A thermostable Cas9 with increased lifetime in human plasma
Abstract
CRISPR-Cas9 is a powerful technology that has enabled genome editing in a wide range of species. However, the currently developed Cas9 homologs all originate from mesophilic bacteria, making them susceptible to degradation and unsuitable for applications requiring cleavage at elevated temperatures. Here, we show that the Cas9 protein from the thermophilic bacterium Geobacillus stearothermophilus (GeoCas9) catalyzes RNA-guided DNA cleavage at elevated temperatures. GeoCas9 is active at temperatures up to 70 °C, compared to 45 °C for Streptococcus pyogenes Cas9 (SpyCas9), which expands the temperature range for CRISPR-Cas9 applications. We also found that GeoCas9 is an effective tool for editing mammalian genomes when delivered as a ribonucleoprotein (RNP) complex. Together with an increased lifetime in human plasma, the thermostable GeoCas9 provides the foundation for improved RNP delivery in vivo and expands the temperature range of CRISPR-Cas9.
- Authors:
-
[2];
[3]
- Univ. of California, Berkeley, CA (United States)
- USDOE Joint Genome Institute (JGI), Walnut Creek, CA (United States)
- Univ. of California, Berkeley, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States)
- Publication Date:
- Research Org.:
- Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). National Energy Research Scientific Computing Center (NERSC)
- Sponsoring Org.:
- USDOE
- OSTI Identifier:
- 1543743
- Resource Type:
- Accepted Manuscript
- Journal Name:
- Nature Communications
- Additional Journal Information:
- Journal Volume: 8; Journal Issue: 1; Journal ID: ISSN 2041-1723
- Publisher:
- Nature Publishing Group
- Country of Publication:
- United States
- Language:
- English
- Subject:
- 59 BASIC BIOLOGICAL SCIENCES; Science & Technology; Other Topics
Citation Formats
Harrington, Lucas B., Paez-Espino, David, Staahl, Brett T., Chen, Janice S., Ma, Enbo, Kyrpides, Nikos C., and Doudna, Jennifer A. A thermostable Cas9 with increased lifetime in human plasma. United States: N. p., 2017.
Web. doi:10.1038/s41467-017-01408-4.
Harrington, Lucas B., Paez-Espino, David, Staahl, Brett T., Chen, Janice S., Ma, Enbo, Kyrpides, Nikos C., & Doudna, Jennifer A. A thermostable Cas9 with increased lifetime in human plasma. United States. https://doi.org/10.1038/s41467-017-01408-4
Harrington, Lucas B., Paez-Espino, David, Staahl, Brett T., Chen, Janice S., Ma, Enbo, Kyrpides, Nikos C., and Doudna, Jennifer A. Fri .
"A thermostable Cas9 with increased lifetime in human plasma". United States. https://doi.org/10.1038/s41467-017-01408-4. https://www.osti.gov/servlets/purl/1543743.
@article{osti_1543743,
title = {A thermostable Cas9 with increased lifetime in human plasma},
author = {Harrington, Lucas B. and Paez-Espino, David and Staahl, Brett T. and Chen, Janice S. and Ma, Enbo and Kyrpides, Nikos C. and Doudna, Jennifer A.},
abstractNote = {CRISPR-Cas9 is a powerful technology that has enabled genome editing in a wide range of species. However, the currently developed Cas9 homologs all originate from mesophilic bacteria, making them susceptible to degradation and unsuitable for applications requiring cleavage at elevated temperatures. Here, we show that the Cas9 protein from the thermophilic bacterium Geobacillus stearothermophilus (GeoCas9) catalyzes RNA-guided DNA cleavage at elevated temperatures. GeoCas9 is active at temperatures up to 70 °C, compared to 45 °C for Streptococcus pyogenes Cas9 (SpyCas9), which expands the temperature range for CRISPR-Cas9 applications. We also found that GeoCas9 is an effective tool for editing mammalian genomes when delivered as a ribonucleoprotein (RNP) complex. Together with an increased lifetime in human plasma, the thermostable GeoCas9 provides the foundation for improved RNP delivery in vivo and expands the temperature range of CRISPR-Cas9.},
doi = {10.1038/s41467-017-01408-4},
journal = {Nature Communications},
number = 1,
volume = 8,
place = {United States},
year = {2017},
month = {11}
}
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Works referenced in this record:
CRISPRTarget: Bioinformatic prediction and analysis of crRNA targets
journal, February 2013
- Biswas, Ambarish; Gagnon, Joshua N.; Brouns, Stan J. J.
