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Title: Ultrasensitive multi-species detection of CRISPR-Cas9 by a portable centrifugal microfluidic platform

Abstract

The discovery of the RNA-guided DNA nuclease CRISPR-Cas9 has enabled the targeted editing of genomes from diverse organisms, but the permanent and inheritable nature of genome modification also poses immense risks. The potential for accidental exposure, malicious use, or undesirable persistence of Cas9 therapeutics and off-target genome effects highlight the need for detection assays. Here we report a centrifugal microfluidic platform for the measurement of both Cas9 protein levels and nuclease activity. Because Cas9 from many bacterial species have been adapted for biotechnology applications, we developed the capability to detect Cas9 from the widely-used S. pyogenes, as well as S. aureus, N. meningitidis, and S. thermophilus using commercially-available antibodies. Further, we show that the phage-derived anti-CRISPR protein AcrIIC1, which binds to Cas9 from several species, can be used as a capture reagent to broaden the species range of detection. Finally, as genome modification generally requires Cas9 nuclease activity, a fluorescence-based sedimentation nuclease assay was also incorporated to allow the sensitive and simultaneous measurement of both Cas9 protein and activity in a single biological sample.

Authors:
 [1];  [1];  [1];  [1];  [1];  [1]; ORCiD logo [1];  [2];  [1]
  1. Sandia National Lab. (SNL-CA), Livermore, CA (United States)
  2. Rochester Inst. of Technology, Rochester, NY (United States)
Publication Date:
Research Org.:
Sandia National Lab. (SNL-CA), Livermore, CA (United States)
Sponsoring Org.:
USDOE National Nuclear Security Administration (NNSA)
OSTI Identifier:
1492349
Alternate Identifier(s):
OSTI ID: 1491208
Report Number(s):
SAND-2019-0320J
Journal ID: ISSN 1759-9660; 671483
Grant/Contract Number:  
AC04-94AL85000; 200186; 204977; NA0003525
Resource Type:
Journal Article: Accepted Manuscript
Journal Name:
Analytical Methods
Additional Journal Information:
Journal Volume: 11; Journal Issue: 5; Journal ID: ISSN 1759-9660
Publisher:
Royal Society of Chemistry
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES

Citation Formats

Phaneuf, Christopher R., Seamon, Kyle J., Eckles, Tyler P., Sinha, Anchal, Schoeniger, Joseph S., Harmon, Brooke, Meagher, Robert J., Abhyankar, Vinay V., and Koh, Chung-Yan. Ultrasensitive multi-species detection of CRISPR-Cas9 by a portable centrifugal microfluidic platform. United States: N. p., 2019. Web. doi:10.1039/C8AY02726A.
Phaneuf, Christopher R., Seamon, Kyle J., Eckles, Tyler P., Sinha, Anchal, Schoeniger, Joseph S., Harmon, Brooke, Meagher, Robert J., Abhyankar, Vinay V., & Koh, Chung-Yan. Ultrasensitive multi-species detection of CRISPR-Cas9 by a portable centrifugal microfluidic platform. United States. doi:10.1039/C8AY02726A.
Phaneuf, Christopher R., Seamon, Kyle J., Eckles, Tyler P., Sinha, Anchal, Schoeniger, Joseph S., Harmon, Brooke, Meagher, Robert J., Abhyankar, Vinay V., and Koh, Chung-Yan. Thu . "Ultrasensitive multi-species detection of CRISPR-Cas9 by a portable centrifugal microfluidic platform". United States. doi:10.1039/C8AY02726A.
@article{osti_1492349,
title = {Ultrasensitive multi-species detection of CRISPR-Cas9 by a portable centrifugal microfluidic platform},
author = {Phaneuf, Christopher R. and Seamon, Kyle J. and Eckles, Tyler P. and Sinha, Anchal and Schoeniger, Joseph S. and Harmon, Brooke and Meagher, Robert J. and Abhyankar, Vinay V. and Koh, Chung-Yan},
abstractNote = {The discovery of the RNA-guided DNA nuclease CRISPR-Cas9 has enabled the targeted editing of genomes from diverse organisms, but the permanent and inheritable nature of genome modification also poses immense risks. The potential for accidental exposure, malicious use, or undesirable persistence of Cas9 therapeutics and off-target genome effects highlight the need for detection assays. Here we report a centrifugal microfluidic platform for the measurement of both Cas9 protein levels and nuclease activity. Because Cas9 from many bacterial species have been adapted for biotechnology applications, we developed the capability to detect Cas9 from the widely-used S. pyogenes, as well as S. aureus, N. meningitidis, and S. thermophilus using commercially-available antibodies. Further, we show that the phage-derived anti-CRISPR protein AcrIIC1, which binds to Cas9 from several species, can be used as a capture reagent to broaden the species range of detection. Finally, as genome modification generally requires Cas9 nuclease activity, a fluorescence-based sedimentation nuclease assay was also incorporated to allow the sensitive and simultaneous measurement of both Cas9 protein and activity in a single biological sample.},
doi = {10.1039/C8AY02726A},
journal = {Analytical Methods},
issn = {1759-9660},
number = 5,
volume = 11,
place = {United States},
year = {2019},
month = {1}
}

Journal Article:
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Works referenced in this record:

Centrifugal Microfluidic Platform for Ultrasensitive Detection of Botulinum Toxin
journal, December 2014

  • Koh, Chung-Yan; Schaff, Ulrich Y.; Piccini, Matthew E.
  • Analytical Chemistry, Vol. 87, Issue 2, p. 922-928
  • DOI: 10.1021/ac504054u

A Programmable Dual-RNA-Guided DNA Endonuclease in Adaptive Bacterial Immunity
journal, June 2012


One-Step Generation of Mice Carrying Mutations in Multiple Genes by CRISPR/Cas-Mediated Genome Engineering
journal, May 2013


RNA-Guided Human Genome Engineering via Cas9
journal, January 2013


Multiplex Genome Engineering Using CRISPR/Cas Systems
journal, January 2013