Journal
ACS CENTRAL SCIENCE
Volume 5, Issue 7, Pages 1241-1249Publisher
AMER CHEMICAL SOC
DOI: 10.1021/acscentsci.9b00340
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Funding
- Defense Advanced Research Projects Agency [HR0011-17-2-0008]
- Caltech Center for Environmental Microbial Interactions (CEMI)
- National Institutes of Health [5T32GM112592]
- Rosen Bioengineering Center at Caltech
- Natural Sciences and Engineering Research Council (NSERC) of Canada
- National Science Foundation Molecular Programming Project [NSF-CCF-1317694]
- Professorial Fellowship at Balliol College (University of Oxford)
- Eastman Visiting Professorship at the University of Oxford
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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 a median conditional response of approximate to 4-fold for an ON -> OFF terminator switch mechanism, approximate to 15-fold for an ON -> OFF splinted switch mechanism, and approximate to 3-fold for an OFF -> ON toehold switch mechanism; the median crosstalk within each cgRNA/trigger library is < 2%, approximate to 2%, and approximate to 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 approximate to 4-fold with median crosstalk of approximate to 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.
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