Portable Bacterial CRISPR Transcriptional Activation Enables Metabolic Engineering in Multiple Bacterial Species

CRISPR-Cas transcriptional programming in bacteria is an emerging tool to regulate gene expression for various applications, including metabolic pathway engineering. Here, we developed and optimized new CRISPR-Cas transcriptional activation (CRISPRa) systems in E. coli and P. putida. We provide a methodology to transfer CRISPRa to a new host by first optimizing expression levels for the CRISPRa system components, and then applying rules for effective CRISPRa based on a systematic characterization of E. coli promoters. We observed a correlation between CRISPR-mediated expression levels in E. coli and P. putida with R² = 0.80. The CRISPRa system can be implemented simultaneously with CRISPR interference (CRISPRi) for transcriptional repression, which enables complex regulation of multi-gene pathways. We demonstrated that CRISPRa can be used to regulate heterologous under synthetic promoters and some selected endogenous genes without further modification of the original sequence. We used this optimized system to regulate multi-gene biosynthetic pathways for biopterin and mevalonate production. This work will enable new metabolic engineering strategies in E. coli and P. putida and pave the way for CRISPR-Cas transcriptional programming in other bacterial species and microbial consortia.