Cell-Free Transcription-Coupled CRISPR/Cas12a Assay for Prototyping Cyanobacterial Promoters.

Cyanobacteria are promising microbial hosts for the production of diverse biofuels and biochemicals. However, compared to other model microbial hosts such as Escherichia coli and yeast, it takes a long time to genetically modify cyanobacteria. One way to efficiently engineer cyanobacteria while minimizing genetic engineering would be to develop a fast, high-throughput prototyping tool for cyanobacteria. In this study, we developed a CRISPR/Cas12a-based assay coupled with cyanobacteria cell-free systems to rapidly prototype promoter characteristics. Using this newly developed assay, we demonstrated cyanobacteria cell-free transcription for the first time and confirmed a positive correlation between the in vitro and in vivo transcription performance. Furthermore, we generated a synthetic promoter library and evaluated the characteristics of promoter subregions by using the assay. Varied promoter strength derived from random mutations were rapidly and effectively measured in a high-throughput way. We believe that this study offers an easily applicable and rapid prototyping platform to characterize promoters for cyanobacterial engineering.