Digital Genome Engineering for High-Throughput Discovery and Strain Optimization Applications

CRISPR-based genome engineering has the potential to accelerate scientific discovery. Current approaches suffer from limitations in scalability, diversity of edit types, and accessibility. Onyx^TM, a benchtop genome engineering platform, overcomes these limitations by providing the capability to generate up to 10,000 precisely edited and trackable strain variants in E. coli in a single run, or up to 6,000 such variants in S. cerevisiae. This enables novel approaches to engineer targets within genes, pathways, and even genome wide. The platform simplifies the complex editing workflow, from design to engineered cell library, by including software, reagents, benchtop instrument and analytics. We present data from three applications using Onyx, from high-throughput target discovery to metabolic engineering, to deconvolution of adaptive laboratory evolution variants. In an application to identify new gene targets that improve glycerol utilization in budding yeast, we generated 7 libraries (>40,000 designs) including truncation of non-essential genes and genome-wide expression modulation with terminator or transcription factor binding site modifications. Using barcodes, we identified >100 novel targets with a glycerol utilization phenotype. In a metabolic engineering application, we generated 200,000 designs across 24 genome-edited libraries in E. coli and screened the resulting strain variants for improved lysine production. We uncovered more than 15 novel genes outside of the known lysine pathway that contribute to increased lysine production. Rapid recombination of the hits through the Onyx workflow resulted in strains with >10,000-fold increase in lysine. Using Onxy libraries, we deconvoluted variants identified in adaptive laboratory evolution studies, including testing growth in media containing several different inhibitors. The Onyx^TM platform will usher in the next era of genomics, enabling researchers to transition from observational studies to precisely engineering living systems at scale. This will have far-reaching benefits for biology, bio-industrial science, agriculture, healthcare, and alternative energy.