CRISPRi Screen Boosts Cyanobacteria Resilience — A Step Toward Industrial Biofuels

Researchers at Oak Ridge National Laboratory's Center for Bioenergy Innovation (CBI) and the University of Colorado used a genome‑wide CRISPR interference (CRISPRi) screen to partially silence genes across Synechococcus cyanobacteria and discovered that broad repression improved growth under industrially relevant stress conditions.

What the team did

Instead of permanently editing DNA, the researchers employed CRISPRi — a gene‑silencing variant of the CRISPR‑Cas system that uses a catalytically inactive Cas9 to block transcription. They built a high‑throughput library of nearly 33,000 guide RNAs (gRNAs), with roughly ten guides per gene, to produce varying levels of knockdown across the cyanobacterial genome. By exposing the population to environmental stress and sequencing survivors, the team linked specific knockdowns to improved or reduced fitness.

Key findings

When exposed to a lower temperature (22 °C versus their typical 37 °C) and to continuous white light instead of a day/night cycle of red and blue wavelengths, cyanobacteria with partial, genome‑wide repression grew better. The authors propose that continuous light disrupted circadian‑clock‑driven protein production, freeing cellular energy that could be redirected toward growth and stress tolerance.

“Partial suppression across the genome altered resource allocation in a way that enhanced tolerance to new environmental conditions,” commented Carrie Eckert, leader of the Synthetic Biology Group at ORNL and co‑author of the study.

Why it matters

This approach provides a faster, broader way to link gene function to desirable traits than traditional one‑gene editing. High‑throughput CRISPRi screens can reveal unexpected genetic strategies for improving microbial robustness, accelerating the optimization of strains used to produce sustainable aviation fuel and other bio‑based chemicals and materials.

CBI has previously engineered Clostridium thermocellum to produce small amounts of sustainable aviation fuel from lignocellulosic biomass — including poplar and switchgrass — and plans to apply CRISPRi insights to microbes and plant cells that must tolerate harsh industrial fermentation conditions.

Broader context and challenges

Microbial fermentation already produces compounds such as acetone, butanol, ethanol, insulin and 2,3‑butanediol, but many processes remain only marginally profitable at scale. Techniques like genome‑wide CRISPRi screening can shorten the path to commercially viable strains by identifying gene manipulations that improve performance under real‑world production stresses.

Next steps: applying the CRISPRi screening strategy to other biofuel‑relevant microbes and integrating top candidate knockdowns into strains for pilot‑scale testing to evaluate productivity, stability and economic feasibility.