A High-Efficacy CRISPRi System And Strong Synthetic Promoters For Alphaproteobacteria And Gammaproteobacteria

The present invention from UW-Madison innovators is a CRISPRi system for studying the genetics of Z. mobilis and lac-regulated synthetic Z. mobilis promoters that are used in the CRISPRi system but also may be research tools for studying gene expression in Z mobilis.

The inventors’ Z. mobilis CRISPRi vectors cause either strong knockdowns (>100-fold) using single guide RNA (sgRNA) spacers that perfectly match target genes, or partial knockdowns using spacers with mismatches. The inventors had to use a specific type of inactive Cas9 protein, had to swap the fluorescent reporter protein they were using with a super folding green fluorescent protein, had to optimize the sgRNAs and had to develop Lac-regulated synthetic promoters improve knockdown efficiency while preventing leaky expression of the CRISPRi components in order to get the controlled gene knockdowns they desired. The innovators demonstrated the efficacy of Z. mobilis CRISPRi by targeting essential genes that are universally conserved in bacteria, key to the efficient metabolism of Z. mobilis, or underlie alcohol tolerance.