- RNA Biology, Vol. 10, Issue 5
Processing-Independent CRISPR RNAs Limit Natural Transformation in Neisseria meningitidis
journal, May 2013
- Zhang, Yan; Heidrich, Nadja; Ampattu, Biju Joseph
- Molecular Cell, Vol. 50, Issue 4
Single-Stranded DNA Cleavage by Divergent CRISPR-Cas9 Enzymes
journal, November 2015
- Ma, Enbo; Harrington, Lucas B.; O’Connell, Mitchell R.
- Molecular Cell, Vol. 60, Issue 3
Temperature effect on CRISPR-Cas9 mediated genome editing
journal, April 2017
- Xiang, Guanghai; Zhang, Xingying; An, Chenrui
- Journal of Genetics and Genomics, Vol. 44, Issue 4
Diversity and evolution of class 2 CRISPR–Cas systems
journal, January 2017
- Shmakov, Sergey; Smargon, Aaron; Scott, David
- Nature Reviews Microbiology, Vol. 15, Issue 3
Genome-scale transcriptional activation by an engineered CRISPR-Cas9 complex
journal, December 2014
- Konermann, Silvana; Brigham, Mark D.; Trevino, Alexandro E.
- Nature, Vol. 517, Issue 7536
Viral Diversity Threshold for Adaptive Immunity in Prokaryotes
journal, December 2012
- Weinberger, Ariel D.; Wolf, Yuri I.; Lobkovsky, Alexander E.
- mBio, Vol. 3, Issue 6
A recombinant polypeptide extends the in vivo half-life of peptides and proteins in a tunable manner
journal, November 2009
- Schellenberger, Volker; Wang, Chia-wei; Geething, Nathan C.
- Nature Biotechnology, Vol. 27, Issue 12
Loop-mediated isothermal amplification of DNA
journal, June 2000
- Notomi, T.
- Nucleic Acids Research, Vol. 28, Issue 12
Broadening the targeting range of Staphylococcus aureus CRISPR-Cas9 by modifying PAM recognition
journal, November 2015
- Kleinstiver, Benjamin P.; Prew, Michelle S.; Tsai, Shengdar Q.
- Nature Biotechnology, Vol. 33, Issue 12
CRISPR/Cas9 in Genome Editing and Beyond
journal, June 2016
- Wang, Haifeng; La Russa, Marie; Qi, Lei S.
- Annual Review of Biochemistry, Vol. 85, Issue 1
Harnessing Type I and Type III CRISPR-Cas systems for genome editing
journal, October 2015
- Li, Yingjun; Pan, Saifu; Zhang, Yan
- Nucleic Acids Research, Vol. 44, Issue 4
Phylogeny of Cas9 determines functional exchangeability of dual-RNA and Cas9 among orthologous type II CRISPR-Cas systems
journal, November 2013
- Fonfara, Ines; Le Rhun, Anaïs; Chylinski, Krzysztof
- Nucleic Acids Research, Vol. 42, Issue 4
Structural analysis of thermostabilizing mutations of cocaine esterase
journal, April 2010
- Narasimhan, Diwahar; Nance, Mark R.; Gao, Daquan
- Protein Engineering, Design and Selection, Vol. 23, Issue 7
Efficient genome editing in the mouse brain by local delivery of engineered Cas9 ribonucleoprotein complexes
journal, February 2017
- Staahl, Brett T.; Benekareddy, Madhurima; Coulon-Bainier, Claire
- Nature Biotechnology, Vol. 35, Issue 5
The Phyre2 web portal for protein modeling, prediction and analysis
journal, May 2015
- Kelley, Lawrence A.; Mezulis, Stefans; Yates, Christopher M.
- Nature Protocols, Vol. 10, Issue 6
Complete genome sequence, metabolic model construction and phenotypic characterization of Geobacillus LC300, an extremely thermophilic, fast growing, xylose-utilizing bacterium
journal, November 2015
- Cordova, Lauren T.; Long, Christopher P.; Venkataramanan, Keerthi P.
