High throughput functional variant screens via in-vivo production of single-stranded DNA

Abstract

Tremendous genetic variation exists in nature, but our ability to create and characterize individual genetic variants remains far more limited in scale. Likewise, engineering proteins and phenotypes requires the introduction of synthetic variants, but design of variants outpaces experimental measurement of variant effect. Here, I first outline the tools available to tackle this challenge, and explore the historical context for this problem. I pose the challenge of a scanning, saturating mutagenesis of the genome, both as a thought experiment and to focus our thinking toward tools that are likely to deliver on such promises. I then introduce Retron Library Recombineering (RLR), my contribution to the set of tools for addressing these challenges. I first optimize Retron Recombineering, demonstrating efficient and continuous generation of precise genomic edits in Escherichia coli, via in-vivo production of single stranded DNA by the targeted reverse-transcription activity of retrons. Greater than 90% editing efficiency can be obtained using this method. Once achieved, this efficiency enables multiplexed applications. Retron Library Recombineering (RLR) becomes a system for high-throughput screens of variants, wherein the association of introduced edits with their retron elements enables a targeted deep sequencing phenotypic output. I use RLR for pooled, quantitative phenotyping of synthesized variants, characterizing antibiotic resistance alleles. I also perform RLR using sheared genomic DNA of an evolved bacterium, experimentally querying millions of sequences for antibiotic resistance variants. In doing so, I demonstrate that RLR is uniquely suited to utilize non-designed sources of variation. Pooled experiments using ssDNA produced in vivo thus present new avenues for exploring variation, both designed and not, across the entire genome. Then, I examine the next steps for retron recombineering. The editing efficiencies obtained in the study of RLR, and possibly new types of engineered retrons, enable exciting new applications. Finally, I review the challenge and promise of functional Retron Recombineering outside of the familiar E. coli.