Applying Precision Genome Editing Technologies to Variant Effect Interpretation

Student: Steven Erwood

Supervisor: Dr. Ronald Cohn

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Meeting ID: 986 1247 7059

Abstract

Over the last two decades, advances in sequencing technologies have enabled routine implementation of genome sequencing in clinical practice. The utility of this sequencing data, however, is undermined by a poor understanding of the functional or clinical consequences of genetics variants found in human genomes. The realization of individualized or precision medicine will require a narrowing of the gap between our ability to read genomes and our ability to interpret their contents. This is being facilitated by the emergence and application of genome editing technologies – particularly those based on the CRISPR-Cas system. These approaches not only allow for the deliberate introduction of virtually any mutation-of-interest into a genome, but also enable complex genome engineering required for the establishment of tractable or relevant model systems. The most recent iteration of CRISPR-Cas development has led to a variety of ‘precision genome editors’ – namely base and prime editors – which permit facile nucleotide editing in a targeted, predictable and highly efficient manner. Here, I describe the implementation of precision genome editors to variant effect interpretation in the pediatric lysosomal storage disorder Niemann-Pick disease type C (NPC). First, I demonstrated the utility of combining base editors with a haploid cell model to rapidly establish models of NPC for molecular characterization. Next, I applied multiplexed prime editing to enable high-throughput variant interpretation in a strategy I call “saturation prime editing”. This was enabled by a methodology I developed based on allele-specific genome editing that allows for targeted “haploidization” of gene loci. Having only a single allele present in a cell – and thus a single allele driving an observed phenotype – allows for accurate mapping of genotype-to-phenotype using next-generation sequencing. By combining saturation prime editing with cell line haploidization, I derived function scores and clinical interpretations for more than 900 different variants in the NPC1 gene underlying NPC. I anticipate the broad application of these strategies will further our understanding of genetic variation and increase the clinical yield of diagnostic or preventative sequencing efforts.