Kealan Exley¹, Zofia D. Jarczynska¹, Linas Tamošaitis², Giovanni Schiesaro², Lars K. Nielsen^{1, 3}, Vijayalakshmi Kandasamy¹

The Inscripta Onyx platform is revolutionizing microbial strain engineering in synthetic biology. The automated benchtop appliance for genome engineering facilitates rapid large-scale CRISPR editing of S. cerevisiae or E. coli to introduce up to 10,000 edits. Using proprietary technology, the MAD7 nuclease can introduce targeted single-nucleotide polymorphisms, insertions, and/or deletions, into the host genome.

With the Inscripta Onyx platform's ability to generate thousands of strain variants within days, it is advantageous to match this pace of library generation to the identification of useful strain variants. Biofoundries possess infrastructure for the execution of high-throughput automated methods and are indispensable for the rapid phenotypical screening and sequencing of strain libraries. By combining the capabilities of the Inscripta Onyx and DTU CFB Biofoundry, for targeting and identifying novel genes respectively, an optimized yeast strain producing cis,cis-muconic acid (ccM) with increased yields was generated within one month. Muconic acid is an important commodity chemical for nylon production. A GFP-biosensor coupled with the ccM-producing yeast strain enabled the online monitoring of ccM production. Using fluorescent assisted selection equipment, such as the PIXL microbial colony picker and FACS, GFP fluorescent ccM-producing yeast strain were isolated. Subsequent in-house sequencing of barcoded editing plasmid DNA from Inscripta tracked the abundance of each edit in the Inscripta library and revealed a number of unique gene targets that improved ccM yields. 

The Inscripta Onyx in conjunction with the integrated infrastructure of a Biofoundry enables the rapid and expansive completion of many iterations of a DBTL cycle. For instance, after analyzing single advantageous gene targets, determined by an Inscripta library, the CFB Biofoundry infrastructure enables multiplex-CRISPR targeting to generate variants with multiple edits for further strain optimization. The Inscripta-generated libraries aid genome discovery and enable forward engineering of strains to improve product titers.