Type III CRISPR-mediated flexible RNA excision with engineered guide RNAs

Current targeted RNA editing techniques are predominantly limited to single-base edits. Here, we introduce SCISSOR for selective cleavage and intramolecular stitches of RNA. Building on the principle that type III CRISPR complex determines target cleavage positions based on guide RNA (gRNA) length in 6-nt increments, we hypothesized that engineering gRNAs with bulge loops could provide a means to bypass this rule, enabling RNA cleavage at non-6-nt intervals and allowing for flexible RNA excision. Through systematic evaluation of gRNAs with various bulge loops at different positions, we established the rules governing the complex for precise and efficient non-6-nt target cleavage and repair. Unexpectedly, we observed that the complex tolerates 1- or 2-nt small bulge loops and accommodates large bulge loops of any length between 6 and 24 nt. Furthermore, excision efficiency can be improved with rationally optimized gRNAs. Consequently, SCISSOR could accomplish nearly any length of short fragment excision. With its capability to modify open reading frames, we demonstrate the potential of SCISSOR in repairing frameshift mutations and introducing frameshift to create immunogenic tumor poly-epitopes. SCISSOR holds promise in RNA therapy and biomedical research.