Digital Genome Engineering: Unlocking the Unlimited Potential of Biology

CRISPR-based genome engineering has the potential to accelerate discovery. Unfortunately, current approaches suffer from limitations in scalability, diversity of edit types, and accessibility. The 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 or up to 6,000 such variants in S. cerevisiae, in a single run. This enables novel approaches to engineering of gene, pathway, and genome-wide targets in S. cerevisiae and E. coli. The platform simplifies the complex editing workflow from design to engineered cell library, by including software, reagents, benchtop instrument and analytics. We present 3 applications including high-throughput target discovery, metabolic engineering and accelerated protein optimization. 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. To accelerate protein optimization we generated a saturation mutagenesis library of 723 designs against the active site of a genome-integrated GFP and identified several engineered variants showing distinct intensity and spectral characteristics. The Onyx^TM platform will usher in the next era of genomics, enabling the researcher to transition from primarily observational studies to truly engineering biology. This will have far-reaching benefits for biology, bio-industrial science, agriculture, healthcare, and alternative energy.