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Conditional Guide RNAs: Programmable Conditional Regulation of CRISPR/Cas Function in Bacteria via Dynamic RNA Nanotechnology

Hanewich-Hollatz, Mikhail H. and Chen, Zhewei and Huang, Jining and Hochrein, Lisa M. and Pierce, Niles A. (2019) Conditional Guide RNAs: Programmable Conditional Regulation of CRISPR/Cas Function in Bacteria via Dynamic RNA Nanotechnology. . (Unpublished)

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A guide RNA (gRNA) directs the function of a CRISPR protein effector to a target gene of choice, providing a versatile programmable platform for engineering diverse modes of synthetic regulation (edit, silence, induce, bind). However, the fact that gRNAs are constitutively active places limitations on the ability to confine gRNA activity to a desired location and time. To achieve programmable control over the scope of gRNA activity, here we apply principles from dynamic RNA nanotechnology to engineer conditional guide RNAs (cgRNAs) whose activity is dependent on the presence or absence of an RNA trigger. These cgRNAs are programmable at two levels, with the trigger-binding sequence controlling the scope of the effector activity and the target-binding sequence determining the subject of the effector activity. We demonstrate molecular mechanisms for both constitutively active cgRNAs that are conditionally inactivated by an RNA trigger (ON→OFF logic) and constitutively inactive cgRNAs that are conditionally activated by an RNA trigger (OFF→ON logic). For each mechanism, automated sequence design is performed using the reaction pathway designer within NUPACK to design an orthogonal library of three cgRNAs that respond to different RNA triggers. In E. coli expressing cgRNAs, triggers, and silencing dCas9 as the protein effector, we observe programmable conditional gene silencing with a median dynamic range of ≈6-fold for an ON→OFF "terminator switch" mechanism, ≈15-fold for an ON→OFF "splinted switch" mechanism, and ≈3.6-fold for an OFF→ON "toehold switch" mechanism; the median crosstalk within each cgRNA library is <2%, <2%, and ≈20% for the three mechanisms. By providing programmable control over both the scope and target of protein effector function, cgRNA regulators offer a promising platform for synthetic biology.

Item Type:Report or Paper (Discussion Paper)
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URLURL TypeDescription Paper Paper
Pierce, Niles A.0000-0003-2367-4406
Additional Information:The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission. bioRxiv preprint first posted online Jan. 21, 2019. We thank S. Qi for the gift of plasmids and the gift of E. coli expressing mRFP and sfGFP, N.J. Porubsky for assistance with reaction pathway engineering using NUPACK, A. Hou and J. Kishi for performing preliminary studies, and R. Phillips for discussions on allosteric regulation. This work was funded by the Defense Advanced Research Projects Agency (HR0011-17-2-0008; the findings are those of the authors and should not be interpreted as representing the official views or policies of the US Government), by the Caltech Center for Environmental Microbial Interactions (CEMI), by the National Institutes of Health (5T32GM112592), by the Rosen Bioengineering Center at Caltech, by the National Science Foundation Molecular Programming Project (NSF-CCF-1317694), by a Professorial Fellowship at Balliol College (University of Oxford), and by the Eastman Visiting Professorship at the University of Oxford. Author Contributions: M.H.H.-H. and Z.C. contributed equally. The authors declare competing financial interests in the form of pending patents.
Group:Rosen Bioengineering Center, Caltech Center for Environmental Microbial Interactions (CEMI)
Funding AgencyGrant Number
Defense Advanced Research Projects Agency (DARPA)HR0011-17-2-0008
Caltech Center for Environmental Microbial Interactions (CEMI)UNSPECIFIED
NIH Predoctoral Fellowship5T32GM112592
Donna and Benjamin M. Rosen Bioengineering CenterUNSPECIFIED
University of OxfordUNSPECIFIED
Subject Keywords:small conditional RNA (scRNA), programmable conditional regulators, allosteric regulators, CRISPR/Cas, dynamic RNA nanotechnology, molecular programming
Record Number:CaltechAUTHORS:20190123-124341981
Persistent URL:
Official Citation:Conditional Guide RNAs: Programmable Conditional Regulation of CRISPR/Cas Function in Bacteria via Dynamic RNA Nanotechnology. Mikhail H. Hanewich-Hollatz, Zhewei Chen, Jining Huang, Lisa M. Hochrein, Niles A. Pierce. bioRxiv 525857; doi:
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:92430
Deposited By: Tony Diaz
Deposited On:23 Jan 2019 20:55
Last Modified:15 May 2019 16:27

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