Reverse Transcriptase Characterization and Engineering for Genome Editing

Abstract

Abstract Templated editing, consisting of a Cas9 enzyme coupled to a reverse transcriptase (RT) directed by a programmable guide RNA to install edits in the genome, offers unprecedented potential for treating genetic diseases. Yet, translation to clinical applications is hindered by unpredictable editing efficiency and limited understanding of how RT properties influence outcomes. This thesis systematically characterized a panel of established and novel RTs, including engineered Moloney Murine Leukemia Virus (MMLV) derivatives, compact bacterial RTs, and AstraZeneca-proprietary candidates, through complementary cellular and in vitro assays to establish rational engineering principles. A HiBiT reporter system enabled high-throughput quantification of templated editing (TE) efficiency in cellulo, while biochemical assays were used to measure polymerization activity, DNA/RNA binding affinity, thermostability, and aggregation propensity. Results suggested that successful templated editing requires optimization of three critical properties: thermostability, protein solubility, and DNA/RNA substrate affinity. Two iterative rational engineering cycles based on this hypothesis highlighted a synergistic relationship between the three properties. Engineered variants that combine solubility, stability and substrate affinity enhancements achieved up to 2.4 times higher editing efficiency compared to the established state-of-the-art benchmarks. By systematically defining the biochemical and biophysical property thresholds required for TE efficiency, this work establishes an evidence-based design framework that transform RT development from empirical screening toward rational, predictive engineering, diminishing optimization timelines and accelerating therapeutic development.