TY - JOUR
T1 - Rapid generation of CRISPR/dCas9-regulated, orthogonally repressible hybrid T7-lac promoters for modular, tuneable control of metabolic pathway fluxes in Escherichia coli
AU - Cress, Brady F.
AU - Jones, J. Andrew
AU - Kim, Daniel C.
AU - Leitz, Quentin D.
AU - Englaender, Jacob A.
AU - Collins, Shannon M.
AU - Linhardt, Robert J.
AU - Koffas, Mattheos A.G.
N1 - Publisher Copyright:
© 2016 The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.
PY - 2016/5/19
Y1 - 2016/5/19
N2 - 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.
AB - 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.
UR - http://www.scopus.com/inward/record.url?scp=84970002343&partnerID=8YFLogxK
U2 - 10.1093/nar/gkw231
DO - 10.1093/nar/gkw231
M3 - Article
C2 - 27079979
AN - SCOPUS:84970002343
SN - 0305-1048
VL - 44
SP - 4472
EP - 4485
JO - Nucleic Acids Research
JF - Nucleic Acids Research
IS - 9
ER -