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

Citation

Hanewich-Hollatz, Mikhail Henning (2020) Conditional Guide RNAs: Programmable Conditional Regulation of CRISPR/Cas Function via Dynamic RNA Nanotechnology. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/NS2B-DJ96. https://resolver.caltech.edu/CaltechTHESIS:11042019-135312842

Abstract

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. There are two possible logical directions for single-input cgRNAs: 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). Using an in vitro assay for cgRNA activity with synthetic trigger, in vitro transcribed cgRNA, and recombinant dCas9, we observe a conditional (ON→OFF logic) response for a set of four allosteric constitutively active cgRNAs with a median ≈6% crosstalk between noncognate cgRNA/trigger pairs. Motivated by the observed lack of conditional response of this mechanism when ported to E. coli, we describe a systematic study of unstructured sequence inserts into the standard gRNA structure and report the conditional response of a set of 34 candidate cgRNAs in living cells. Molecular mechanisms for both ON→OFF and OFF→ON cgRNAs are demonstrated in E. coli. For each mechanism, automated sequence design is performed using the reaction pathway designer within NUPACK to produce an orthogonal library of 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 ≈15-fold for a library of three orthogonal ON→OFF "splinted switch" cgRNA/trigger pairs, and ≈3-fold for a library of three orthogonal OFF→ON "toehold switch" cgRNA/trigger pairs; the median crosstalk within each library is <2% and ≈20% for the two mechanisms, respectively. By providing programmable control over both the scope and target of protein effector function, cgRNA regulators offer a promising platform for conditional gene regulation and synthetic biology.

Thesis Files