Development of novel precision genome editing tools and strategies for functional investigation of genetic variants

University Of California, San Diego, La Jolla CA

Investigators

Linked publications & trials

Abstract

Komor – Project Summary/Abstract Advances in sequencing technologies have made the detection of genetic variants in patients increasingly routine, and identification of clinically actionable genes and pathogenic mutations has revolutionized the field of precision medicine. However, less than 0.5% of the 759 million genetic variants (96% of which are single nucleotide variants, or SNVs) currently in the Genome Aggregation Database have a defined clinical interpretation, highlighting the need for new strategies to functionally characterize their impact in higher throughput. New methods capable of interpreting the full range of human genetic diversity in high throughput would have the potential to advance the field of precision medicine in a more equitable manner. A major goal of our research program is to combat this “variant interpretation problem” through the development of new precision genome editing tools and strategies and their application in generating cellular models of human genetic variants. We focus here on base editors (BEs), which utilize single-stranded DNA (ssDNA)-specific editing enzymes tethered to a catalytically impaired Cas9 (Cas9n) protein to install SNVs with high efficiency and precision. Current BEs can facilitate the introduction of C∙G to T∙A base pairs (called cytosine BEs, or CBEs) or A∙T to G∙C base pairs (called adenine BEs, or ABEs) using cytidine or adenosine deaminase enzymes, respectively. There are a variety of opportunities upon which to improve, optimize, and expand the technology, which we seek to address with this MIRA proposal. Specifically, we aim to expand the genome editing toolbox through the study and characterization of nucleic acid editing enzymes (Direction 1), development of novel directed evolution platforms (Direction 2), and the improvement and engineering of current editing tools (Direction 3). Direction 1 research aims to use new machine learning methods in combination with next-generation sequencing-based profiling assays to characterize the RNA editing profiles of a diverse set of nucleic acid editing enzymes. These data will be of high interest to the nucleic acid editing community and provide us with ideal starting points for our directed evolution efforts. Direction 2 research seeks to develop mammalian cell-based directed evolution platforms for genome editors, as we have found that inherent differences between bacteria (the host for most directed evolution platforms) and mammalian cells have caused setbacks in our development of new genome editing tools. This modular system will then be applied to evolve new BE tools and improve upon existing ones. Finally, Direction 3 research aims to improve upon existing BEs by eliminating bystander editing (when multiple Cs or As are edited concurrently) and the propensity of CBEs to install C∙G to non-T∙A edits. While we currently have the tools to begin work in all three areas, our research Directions are designed such that progress in any one Direction can be integrated into the other Directions to exponentially advance the research. The successful completion of the proposed work will produce new genome editors that will be of immediate use to basic science researchers, and can act as starting points for the development of therapeutics.