Genome engineering has the potential to fundamentally revolutionize our capabilities in developing organisms to produce novel proteins and compounds, detect environmental contaminates, and interact with the natural world in new ways. Development of tools to hasten progress in genome engineering is critically needed to enable new biochemical discoveries and generate novel strains customized to the application of interest. CRISPR has become a core tool to facilitate genome editing, but first-generation CRISPR technologies have been limited in scalability, accessibility, edit variety, and ease of use, restricting the potential of the technology. Inscripta’s Onyx platform addresses these limitations by leveraging software and benchtop automation to enable high efficiency, massively parallel, precision-engineered edits to S. cerevisiae and E. coli genomes. We present a protein production and engineering application in E. coli leveraging this platform technology, showing how, in just a few weeks, a saturation mutagenesis library was generated against an integrated green fluorescent protein (GFP), with several engineered variants showing distinct intensity and spectral characteristics, including several literature-validated spectral variants as well as novel variants. Further, we generated a library of all predicted E. coli promoters and ribosome binding sites (RBS) 5′ of the integrated GFP start codon, identifying bins of low, medium, and high strength from minimal and rich media growth. These high-throughput profiling experiments yield insights into genetic parts regulation and provide a measure of native sequence strengths. Together, these applications demonstrate the power of the Onyx platform to improve heterologous protein engineering and expression workflows while generating new biochemical insights.

Rapid multiplexed genome editing for improved heterologous protein engineering and expression in E. coli

ACS Fall 2021

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