Rapid generation of CRISPR/dCas9-regulated, orthogonally repressible hybrid T7-lac promoters for modular, tuneable control of metabolic pathway fluxes inEscherichia coli

Abstract

Robust gene circuit construction requires use of promoters exhibiting low crosstalk. Orthogonal promoters have been engineered utilizing an assortment of natural and synthetic transcription factors, but design of large orthogonal promoter-repressor sets is complicated, labor-intensive, and often results in unanticipated crosstalk. The specificity and ease of targeting the RNA-guided DNA-binding protein dCas9 to any 20 bp user-defined DNA sequence makes it a promising candidate for orthogonal promoter regulation. Here, we rapidly construct orthogonal variants of the classic T7-lac promoter using site-directed mutagenesis, generating a panel of inducible hybrid promoters regulated by both LacI and dCas9. Remarkably, orthogonality is mediated by only two to three nucleotide mismatches in a narrow window of the RNA:DNA hybrid, neighboring the protospacer adjacent motif. We demonstrate that, contrary to many reports, one PAM-proximal mismatch is insufficient to abolish dCas9-mediated repression, and we show for the first time that mismatch tolerance is a function of target copy number. Finally, these promoters were incorporated into the branched violacein biosynthetic pathway as dCas9-dependent switches capable of throttling and selectively redirecting carbon flux in Escherichia coli. We anticipate this strategy is relevant for any promoter and will be adopted for many applications at the interface of synthetic biology and metabolic engineering.

CRISPR interference (CRISPRi) for gene regulation and succinate production in cyanobacterium S. elongatus PCC 7942

“…CRISPRi holds great promise for a wide range of applications in microorganisms, including bacterial cell growth control [35], genetic screen [25, 36], synthetic biology module development [37, 38] or metabolic networks control in various microorganisms such as E. coli [24, 39, 40], mycobacteria [41], Bacillus subtilis [42], Corynebacterium glutamicum [43], Clostridium beijerinckii [44], yeast [45] and cyanobacteria [7]. In particular, a number of recent studies have exploited CRISPRi to regulate the metabolic pathways in E. coli for enhanced production of various biotechnological products including poly(3-hydroxybutyrate- co -4-hydroxybutyrate) [23], terpenoid [8], pinosylvin [46], flavonoid [47] and mevalonate [48].…”

Transcriptional regulation with CRISPR-Cas9: principles, advances, and applications

“…This property can be leveraged to achieve transcriptional regulation while avoiding DNA cleavage using nucleolytically active Cas9 guided by sgRNA with mismatches or truncations within the guide sequence [8,48]. Mismatch sensitivity is further affected by the concentration of dCas9/sgRNA complex relative to the target DNA [49], which itself depends on the nature and relative amounts of sgRNAs expressed [50]. In eukaryotes, efficiency of regulation is diminished by guanine enrichment and adenine depletion of the guide sequence since it decreases sgRNA stability [51].…”

“…2(b), bottom); the output of a synthetic NOT[NOR(A,B)] logic circuit was connected to native E. coli network via an sgRNA directing repression of an endogenous transcription factor gene. In a separate study, a panel of orthogonal T7 RNA polymerase driven dCas9-regulatable hybrid promoters was designed, tested, and used to control a reconstituted metabolic pathway [49]. …”

“…This has been demonstrated in E. coli and C. glutamicum , where multiplexed promoter repression resulted in increased yields of naringenin [66], 4-hydroxybutyrate [67], as well as lysine and glutamate [16]. Such an approach has also been successfully utilized in E. coli [49] and S. cerevisiae [24] to increase the flux in the violacein metabolic pathway (Fig. 3(b), bottom).…”

CRISPRi-mediated metabolic engineering of E. coli for O-methylated anthocyanin production

“…Although it is not clear why this occurred, we speculate that previously reported growth inhibition associated with expression of dCas9 and crRNA/gRNA [48] slightly altered the production capacity or induction optimum for this strain. Regardless, compared to the non-targeting spacer (previously used as a negative control in E. coli by other groups [49] and ours [32, 50]), both anti- metJ spacers improved production of P3G by approximately twofold (Fig. 5c), representing approximately 20-fold improvement over the best strain from the initial screen.…”