TY - JOUR
T1 - Orthogonal control of gene expression in plants using synthetic promoters and CRISPR-based transcription factors
AU - Kar, Shaunak
AU - Bordiya, Yogendra
AU - Rodriguez, Nestor
AU - Kim, Junghyun
AU - Gardner, Elizabeth C.
AU - Gollihar, Jimmy D.
AU - Sung, Sibum
AU - Ellington, Andrew D.
N1 - Funding Information:
This work was supported by the Defense Advanced Research Projects Agency (DARPA) agreement HR00111820048 to AE and SS. The content of the information does not necessarily reflect the position or the policy of the Government, and no official endorsement should be inferred. This work was also supported by Welch Foundation grant (F-1654) to ADE.
Publisher Copyright:
© 2022, The Author(s).
PY - 2022/3/29
Y1 - 2022/3/29
N2 - Background: The construction and application of synthetic genetic circuits is frequently improved if gene expression can be orthogonally controlled, relative to the host. In plants, orthogonality can be achieved via the use of CRISPR-based transcription factors that are programmed to act on natural or synthetic promoters. The construction of complex gene circuits can require multiple, orthogonal regulatory interactions, and this in turn requires that the full programmability of CRISPR elements be adapted to non-natural and non-standard promoters that have few constraints on their design. Therefore, we have developed synthetic promoter elements in which regions upstream of the minimal 35S CaMV promoter are designed from scratch to interact via programmed gRNAs with dCas9 fusions that allow activation of gene expression. Results: A panel of three, mutually orthogonal promoters that can be acted on by artificial gRNAs bound by CRISPR regulators were designed. Guide RNA expression targeting these promoters was in turn controlled by either Pol III (U6) or ethylene-inducible Pol II promoters, implementing for the first time a fully artificial Orthogonal Control System (OCS). Following demonstration of the complete orthogonality of the designs, the OCS was tied to cellular metabolism by putting gRNA expression under the control of an endogenous plant signaling molecule, ethylene. The ability to form complex circuitry was demonstrated via the ethylene-driven, ratiometric expression of fluorescent proteins in single plants. Conclusions: The design of synthetic promoters is highly generalizable to large tracts of sequence space, allowing Orthogonal Control Systems of increasing complexity to potentially be generated at will. The ability to tie in several different basal features of plant molecular biology (Pol II and Pol III promoters, ethylene regulation) to the OCS demonstrates multiple opportunities for engineering at the system level. Moreover, given the fungibility of the core 35S CaMV promoter elements, the derived synthetic promoters can potentially be utilized across a variety of plant species.
AB - Background: The construction and application of synthetic genetic circuits is frequently improved if gene expression can be orthogonally controlled, relative to the host. In plants, orthogonality can be achieved via the use of CRISPR-based transcription factors that are programmed to act on natural or synthetic promoters. The construction of complex gene circuits can require multiple, orthogonal regulatory interactions, and this in turn requires that the full programmability of CRISPR elements be adapted to non-natural and non-standard promoters that have few constraints on their design. Therefore, we have developed synthetic promoter elements in which regions upstream of the minimal 35S CaMV promoter are designed from scratch to interact via programmed gRNAs with dCas9 fusions that allow activation of gene expression. Results: A panel of three, mutually orthogonal promoters that can be acted on by artificial gRNAs bound by CRISPR regulators were designed. Guide RNA expression targeting these promoters was in turn controlled by either Pol III (U6) or ethylene-inducible Pol II promoters, implementing for the first time a fully artificial Orthogonal Control System (OCS). Following demonstration of the complete orthogonality of the designs, the OCS was tied to cellular metabolism by putting gRNA expression under the control of an endogenous plant signaling molecule, ethylene. The ability to form complex circuitry was demonstrated via the ethylene-driven, ratiometric expression of fluorescent proteins in single plants. Conclusions: The design of synthetic promoters is highly generalizable to large tracts of sequence space, allowing Orthogonal Control Systems of increasing complexity to potentially be generated at will. The ability to tie in several different basal features of plant molecular biology (Pol II and Pol III promoters, ethylene regulation) to the OCS demonstrates multiple opportunities for engineering at the system level. Moreover, given the fungibility of the core 35S CaMV promoter elements, the derived synthetic promoters can potentially be utilized across a variety of plant species.
KW - Modular cloning
KW - Orthogonal promoter
KW - Plant synthetic biology
KW - Synthetic transcription factor
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U2 - 10.1186/s13007-022-00867-1
DO - 10.1186/s13007-022-00867-1
M3 - Article
C2 - 35351174
AN - SCOPUS:85127316774
VL - 18
SP - 42
JO - Plant Methods
JF - Plant Methods
SN - 1746-4811
IS - 1
M1 - 42
ER -