Alternative genome editing
Over the past years, Cas9 and Cas12a nucleases have been adopted as robust genome editing and transcriptome manipulation tools. To recognize and cleave a dsDNA target, both Cas9 and Cas12 require a short sequence, termed the protospacer adjacent motif (PAM), in the vicinity of a DNA sequence targeted by the gRNA. The PAM requirement imposes a serious bottleneck in therapeutic editing where often a precise targeting close to the mutation site is required. If there is no PAM near the target site, Cas9 and Cas12a proteins cannot access it. Next, there are difficulties with the delivery of Cas9 and Cas12a into tissues or cells to achieve therapeutic effects. AAVs often would be a preferable choice however, these vectors have their own cargo packaging limit thus small size Cas nuclease variants are highly desirable. Here we aim to assess the therapeutic potential of novel Cas9 orthologues and variants that cut close to the desired mutation sites in EYS and USH2A, and explore the gene editing potential of the miniature Cas12f nucleases 50 in HEK293 cells. The most promising Cas nucleases will be selected and further optimized in either wild type, or patient derived retinal organoids, in collaboration with partners within the consortium.