Rapid target discovery in microbes using massively parallel genome engineering

Target discovery in biological systems relies on access to relevant genetic diversity.  Historically, genetic diversity has been sourced through natural variation, classical mutagenesis approaches, or more recently using molecular genetic tools such as zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), meganucleases and CRISPR nucleases to deliver targeted genetic manipulations.  While target discovery has been performed using each of these approaches, the ability to generate large populations of cells with precisely targeted and controlled genetic variation throughout the entire genome has never been an option in this field of study.
Inscripta’s Onyx platform now makes this possible.
Onyx allows you to design your experiments, generate thousands of unique edited cells, assess and test your library quality, interrogate genotypes in your engineered cell populations, and rapidly analyze and identify novel genotype-phenotype associations.
The scope of discovery that once took years to decades to achieve can now be accomplished in a matter of months to years. With rapid genome engineering and genome-wide CRISPR library screening you can create and test more ideas, and discover and exploit new biology in areas such as biomass hydrolysate component growth inhibition, antibiotic resistance, and adaptive laboratory evolution (ALE), for rapid target discovery.  


Microbial genome engineering workflow using the Onyx benchtop platform:
Design: edit library is designed 
using InscriptaDesigner™ software.
Engineer cells: cells are engineered using Onyx system and Onyx genome engineering chemistry.
Genotype: cells are genotyped using Onyx assays.
Analyze and learn: results are analyzed using InscriptaResolver™ software.

Application Note


Inscripta’s application note on Massively parallel genome engineering followed by pooled growth selections for rapid target discovery in microbes details the generation of precision-engineered E. coli populations and analysis of pooled growth selection in the presence of inhibitory compounds.

An E. coli strain was engineered, for genome-wide protein truncations and genome-wide insertion of promoters of varying strengths. Only high-quality designs were retained, leading to a population of 3,676 genes with promoter designs and 7,933 knockout designs. The resulting six engineered cell libraries were pooled and grown in the presence of four compounds known to inhibit E. coli growth to understand variant function in the face of selective pressure. Using Inscripta's rapid barcode sequencing assay, hundreds (in some cases, thousands) of edits that were significantly enriched or depleted in response to each inhibitory compound were identified.

The availability of a ladder of gene expression variants paired with the concomitant knockout strain for nearly every gene makes it possible to obtain a deeply nuanced view into the mechanisms of inhibitor tolerance in E. coli.