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

Cox, Kurt J.; Subramanian, Hari K.  K.; Samaniego, Christian Cuba; Franco, Elisa; Choudhary, Amit

doi:10.1039/c8sc03426e

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

Journal Article · 16 January 2019 · Chemical Science

DOI: https://doi.org/10.1039/c8sc03426e · OSTI ID:1611416

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Broad Inst. of MIT and Harvard; Cambridge, MA (United States); DOE/OSTI
Univ. of California, Riverside, CA (United States)
Broad Inst. of MIT and Harvard; Cambridge, MA (United States)

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.

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Research Organization:: Johns Hopkins Univ., Baltimore, MD (United States)

Sponsoring Organization:: USDOE Office of Science (SC)

Grant/Contract Number:: SC0010426

OSTI ID:: 1611416

Journal Information:: Chemical Science, Journal Name: Chemical Science Journal Issue: 9 Vol. 10; ISSN 2041-6520; ISSN CSHCBM

Publisher:: Royal Society of ChemistryCopyright Statement

Country of Publication:: United States

Language:: English

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