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Published July 24, 2019 | Submitted + Published + Supplemental Material
Journal Article Open

Conditional Guide RNAs: Programmable Conditional Regulation of CRISPR/Cas Function in Bacterial and Mammalian Cells via Dynamic RNA Nanotechnology


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 a median conditional response of ≈4-fold for an ON → OFF "terminator switch" mechanism, ≈15-fold for an ON → OFF "splinted switch" mechanism, and ≈3-fold for an OFF → ON "toehold switch" mechanism; the median crosstalk within each cgRNA/trigger library is <2%, ≈2%, and ≈20% for the three mechanisms. To test the portability of cgRNA mechanisms prototyped in bacteria to mammalian cells, as well as to test generalizability to different effector functions, we implemented the terminator switch in HEK 293T cells expressing inducing dCas9 as the protein effector, observing a median ON → OFF conditional response of ≈4-fold with median crosstalk of ≈30% for three orthogonal cgRNA/trigger pairs. By providing programmable control over both the scope and target of protein effector function, cgRNA regulators offer a promising platform for synthetic biology.

Additional Information

© 2019 American Chemical Society. This is an open access article published under an ACS AuthorChoice License, which permits copying and redistribution of the article or any adaptations for non-commercial purposes. Received: April 3, 2019; Published: June 4, 2019. We thank S. Qi for the gift of E. coli expressing mRFP and sfGFP, N. J. Porubsky for assistance with reaction pathway engineering using NUPACK, J. S. Bois for discussions on data analysis, 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 Natural Sciences and Engineering Research Council (NSERC) of Canada, 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. cgRNA project conceived by N.A.P. cgRNA mechanism invention by all coauthors. Exploratory mechanism studies by M.H.H.-H., Z.C., and J.H. in bacteria. Computational sequence design by M.H.H.-H. (splinted switch and toehold switch for bacteria, terminator switch for mammalian) and Z.C. (terminator switch for bacteria). Experimental design and data presentation approach developed by all coauthors. Presented cgRNA mechanisms prototyped and optimized in bacteria by Z.C. (terminator switch and toehold switch) and M.H.H.-H. (splinted switch and toehold switch), extended to mammalian cells by L.M.H. (terminator switch). Final data collected by M.H.H.-H. (bacterial) and L.M.H. (mammalian). Paper written by M.H.H.-H. and N.A.P. Supporting Information written by M.H.H.-H., L.M.H., and N.A.P. Paper edited and approved by all coauthors. The authors declare the following competing financial interest(s): Filed patents.

Attached Files

Published - acscentsci.9b00340.pdf

Submitted - 525857.full.pdf

Supplemental Material - oc9b00340_si_001.pdf


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