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A technique adapted from CRISPR to control and activate a wide range of genes, at significantly greater levels than seen with other methods
About
Background Advances in reading, writing and editing DNA are helping to drive the synthetic biology industry. Beyond gene editing, CRISPR-Cas systems have been developed for targeted and programmable gene regulation. However, until now, the limitation of existing techniques in bacteria have prevented useful products to be made cost effectively and efficiently. This novel system is therefore a powerful and versatile synthetic biology tool for diverse research and industrial applications Technology Overview Edinburgh researchers have designed, constructed and characterised a CRISPRa system based on a eukaryote-like activation mechanism in bacteria, which shows strong activity, superior dynamic range and good tolerance to a wide range of UAS locations (at least 40 bp) (Figure 1). The system utilises dxCas9 (mutation of dCas9 based on xCas93.7) which increased the output dynamic range and further permitted use of non-canonical PAM. The system allows a multi-gene expression profile screening platform engineering approach; only one pathway circuit needs to be constructed to generate multiple expression profiles, overcoming the bottle-neck of conventional library construction approaches (Figure 2). Further Details: Engineered CRISPRa enables programmable eukaryote-like gene activation in bacteria. Liu et al, Nature Communications 2019, 10: 3693
Key Benefits
Overcomes low dynamic ranges of activation output seen in existing CRISPRi and CRISPRa approaches to mediate gene regulation. Levels of gene activation >100-fold higher. System supports multi-input activation Device works in E.coli and other species of bacteria Device can activate multiple wild type σ54-dependent promoters (PpspA, PhrpL, PnifH and PnifJ promoters) (Figure 3) Tuneable gene expression; ability to scale the global expression level of all target genes proportionally by tuning the activator induction level Reusable metabolic pathway screening tool; multi-gene expression profile diversities stored on a universally applicable library.
Applications
Potentially powerful tool for programming bacteria for diverse applications in industry and research: Tool to understand gene function, especially difficult to activate genes Tool to understand cellular behaviours Tool to tune and control metabolic pathway construction for the production of high value products using synthetic biology approaches Reusuable library to activate multiple genes to produce high yields of products efficiently and effectively