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Title: Versatile High-Throughput Fluorescence Assay for Monitoring Cas9 Activity

Abstract

Here, the RNA-guided DNA nuclease Cas9 is now widely used for the targeted modification of genomes of human cells and various organisms. Despite the extensive use of Clustered Regularly Interspaced Palindromic Repeats (CRISPR) systems for genome engineering and the rapid discovery and engineering of new CRISPR-associated nucleases, there are no high-throughput assays for measuring enzymatic activity. The current laboratory and future therapeutic uses of CRISPR technology have a significant risk of accidental exposure or clinical off-target effects, underscoring the need for therapeutically effective inhibitors of Cas9. Here, we develop a fluorescence assay for monitoring Cas9 nuclease activity and demonstrate its utility with S. pyogenes (Spy), S. aureus (Sau), and C. jejuni (Cje) Cas9. The assay was validated by quantitatively profiling the species specificity of published anti-CRISPR (Acr) proteins, confirming the reported inhibition of Spy Cas9 by AcrIIA4 and Cje Cas9 by AcrIIC1 and no inhibition of Sau Cas9 by either anti-CRISPR. To identify drug-like inhibitors, we performed a screen of 189 606 small molecules for inhibition of Spy Cas9. Of 437 hits (0.2% hit rate), six were confirmed as Cas9 inhibitors in a direct gel electrophoresis secondary assay. The high-throughput nature of this assay makes it broadly applicable for themore » discovery of additional Cas9 inhibitors or the characterization of Cas9 enzyme variants.« less

Authors:
 [1];  [1];  [1];  [1]; ORCiD logo [1]
  1. Sandia National Lab. (SNL-CA), Livermore, CA (United States)
Publication Date:
Research Org.:
Sandia National Lab. (SNL-NM), Albuquerque, NM (United States)
Sponsoring Org.:
USDOE National Nuclear Security Administration (NNSA)
OSTI Identifier:
1457411
Report Number(s):
SAND-2018-2676J
Journal ID: ISSN 0003-2700; 661465
Grant/Contract Number:  
AC04-94AL85000
Resource Type:
Accepted Manuscript
Journal Name:
Analytical Chemistry
Additional Journal Information:
Journal Volume: 90; Journal Issue: 11; Journal ID: ISSN 0003-2700
Publisher:
American Chemical Society (ACS)
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES

Citation Formats

Seamon, Kyle Jeffrey, Light, Yooli Kim, Saada, Edwin A., Schoeniger, Joseph S., and Harmon, Brooke Nicole. Versatile High-Throughput Fluorescence Assay for Monitoring Cas9 Activity. United States: N. p., 2018. Web. doi:10.1021/acs.analchem.8b01155.
Seamon, Kyle Jeffrey, Light, Yooli Kim, Saada, Edwin A., Schoeniger, Joseph S., & Harmon, Brooke Nicole. Versatile High-Throughput Fluorescence Assay for Monitoring Cas9 Activity. United States. doi:10.1021/acs.analchem.8b01155.
Seamon, Kyle Jeffrey, Light, Yooli Kim, Saada, Edwin A., Schoeniger, Joseph S., and Harmon, Brooke Nicole. Mon . "Versatile High-Throughput Fluorescence Assay for Monitoring Cas9 Activity". United States. doi:10.1021/acs.analchem.8b01155. https://www.osti.gov/servlets/purl/1457411.
@article{osti_1457411,
title = {Versatile High-Throughput Fluorescence Assay for Monitoring Cas9 Activity},
author = {Seamon, Kyle Jeffrey and Light, Yooli Kim and Saada, Edwin A. and Schoeniger, Joseph S. and Harmon, Brooke Nicole},
abstractNote = {Here, the RNA-guided DNA nuclease Cas9 is now widely used for the targeted modification of genomes of human cells and various organisms. Despite the extensive use of Clustered Regularly Interspaced Palindromic Repeats (CRISPR) systems for genome engineering and the rapid discovery and engineering of new CRISPR-associated nucleases, there are no high-throughput assays for measuring enzymatic activity. The current laboratory and future therapeutic uses of CRISPR technology have a significant risk of accidental exposure or clinical off-target effects, underscoring the need for therapeutically effective inhibitors of Cas9. Here, we develop a fluorescence assay for monitoring Cas9 nuclease activity and demonstrate its utility with S. pyogenes (Spy), S. aureus (Sau), and C. jejuni (Cje) Cas9. The assay was validated by quantitatively profiling the species specificity of published anti-CRISPR (Acr) proteins, confirming the reported inhibition of Spy Cas9 by AcrIIA4 and Cje Cas9 by AcrIIC1 and no inhibition of Sau Cas9 by either anti-CRISPR. To identify drug-like inhibitors, we performed a screen of 189 606 small molecules for inhibition of Spy Cas9. Of 437 hits (0.2% hit rate), six were confirmed as Cas9 inhibitors in a direct gel electrophoresis secondary assay. The high-throughput nature of this assay makes it broadly applicable for the discovery of additional Cas9 inhibitors or the characterization of Cas9 enzyme variants.},
doi = {10.1021/acs.analchem.8b01155},
journal = {Analytical Chemistry},
number = 11,
volume = 90,
place = {United States},
year = {2018},
month = {5}
}

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Cited by: 5 works
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Figures / Tables:

Figure 1 Figure 1: Comparison of Quenched FRET Substrates for Measuring Cas9 Activity. Schematic representations of the “same-strand” (A) and “opposite-strand” (B) substrates showing the relative locations of the fluorophore, quenchers, and HNH and RuvC cleavage sites. The fluorescence of the “same-strand” (C) and “opposite-strand” (D) substrates when incubated with an excessmore » of the wt or dCas9 and sgRNA before and after addition of a Gdn-HCl quench. The fluorescence of the “same-strand” (E) and “opposite-strand” (F) substrates when incubated with an excess of the indicated Cas9 and sgRNA after the addition of 4M Gdn-HCl quench. Data are the average and standard deviation of three replicate wells.« less

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