CaltechAUTHORS
  A Caltech Library Service

High-Performance Allosteric Conditional Guide RNAs for Mammalian Cell-Selective Regulation of CRISPR/Cas

Hochrein, Lisa M. and Li, Heyun and Pierce, Niles A. (2021) High-Performance Allosteric Conditional Guide RNAs for Mammalian Cell-Selective Regulation of CRISPR/Cas. ACS Synthetic Biology, 10 (5). pp. 964-971. ISSN 2161-5063. doi:10.1021/acssynbio.1c00037. https://resolver.caltech.edu/CaltechAUTHORS:20210503-115702708

[img]
Preview
PDF (Methods, sequences, plasmids, schematics, flow cytometry replicates, and additional studies) - Supplemental Material
See Usage Policy.

8MB

Use this Persistent URL to link to this item: https://resolver.caltech.edu/CaltechAUTHORS:20210503-115702708

Abstract

The activity of a conditional guide RNA (cgRNA) is dependent on the presence or absence of an RNA trigger, enabling cell-selective regulation of CRISPR/Cas function. cgRNAs are programmable at two levels, with the target-binding sequence controlling the target of Cas activity (edit, silence, or induce a gene of choice) and the trigger-binding sequence controlling the scope of Cas activity (subset of cells expressing the trigger RNA). Allosteric cgRNA mechanisms enable independent design of the target and trigger sequences, providing the flexibility to select the regulatory target and scope independently. Building on prior advances in dynamic RNA nanotechnology that demonstrated the cgRNA concept, here we set the goal of engineering high-performance allosteric cgRNA mechanisms for the mammalian setting, pursuing both ON → OFF logic (conditional inactivation by an RNA trigger) and OFF → ON logic (conditional activation by an RNA trigger). For each mechanism, libraries of orthogonal cgRNA/trigger pairs were designed using NUPACK. In HEK 293T cells expressing cgRNAs, triggers, and inducing dCas9: (1) a library of four ON → OFF “terminator switch” cgRNAs exhibit a median fold-change of ≈50×, a median fractional dynamic range of ≈20%, and a median crosstalk modulus of ≈9%; (2) a library of three OFF → ON “split-terminator switch” cgRNAs exhibit a median fold-change of ≈150×, a median fractional dynamic range of ≈50%, and a median crosstalk modulus of ≈4%. Further, we demonstrate that xrRNA elements that protect viral RNAs from degradation by exoribonucleases can dramatically enhance the performance of RNA synthetic biology. The high-performance allosteric cgRNAs demonstrated here for ON → OFF and OFF → ON logic in mammalian cells provide a foundation for pursuing applications of programmable cell-selective regulation.


Item Type:Article
Related URLs:
URLURL TypeDescription
https://doi.org/10.1021/acssynbio.1c00037DOIArticle
ORCID:
AuthorORCID
Pierce, Niles A.0000-0003-2367-4406
Additional Information:© 2021 American Chemical Society. Received: January 26, 2021; Published: April 30, 2021. We thank Z. Chen, M. H. Hanewich-Hollatz, J. Huang, and P. W. K. Rothemund for helpful discussions. 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 U.S. Government), by the National Aeronautics and Space Administration (7000000323), by the Rosen Bioengineering Center at Caltech, by the Beckman Institute at Caltech (Programmable Molecular Technology Center, PMTC), and by a Beckman-Gray Graduate Fellowship. The authors declare the following competing financial interest(s): Patent applications filed by the California Institute of Technology.
Group:Rosen Bioengineering Center
Funders:
Funding AgencyGrant Number
Defense Advanced Research Projects Agency (DARPA)HR0011-17-2-0008
NASA7000000323
Donna and Benjamin M. Rosen Bioengineering CenterUNSPECIFIED
Caltech Beckman InstituteUNSPECIFIED
Subject Keywords:Allosteric cgRNAs, Small conditional RNAs, Dynamic RNA nanotechnology, RNA degradation, Molecular programming, Synthetic biology
Issue or Number:5
DOI:10.1021/acssynbio.1c00037
Record Number:CaltechAUTHORS:20210503-115702708
Persistent URL:https://resolver.caltech.edu/CaltechAUTHORS:20210503-115702708
Official Citation:High-Performance Allosteric Conditional Guide RNAs for Mammalian Cell-Selective Regulation of CRISPR/Cas. Lisa M. Hochrein, Heyun Li, and Niles A. Pierce. ACS Synthetic Biology 2021 10 (5), 964-971; DOI: 10.1021/acssynbio.1c00037
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:108927
Collection:CaltechAUTHORS
Deposited By: George Porter
Deposited On:07 May 2021 21:04
Last Modified:26 May 2021 18:54

Repository Staff Only: item control page