A thermostable Cas9 with increased lifetime in human plasma

doi:10.1038/s41467-017-01408-4

Title: 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:

Harrington, Lucas B. ^[1];

^[2]; Staahl, Brett T. ^[1]; Chen, Janice S. ^[1]; Ma, Enbo ^[1]; Kyrpides, Nikos C. ^[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:: 2017-11-10

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.

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                    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.  doi:10.1038/s41467-017-01408-4.

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                    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.  doi:10.1038/s41467-017-01408-4.  https://www.osti.gov/servlets/purl/1543743.

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@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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DOI: 10.1038/s41467-017-01408-4

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Works referencing / citing this record:

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
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Title: A thermostable Cas9 with increased lifetime in human plasma

Abstract

Citation Formats

CRISPRTarget: Bioinformatic prediction and analysis of crRNA targets journal, February 2013

Processing-Independent CRISPR RNAs Limit Natural Transformation in Neisseria meningitidis journal, May 2013

Single-Stranded DNA Cleavage by Divergent CRISPR-Cas9 Enzymes journal, November 2015

Diversity and evolution of class 2 CRISPR–Cas systems journal, January 2017

Efficient Genome Editing of a Facultative Thermophile Using Mesophilic spCas9 journal, February 2017

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

Genome-scale transcriptional activation by an engineered CRISPR-Cas9 complex journal, December 2014

Viral Diversity Threshold for Adaptive Immunity in Prokaryotes journal, December 2012

A recombinant polypeptide extends the in vivo half-life of peptides and proteins in a tunable manner journal, November 2009

Loop-mediated isothermal amplification of DNA journal, June 2000

CRISPR/Cas9 in Genome Editing and Beyond journal, June 2016

Harnessing Type I and Type III CRISPR-Cas systems for genome editing journal, October 2015

Phylogeny of Cas9 determines functional exchangeability of dual-RNA and Cas9 among orthologous type II CRISPR-Cas systems journal, November 2013

Fast gapped-read alignment with Bowtie 2 journal, March 2012

Structural analysis of thermostabilizing mutations of cocaine esterase journal, April 2010

The Phyre2 web portal for protein modeling, prediction and analysis journal, May 2015

Complete genome sequence, metabolic model construction and phenotypic characterization of Geobacillus LC300, an extremely thermophilic, fast growing, xylose-utilizing bacterium journal, November 2015

Strategies for extended serum half-life of protein therapeutics journal, December 2011

Genome Editing: A New Approach to Human Therapeutics journal, January 2016

CRISPR Immunity Drives Rapid Phage Genome Evolution in Streptococcus thermophilus journal, April 2015

Rational design of a split-Cas9 enzyme complex journal, February 2015

Efficient genome engineering in human pluripotent stem cells using Cas9 from Neisseria meningitidis journal, August 2013

Classification and evolution of type II CRISPR-Cas systems journal, April 2014

How Do Thermophilic Proteins and Proteomes Withstand High Temperature? journal, July 2011

IMG: the integrated microbial genomes database and comparative analysis system journal, December 2011

CAS9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering journal, August 2013

In vivo genome editing using Staphylococcus aureus Cas9 journal, April 2015

Orthogonal Cas9 proteins for RNA-guided gene regulation and editing journal, September 2013

Internal guide RNA interactions interfere with Cas9-mediated cleavage journal, June 2016

Extremely thermophilic microorganisms as metabolic engineering platforms for production of fuels and industrial chemicals journal, November 2015

New CRISPR–Cas systems from uncultivated microbes journal, December 2016

CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III journal, March 2011

An improved zinc-finger nuclease architecture for highly specific genome editing journal, July 2007

Temperature effect on CRISPR-Cas9 mediated genome editing journal, April 2017

Efficient genome editing in the mouse brain by local delivery of engineered Cas9 ribonucleoprotein complexes journal, February 2017

Engineered CRISPR-Cas9 nucleases with altered PAM specificities journal, June 2015

