Characterizing a thermostable Cas9 for bacterial genome editing and silencing

Ioannis Mougiakos, Prarthana Mohanraju, Elleke F. Bosma, Valentijn Vrouwe, Max Finger Bou, Mihris I.S. Naduthodi, Alex Gussak, Rudolf B.L. Brinkman, Richard Van Kranenburg, John Van Der Oost*

*Corresponding author for this work

Research output: Contribution to journalArticleAcademicpeer-review

29 Citations (Scopus)

Abstract

CRISPR-Cas9-based genome engineering tools have revolutionized fundamental research and biotechnological exploitation of both eukaryotes and prokaryotes. However, the mesophilic nature of the established Cas9 systems does not allow for applications that require enhanced stability, including engineering at elevated temperatures. Here we identify and characterize ThermoCas9 from the thermophilic bacterium Geobacillus thermodenitrificans T12. We show that in vitro ThermoCas9 is active between 20 and 70 °C, has stringent PAM-preference at lower temperatures, tolerates fewer spacer-protospacer mismatches than SpCas9 and its activity at elevated temperatures depends on the sgRNA-structure. We develop ThermoCas9-based engineering tools for gene deletion and transcriptional silencing at 55 °C in Bacillus smithii and for gene deletion at 37 °C in Pseudomonas putida. Altogether, our findings provide fundamental insights into a thermophilic CRISPR-Cas family member and establish a Cas9-based bacterial genome editing and silencing tool with a broad temperature range.
Original languageEnglish
Article number1647
JournalNature Communications
Volume8
Issue number1
DOIs
Publication statusPublished - 1 Dec 2017

Fingerprint

Bacterial Genomes
editing
genome
deletion
Clustered Regularly Interspaced Short Palindromic Repeats
Genes
engineering
genes
Temperature
Gene Deletion
prokaryotes
eukaryotes
pulse amplitude modulation
pseudomonas
Geobacillus
Bacillus
exploitation
spacers
Pulse amplitude modulation
bacteria

Cite this

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title = "Characterizing a thermostable Cas9 for bacterial genome editing and silencing",
abstract = "CRISPR-Cas9-based genome engineering tools have revolutionized fundamental research and biotechnological exploitation of both eukaryotes and prokaryotes. However, the mesophilic nature of the established Cas9 systems does not allow for applications that require enhanced stability, including engineering at elevated temperatures. Here we identify and characterize ThermoCas9 from the thermophilic bacterium Geobacillus thermodenitrificans T12. We show that in vitro ThermoCas9 is active between 20 and 70 °C, has stringent PAM-preference at lower temperatures, tolerates fewer spacer-protospacer mismatches than SpCas9 and its activity at elevated temperatures depends on the sgRNA-structure. We develop ThermoCas9-based engineering tools for gene deletion and transcriptional silencing at 55 °C in Bacillus smithii and for gene deletion at 37 °C in Pseudomonas putida. Altogether, our findings provide fundamental insights into a thermophilic CRISPR-Cas family member and establish a Cas9-based bacterial genome editing and silencing tool with a broad temperature range.",
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Characterizing a thermostable Cas9 for bacterial genome editing and silencing. / Mougiakos, Ioannis; Mohanraju, Prarthana; Bosma, Elleke F.; Vrouwe, Valentijn; Finger Bou, Max; Naduthodi, Mihris I.S.; Gussak, Alex; Brinkman, Rudolf B.L.; Van Kranenburg, Richard; Van Der Oost, John.

In: Nature Communications, Vol. 8, No. 1, 1647, 01.12.2017.

Research output: Contribution to journalArticleAcademicpeer-review

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AU - Mougiakos, Ioannis

AU - Mohanraju, Prarthana

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AU - Vrouwe, Valentijn

AU - Finger Bou, Max

AU - Naduthodi, Mihris I.S.

AU - Gussak, Alex

AU - Brinkman, Rudolf B.L.

AU - Van Kranenburg, Richard

AU - Van Der Oost, John

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AB - CRISPR-Cas9-based genome engineering tools have revolutionized fundamental research and biotechnological exploitation of both eukaryotes and prokaryotes. However, the mesophilic nature of the established Cas9 systems does not allow for applications that require enhanced stability, including engineering at elevated temperatures. Here we identify and characterize ThermoCas9 from the thermophilic bacterium Geobacillus thermodenitrificans T12. We show that in vitro ThermoCas9 is active between 20 and 70 °C, has stringent PAM-preference at lower temperatures, tolerates fewer spacer-protospacer mismatches than SpCas9 and its activity at elevated temperatures depends on the sgRNA-structure. We develop ThermoCas9-based engineering tools for gene deletion and transcriptional silencing at 55 °C in Bacillus smithii and for gene deletion at 37 °C in Pseudomonas putida. Altogether, our findings provide fundamental insights into a thermophilic CRISPR-Cas family member and establish a Cas9-based bacterial genome editing and silencing tool with a broad temperature range.

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