RNA sequencing data after simultaneous transactivation of M-opsin and knockout of rhodopsin upon subretinal injection of mice with the REVeRT dual AAV system

Large genes including several CRISPR-Cas modules, such as gene activators (CRISPRa), require dual adeno-associated viral (AAV) vectors for efficient in vivo delivery and expression. Current dual AAV vector approaches have important limitations, e.g., low reconstitution efficiency, production of alien proteins, or low flexibility in split site selection. Here, we present a dual AAV vector technology based on reconstitution via mRNA trans-splicing (REVeRT). REVeRT is flexible in split site selection and can efficiently reconstitute different split genes in numerous in vitro models, in human organoids and in vivo. Furthermore, REVeRT can functionally reconstitute a CRISPRa module targeting genes in various mouse tissues and organs in single or multiplexed approaches upon different routes of administration. Finally, supplementation of ABCA4 (6.8 kb) via REVeRT improves retinal degeneration and function in a mouse model of inherited blindness. Due to its flexibility and efficiency REVeRT harbors great potential for basic research and clinical applications. Overall design: Examination of two different groups of mice heterozygous for the P23H mutation subretinal injected at post natal day 21 with dual AAV encoding for Cas9-VPR and sgRNAs for either the knockout of Rhodopsin and transactivation of M-opsin (sgRho-3xsgOpn1mw, n = 3) or a control sgRNAs targeting the bacterial lacZ locus (sglacZ, n = 4). Retinas were isolated 4 weeks after subretinal injection.