BACKGROUND AND OBJECTIVES: The variety of ocular cell types involved in inherited retinal disease (IRD) necessitates the use of gene editing therapeutics which have generalizable components. In our study, we investigate the generalizable characteristics of non-engineered pegRNA design (PE2) for efficient, proof-in-principle gene correction of over 21 genes implicated in IRDs and associated syndromes. We use a single-transgene oligopool approach, comprising approximately 12,000 uniquely barcoded pegRNAs that target a synthetically integrated, 50 bp sequence motif, which faithfully recapitulate the disease context of their various counterpart IRDs. Using this approach, we perform a high throughput, pooled analysis of pegRNA characteristics across non- and ocular cell types to propose a cell-line agnostic set of pegRNA design guidelines.
RESULTS: Briefly, we find that non-engineered pegRNA 3' extensions should mediate substitution-type edits and that the desired edit should be placed within five nucleotides upstream of the nick site induced by the Cas-endonuclease. Further, PBS and RTT lengths of at least 12 and 14 nucleotides, respectively, should be used and each non-engineered pegRNA 3' extension should obviate an initial templating cytosine nucleotide.
CONCLUSION: We establish a set of recommendations for the generalizable design of the non-engineered pegRNA 3' extension for the correction of several IRDs, enabling overall simplification of design parameters for PE2-based systems.