Broadening the targeting range of Staphylococcus aureus CRISPR-Cas9 by modifying PAM recognition journal, November 2015

Characterization of Staphylococcus aureus Cas9: a smaller Cas9 for all-in-one adeno-associated virus delivery and paired nickase applications journal, November 2015

CRISPR Interference–Potential Application in Retinal Disease journal, March 2020

CRISPRTarget: Bioinformatic prediction and analysis of crRNA targets
journal, February 2013

Processing-Independent CRISPR RNAs Limit Natural Transformation in Neisseria meningitidis
journal, May 2013

Single-Stranded DNA Cleavage by Divergent CRISPR-Cas9 Enzymes
journal, November 2015

Diversity and evolution of class 2 CRISPR–Cas systems
journal, January 2017

Efficient Genome Editing of a Facultative Thermophile Using Mesophilic spCas9
journal, February 2017

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

Genome-scale transcriptional activation by an engineered CRISPR-Cas9 complex
journal, December 2014

Viral Diversity Threshold for Adaptive Immunity in Prokaryotes
journal, December 2012

A recombinant polypeptide extends the in vivo half-life of peptides and proteins in a tunable manner
journal, November 2009

Loop-mediated isothermal amplification of DNA
journal, June 2000

CRISPR/Cas9 in Genome Editing and Beyond
journal, June 2016

Harnessing Type I and Type III CRISPR-Cas systems for genome editing
journal, October 2015

Phylogeny of Cas9 determines functional exchangeability of dual-RNA and Cas9 among orthologous type II CRISPR-Cas systems
journal, November 2013

Fast gapped-read alignment with Bowtie 2
journal, March 2012

Structural analysis of thermostabilizing mutations of cocaine esterase
journal, April 2010

The Phyre2 web portal for protein modeling, prediction and analysis
journal, May 2015

Complete genome sequence, metabolic model construction and phenotypic characterization of Geobacillus LC300, an extremely thermophilic, fast growing, xylose-utilizing bacterium
journal, November 2015

Strategies for extended serum half-life of protein therapeutics
journal, December 2011

Genome Editing: A New Approach to Human Therapeutics
journal, January 2016

CRISPR Immunity Drives Rapid Phage Genome Evolution in Streptococcus thermophilus
journal, April 2015

Rational design of a split-Cas9 enzyme complex
journal, February 2015

Efficient genome engineering in human pluripotent stem cells using Cas9 from Neisseria meningitidis
journal, August 2013

Classification and evolution of type II CRISPR-Cas systems
journal, April 2014

How Do Thermophilic Proteins and Proteomes Withstand High Temperature?
journal, July 2011

IMG: the integrated microbial genomes database and comparative analysis system
journal, December 2011

CAS9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering
journal, August 2013

In vivo genome editing using Staphylococcus aureus Cas9
journal, April 2015

Orthogonal Cas9 proteins for RNA-guided gene regulation and editing
journal, September 2013

Internal guide RNA interactions interfere with Cas9-mediated cleavage
journal, June 2016

Extremely thermophilic microorganisms as metabolic engineering platforms for production of fuels and industrial chemicals
journal, November 2015

New CRISPR–Cas systems from uncultivated microbes
journal, December 2016

CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III
journal, March 2011

An improved zinc-finger nuclease architecture for highly specific genome editing
journal, July 2007

Temperature effect on CRISPR-Cas9 mediated genome editing
journal, April 2017

Efficient genome editing in the mouse brain by local delivery of engineered Cas9 ribonucleoprotein complexes
journal, February 2017

Engineered CRISPR-Cas9 nucleases with altered PAM specificities
journal, June 2015

Broadening the targeting range of Staphylococcus aureus CRISPR-Cas9 by modifying PAM recognition
journal, November 2015

Characterization of Staphylococcus aureus Cas9: a smaller Cas9 for all-in-one adeno-associated virus delivery and paired nickase applications
journal, November 2015

CRISPR Interference–Potential Application in Retinal Disease
journal, March 2020