACS. Using this methodology we identified allele-specific guides for BEST1, PRPF8, and PRPH2. The rate of allele-specific targeting by Cas9 was then assessed for each sgRNA in patient-derived and control fibroblasts by next-generation sequencing (NGS).
Results: In patient fibroblasts, we saw up to 16.3% indel formation for PRPF8, 14% for BEST1, and 15% for PRPH2. The low rates of indel formation in wildtype alleles (<2.5%) suggest specific targeting of pathogenic mutations. In addition, 76-94% of the total indels formed were out-of-frame in nature, a promising sign that Cas9 cleavage is likely to produce a loss or insertion of bases that disrupts the coding sequence.
Conclusions: This study demonstrates that CRISPR genome editing has single base specificity and can be used to selectively knock down mutant alleles even when the mutation does not create a new protospacer adjacent motif (PAM) sequence. The RNA-guided Cas9 nuclease is a potentially useful tool for targeted therapy in IRDs. This work is a proof of concept that genome editing tools could be used to provide long-term suppression of GOF alleles to treat dominant genetic diseases.
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