Using RNA-mediated Genome Editing to Create an Animal Model of Retinal Dystrophy for Analysis of in vivo CRISPR/CAS9 Treatment Efficacy.

Abstract

Purpose: Mutations in CEP290 are the most common cause of LCA, a severe form of inherited retinal dystrophy for which no treatment exists. Although autosomal recessive CEP290 LCA appeared to be a good candidate for gene-therapy, its large size coupled with over expression toxicity have made development of clinical gene augmentation approaches difficult. In vivo CRISPR-based genome editing, which A) use reagents that can be delivered via clinically relevant viruses and B) leave the endogenous genetic regulatory elements intact, have emerged as a new potential treatment strategy. The purpose of this study is to employ the CRISPR/Cas genome editing technology to generate a murine model carrying a deep intronic splice mutation in Cep290, similar to the most common mutation observed (IVS26 + 1655 A > G) in patients with CEP290-associated LCA, which can be used to test in vivo CRISPR-based treatment strategies.

Methods: Sequence from the human CEP290 intron 26 was aligned to the homologous murine Cep290 sequence (mmIVS26) using ClustalW. Small guide RNAs were designed using the CRISPR Design Tool (crispr.mit.edu) and cloned into the bicistronic CRISPR/Cas9 expression plasmid. Efficiency of CRISPR/Cas9 modification was assessed via Surveyor nuclease assay and TA cloning/Sanger sequencing. Donor template containing ~500 bases of mmIVS26 homology flanking the 5’ splice donor site of murine IVS26 was cloned and co-delivered with the bicistronic CRISPR/Cas9 expression plasmid into murine iPSCs. Homology-directed repair was detected via PCR and Cep290 transcript analysis was performed by rt-PCR.

Results: Six small guide RNAs were chosen to test based on the ClustalW alignments. Of these, three successfully and efficiently modified the mmIVS26 locus as determined by Surveyor and TA cloning/Sanger sequencing assays. Following co-delivery of CRISPR/Cas9-expressing plasmid with donor template containing the 5’ splice donor site (creating the deep intronic mutation), successful homology-directed repair was detected. Cep290 expression analysis indicated a reduction in the amount of transcript in cells treated with both the sgRNA/Cas9 and donor template plasmids as compared to cells treated with the donor template alone.

Conclusions: These results provide a foundation on which to build an animal model of CEP290 LCA that can be used to evaluate in vivo genome editing-based therapies.