- Metabolic Engineering, Vol. 32
Strategies for extended serum half-life of protein therapeutics
journal, December 2011
- Kontermann, Roland E.
- Current Opinion in Biotechnology, Vol. 22, Issue 6
Genome Editing: A New Approach to Human Therapeutics
journal, January 2016
- Porteus, Matthew
- Annual Review of Pharmacology and Toxicology, Vol. 56, Issue 1
CRISPR Immunity Drives Rapid Phage Genome Evolution in Streptococcus thermophilus
journal, April 2015
- Paez-Espino, David; Sharon, Itai; Morovic, Wesley
- mBio, Vol. 6, Issue 2
Efficient genome engineering in human pluripotent stem cells using Cas9 from Neisseria meningitidis
journal, August 2013
- Hou, Z.; Zhang, Y.; Propson, N. E.
- Proceedings of the National Academy of Sciences, Vol. 110, Issue 39
Classification and evolution of type II CRISPR-Cas systems
journal, April 2014
- Chylinski, Krzysztof; Makarova, Kira S.; Charpentier, Emmanuelle
- Nucleic Acids Research, Vol. 42, Issue 10
IMG: the integrated microbial genomes database and comparative analysis system
journal, December 2011
- Markowitz, V. M.; Chen, I. -M. A.; Palaniappan, K.
- Nucleic Acids Research, Vol. 40, Issue D1
Characterization of Staphylococcus aureus Cas9: a smaller Cas9 for all-in-one adeno-associated virus delivery and paired nickase applications
journal, November 2015
- Friedland, Ari E.; Baral, Reshica; Singhal, Pankhuri
- Genome Biology, Vol. 16, Issue 1
Engineered CRISPR-Cas9 nucleases with altered PAM specificities
journal, June 2015
- Kleinstiver, Benjamin P.; Prew, Michelle S.; Tsai, Shengdar Q.
- Nature, Vol. 523, Issue 7561
Orthogonal Cas9 proteins for RNA-guided gene regulation and editing
journal, September 2013
- Esvelt, Kevin M.; Mali, Prashant; Braff, Jonathan L.
- Nature Methods, Vol. 10, Issue 11
CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III
journal, March 2011
- Deltcheva, Elitza; Chylinski, Krzysztof; Sharma, Cynthia M.
- Nature, Vol. 471, Issue 7340
Efficient Genome Editing of a Facultative Thermophile Using Mesophilic spCas9
journal, February 2017
- Mougiakos, Ioannis; Bosma, Elleke F.; Weenink, Koen
- ACS Synthetic Biology, Vol. 6, Issue 5
A Programmable Dual-RNA-Guided DNA Endonuclease in Adaptive Bacterial Immunity
journal, June 2012
- Jinek, M.; Chylinski, K.; Fonfara, I.
- Science, Vol. 337, Issue 6096, p. 816-821
Fast gapped-read alignment with Bowtie 2
journal, March 2012
- Langmead, Ben; Salzberg, Steven L.
- Nature Methods, Vol. 9, Issue 4
Rational design of a split-Cas9 enzyme complex
journal, February 2015
- Wright, Addison V.; Sternberg, Samuel H.; Taylor, David W.
- Proceedings of the National Academy of Sciences, Vol. 112, Issue 10
How Do Thermophilic Proteins and Proteomes Withstand High Temperature?
journal, July 2011
- Sawle, Lucas; Ghosh, Kingshuk
- Biophysical Journal, Vol. 101, Issue 1
CAS9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering
journal, August 2013
- Mali, Prashant; Aach, John; Stranges, P. Benjamin
- Nature Biotechnology, Vol. 31, Issue 9
In vivo genome editing using Staphylococcus aureus Cas9
journal, April 2015
- Ran, F. Ann; Cong, Le; Yan, Winston X.
- Nature, Vol. 520, Issue 7546
An improved zinc-finger nuclease architecture for highly specific genome editing
journal, July 2007
- Miller, Jeffrey C.; Holmes, Michael C.; Wang, Jianbin
- Nature Biotechnology, Vol. 25, Issue 7
Internal guide RNA interactions interfere with Cas9-mediated cleavage
journal, June 2016
- Thyme, Summer B.; Akhmetova, Laila; Montague, Tessa G.
