Jump to main content
Jump to site search


A universal method for sensitive and cell-free detection of CRISPR-associated nucleases

Author affiliations

Abstract

A multitude of biological applications for CRISPR-associated (Cas) nucleases have propelled the development of robust cell-based methods for quantitation of on- and off-target activities of these nucleases. However, emerging applications of these nucleases require cell-free methods that are simple, sensitive, cost effective, high throughput, multiplexable, and generalizable to all classes of Cas nucleases. Current methods for cell-free detection are cumbersome, expensive, or require sophisticated sequencing technologies, hindering their widespread application beyond the field of life sciences. Developing such cell-free assays is challenging for multiple reasons, including that Cas nucleases are single-turnover enzymes that must be present in large excess over their substrate and that different classes of Cas nucleases exhibit wildly different operating mechanisms. Here, we report the development of a cell-free method wherein Cas nuclease activity is amplified via an in vitro transcription reaction that produces a fluorescent RNA:small-molecule adduct. We demonstrate that our method is sensitive, detecting activity from low nanomolar concentrations of several families of Cas nucleases, and can be conducted in a high-throughput microplate fashion with a simple fluorescent-based readout. We provide a mathematical framework for quantifying the activities of these nucleases and demonstrate two applications of our method, namely the development of a logic circuit and the characterization of an anti-CRISPR protein. We anticipate our method will be valuable to those studying Cas nucleases and will allow the application of Cas nuclease beyond the field of life sciences.

Graphical abstract: A universal method for sensitive and cell-free detection of CRISPR-associated nucleases

Back to tab navigation

Supplementary files

Publication details

The article was received on 01 Aug 2018, accepted on 28 Dec 2018 and first published on 16 Jan 2019


Article type: Edge Article
DOI: 10.1039/C8SC03426E
Citation: Chem. Sci., 2019, Advance Article
  • Open access: Creative Commons BY-NC license
  •   Request permissions

    A universal method for sensitive and cell-free detection of CRISPR-associated nucleases

    K. J. Cox, H. K. K. Subramanian, C. C. Samaniego, E. Franco and A. Choudhary, Chem. Sci., 2019, Advance Article , DOI: 10.1039/C8SC03426E

    This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. Material from this article can be used in other publications provided that the correct acknowledgement is given with the reproduced material and it is not used for commercial purposes.

    Reproduced material should be attributed as follows:

    • For reproduction of material from NJC:
      [Original citation] - Published by The Royal Society of Chemistry (RSC) on behalf of the Centre National de la Recherche Scientifique (CNRS) and the RSC.
    • For reproduction of material from PCCP:
      [Original citation] - Published by the PCCP Owner Societies.
    • For reproduction of material from PPS:
      [Original citation] - Published by The Royal Society of Chemistry (RSC) on behalf of the European Society for Photobiology, the European Photochemistry Association, and RSC.
    • For reproduction of material from all other RSC journals:
      [Original citation] - Published by The Royal Society of Chemistry.

    Information about reproducing material from RSC articles with different licences is available on our Permission Requests page.

Search articles by author

Spotlight

Advertisements