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];
- Sandia National Lab. (SNL-CA), Livermore, CA (United States)
- 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:
- 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. https://doi.org/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. https://doi.org/10.1039/C8AY02726A. https://www.osti.gov/servlets/purl/1492349.
@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},
number = 5,
volume = 11,
place = {United States},
year = {Thu Jan 03 00:00:00 EST 2019},
month = {Thu Jan 03 00:00:00 EST 2019}
}
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Works referencing / citing this record:
Anti‐CRISPRs: The natural inhibitors for CRISPR‐Cas systems
journal, May 2019
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- Animal Models and Experimental Medicine