- Nature Communications, Vol. 7, Issue 1
Extremely thermophilic microorganisms as metabolic engineering platforms for production of fuels and industrial chemicals
journal, November 2015
- Zeldes, Benjamin M.; Keller, Matthew W.; Loder, Andrew J.
- Frontiers in Microbiology, Vol. 6
New CRISPR–Cas systems from uncultivated microbes
journal, December 2016
- Burstein, David; Harrington, Lucas B.; Strutt, Steven C.
- Nature, Vol. 542, Issue 7640
A Highly Resistant Thermophilic Organism
journal, January 1920
- Donk, P. J.
- Journal of Bacteriology, Vol. 5, Issue 4
Works referencing / citing this record:
Under Diffusion Control: from Structuring Matter to Directional Motion
journal, June 2018
- Cera, Luca; Schalley, Christoph A.
- Advanced Materials, Vol. 30, Issue 38
Targeted gene disruption by CRISPR/xCas9 system in Drosophila melanogaster
journal, May 2020
- Ni, Xu‐yang; Zhou, Zhen‐dong; Huang, Jia
- Archives of Insect Biochemistry and Physiology, Vol. 104, Issue 1
Building a genome engineering toolbox in nonmodel prokaryotic microbes
journal, May 2018
- Freed, Emily; Fenster, Jacob; Smolinski, Sharon L.
- Biotechnology and Bioengineering, Vol. 115, Issue 9
Peculiarities and biotechnological potential of environmental adaptation by Geobacillus species
journal, October 2018
- Suzuki, Hirokazu
- Applied Microbiology and Biotechnology, Vol. 102, Issue 24
Repurposing CRISPR-Cas12b for mammalian genome engineering
journal, November 2018
- Teng, Fei; Cui, Tongtong; Feng, Guihai
- Cell Discovery, Vol. 4, Issue 1
Continuous evolution of SpCas9 variants compatible with non-G PAMs
journal, February 2020
- Miller, Shannon M.; Wang, Tina; Randolph, Peyton B.
- Nature Biotechnology, Vol. 38, Issue 4
Ultrasensitive multi-species detection of CRISPR-Cas9 by a portable centrifugal microfluidic platform
journal, January 2019
- Phaneuf, Christopher R.; Seamon, Kyle J.; Eckles, Tyler P.
- Analytical Methods, Vol. 11, Issue 5
Editor's cut: DNA cleavage by CRISPR RNA-guided nucleases Cas9 and Cas12a
journal, December 2019
- Swartjes, Thomas; Staals, Raymond H. J.; van der Oost, John
- Biochemical Society Transactions, Vol. 48, Issue 1
Nucleic acid cleavage with a hyperthermophilic Cas9 from an uncultured Ignavibacterium
journal, October 2019
- Schmidt, Stephanie Tzouanas; Yu, Feiqiao Brian; Blainey, Paul C.
- Proceedings of the National Academy of Sciences, Vol. 116, Issue 46
DNA targeting by Clostridium cellulolyticum CRISPR–Cas9 Type II-C system
journal, January 2020
- Fedorova, Iana; Arseniev, Anatolii; Selkova, Polina
- Nucleic Acids Research, Vol. 48, Issue 4
CRISPR-Cas9: A cornerstone for the evolution of precision medicine
journal, July 2018
- Razzouk, Sleiman
- Annals of Human Genetics, Vol. 82, Issue 6
Minimal PAM specificity of a highly similar SpCas9 ortholog
journal, October 2018
- Chatterjee, Pranam; Jakimo, Noah; Jacobson, Joseph M.
- Science Advances, Vol. 4, Issue 10
Programmed DNA destruction by miniature CRISPR-Cas14 enzymes
journal, October 2018
- Harrington, Lucas B.; Burstein, David; Chen, Janice S.
- Science, Vol. 362, Issue 6416
A study on endonuclease BspD6I and its stimulus-responsive switching by modified oligonucleotides
journal, November 2018
- Abrosimova, Liudmila A.; Migur, Anzhela Yu.; Kubareva, Elena A.
- PLOS ONE, Vol. 13, Issue 11
CRISPR Interference–Potential Application in Retinal Disease
journal, March 2020
- Peddle, Caroline F.; Fry, Lewis E.; McClements, Michelle E.
- International Journal of Molecular Sciences, Vol. 21, Issue 7