Engineering of PPR base editors to repair pathogenic SNPs at the level of RNA

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Abstract Many pathogenic T-to-C SNPs have been identified in humans including several in the mitochondrial genome linked to Leigh syndrome. The mitochondrial genome is especially difficult to manipulate using existing gene editing technologies due to inefficient transfer of guide RNAs through the mitochondrial membranes. The organelle genomes of most land plants contain hundreds of ancient T-to-C mutations that are “repaired” by C-to-U RNA editing before translation to produce functional proteins. The sufficient editing apparatus in plants has been recently discovered to be comprised of a single protein with an RNA binding PPR tract domain and a C-terminal catalytic domain called the DYW domain. The PPR domains follow a combinatorial code where two polar amino acid positions strongly influence the ribobase recognized. Changes in the polar amino acids have been correlated with predictable changes in RNA substrate specificity making the PPR domains programable. This grant aims to reprogram plant PPR editing factors to recognize human SNPs. In the first aim of the proposal, the editing factor PPR65 will be manipulated through engineered amino acid changes in the PPR domains to target mitochondrial pathogenic SNPs. In a second aim, local sequence requirements imposed by the enzymatic domain will be investigated and DYW domain swapping experiments should identify a catalytic domain with the least sequence bias. Catalytic sequence bias could potentially limit application of repair of human SNPs and the diversity of targeted sequences in higher plants suggest such bias is not universal. Both aims seek to apply the plant RNA editing machinery to make specific base edits to improve human health. Advantages in using the plant system include the prevention of permanent off-target effects through RNA recognition by the PPR tract and a fully proteinaceous, compact structure that can theoretically be efficiently delivered to mitochondria. This project will also provide research opportunities for six under- represented minority students in biochemistry each semester. Primary research will foster excitement for biochemistry and lead to a greater equity into the backgrounds of students prepared for STEM careers.