Project Summary: While gene editing technologies have revolutionized the ability to programmably edit DNA with high efficiency in diverse tissues, there remain several challenges with DNA editing, including permanent off-targets, concern for permanent correction of certain diseases, and some diseases being better targeted by other modalities than gene editing. For example, treatment of triplet repeat disorders with gene editing remains difficult, due to the difficulty of targeting repeat regions in the genome and the need to make large and precise deletions, without causing off-target genome rearrangements and other undesired effects on the genome. RNA modifications, however, may offer a better approach with notable features: 1) temporal and reversible modification of genetic diseases, 2) minimal off-targets which are reversible and less harmful, and 3) more versatile editing beyond genome editing. For example, with triplet repeat disorders, an RNA writing strategy could allow for collapse of the repeats to the exact desired number, an approach that would be more successful than gene editing or RNA knockdown strategies that have failed. To accomplish RNA writing, which involves all possible base edits (transitions and transversions), small or large insertions, and small or large replacements (e.g. exon swapping), some approaches have been developed, such as trans-splicing, but with limited success. Trans-splicing relies on the recruitment of an RNA template to a pre-mRNA without any active targeting domains and involves competition with the cis target. As a result, programmable trans-splicing has had low efficiency. We hypothesized that combining trans-splicing with programmable RNA guided CRISPR systems could help boost the efficiency of the trans-splicing mechanism, enabling any potential type of RNA edit, insertion, deletion, or replacement to be incorporated into endogenous transcripts. While we and others have characterized novel programmable RNA targeting CRISPR systems, such as Cas13, and developed tools from these systems, use of these tools have been limited in cellular systems due to a non-promiscuous cleavage activity known as collateral activity. While Cas13 has been shown to have specific RNA cleavage activity in some cell types, other cell types have had significant collateral cleavage of cellular RNAs, leading to toxicity in cell models. The proposed work will address these needs by combining biochemical characterization, structural characterization, and enzyme engineering to develop new RNA targeting CRISPR nucleases without collateral activity, such as the novel CRISPR-Cas7-11 enzyme, for specific RNA writing tools in conjunction with trans-splicing to enable any possible RNA edit. Beyond optimizing the RNA writing technology via trans-splicing optimization using RNA and protein engineering, we will showcase RNA writing’s therapeutic potential by correcting triplet repeat disorders in iPSC-derived human neurons. The developed tec...