Industrial Biotechnology

High Performing Strain Development

Due to their exceptional natural characteristics yeasts, and other microbes play an important role as the vector for bioproduction of biofuels, food additives and proteins.

By applying Cas-CLOVER and piggyBac gene editing technology to microbial strain engineering, these traits can be further leveraged for commercial manufacturing, product discovery and development.

Vast Opportunities with Cas-CLOVER & piggyBac

PiggyBac has demonstrated effective gene integration & expression capabilities across species, including those with less robust genetic tools.
Cas-CLOVER enables high fidelity editing of metabolic targets, where off-targets won't mask or select out important traits.

Demeetra AgBio, Inc.

PiggyBac: A Simple, Versatile & Efficient Tool

Demeetra AgBio, Inc.
  • Step 1 - Clone cargo into transposon vector
  • Step 2 - Co-transfect transposon and transposase into cells
  • Step 3 - Transposes cuts out cargo and pastes into random TTAA sites
  • Step 4 - Screen clones for the desired insert
  • Step 5 - (optional): Use Excision-only piggyBac for Footprint-Free gene editing

Key Features

PiggyBac is known for its high-efficiency. It is stable and provides high expression. From small to large gene integration (200KB+), you can engineer entire metabolic pathways with one event. It is also a scar-less removal for phenotype reversion.

Overview of PiggyBac In Yeast

PiggyBac is potentially active in all yeast strains. Unlike CRISPR, one piggyBac vector can rapidly develop mutant libraries for loss (gene-trapping) and gain of function (enhancer-trapping) across species. With piggyBac, genes are easily mapped, and phenotype reversion is seamless. Unlike CRISPR, piggyBac IP is issued.

S. pombe Mutagenesis

Ura4+ selectable marker is flanked by piggyBac ITRs. PiggyBac transposase “PBase” results in high-efficiency transposition. Although S. pombe is a powerful genetic system, chemical mutagenesis requires extensive mapping, and CRISPR requires the design and testing of individual guide RNAs for each target. PiggyBac, on the other hand, integrates efficiently with rapid genetic mapping to the ITRs.

Cas-CLOVER High-Fidelity Targeting In Yeasts

The Cas-CLOVER gene editing system utilizes a truly dimeric Clo51 nucleus domain, recruited by a pair of guide RNAs (gRNAs) to introduce targeted mutations while removing the risk of off-targets. Cas-CLOVER was used to disrupt a pathway in yeast resulting in the accumulation of red pigment in vacuole resulting with up to 90% targeted knockout efficiency.

Demeetra AgBio, Inc.

Citations

  • Li et al. Nuc Acid Res. (2011) A piggyBac transposon-based mutagenesis system for the fission yeast Schizosaccharomyces pombe
  • Cai, P., Gao, J., & Zhou, Y. (2019). CRISPR-mediated genome editing in non-conventional yeasts for biotechnological applications. Microbial Cell Factories, 18(1), 1–12. https://doi.org/10.1186/s12934-019-1112-2