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Pooled cultivation of large and diverse populations of genetic variants against a range of growth conditions is a proven investigative approach for understanding complex biological systems. By leveraging new and powerful high-throughput genome engineering tools, researchers will have the opportunity to both expand the scope and accelerate the pace of target discovery using pooled growth selection approaches. One such genome engineering tool is the Onyx platform from Inscripta™, which enables genome-wide CRISPR editing at scale in an automated benchtop device.

Here we describe an application of the Onyx platform for massively parallel and targeted strain engineering in Escherichia coli to generate a genetically diversified seed population for pooled cultivations under selective pressure (diagram). Engineered strain populations were grown in the presence of one of four known growth-inhibitory compounds: furfural, hydroxymethyl-furfural, vanillin, and syringic acid.

Genome-wide engineering was performed on E. coli strain MG1655 using Onyx technology. A total of six individual strain engineering oligonucleotide libraries were designed to target all 4,336 annotated protein-encoding genes in the genome for knockout, or for expression modulation by insertion of five small constitutive synthetic promoters of defined expression strengths. Each library design construct included an edit-specific trackable barcode sequence.

Microbial genome engineering workflow using the Onyx Benchtop Platform. Design: Quick and easy edit library design with InscriptaDesigner™ Software Engineer cells: Fully automated genome engineering using Onyx Instrument and
– Onyx Genome Engineering Kit
Genotype: Onyx Cell Library characterization using Inscripta’s Genotyping Assays
Analyze and learn: Automated genotype-phenotype relationship analysis using
– InscriptaResolver™ Software

Analysis of the strain-variant enrichment and depletion profiles across knockout and promoter library types, as well as by biomass hydrolysate inhibitory compound, allowed for parsing the behavior of engineered strain variants within the experimental condition. For example, a total of 2,404 promoter edits and 1,405 knockouts across 1,313 and 1,076 genes, respectively, were significantly enriched or depleted in response to furfural. Genes previously identified for their roles in furfural tolerance were observed among the edited loci, providing a subset of validation data within our larger data set.

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. This extensive and precise strain-engineering strategy enables rapid discovery and ranking of loci both sensitive and resistant to the applied cultivation conditions.

The ability to engineer the entire genome of microorganisms at a massively parallel scale using the Onyx platform will reshape the manner in which researchers perform pooled cultivation experiments and generate data sets.


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