DNA-guided DNA cleavage at moderate temperatures by Clostridium butyricum Argonaute

Jorrit W. Hegge; Daan C. Swarts; Stanley D. Chandradoss; Tao Ju Cui; Jeroen Kneppers; Martin Jinek; Chirlmin Joo; John Van Der Oost

doi:10.1093/nar/gkz306

DNA-guided DNA cleavage at moderate temperatures by Clostridium butyricum Argonaute

Jorrit W. Hegge, Daan C. Swarts, Stanley D. Chandradoss, Tao Ju Cui, Jeroen Kneppers, Martin Jinek, Chirlmin Joo, John Van Der Oost

Department of Physics

Research output: Contribution to journal › Article › peer-review

124 Scopus citations

Abstract

Prokaryotic Argonaute proteins (pAgos) constitute a diverse group of endonucleases of which some mediate host defense by utilizing small interfering DNA guides (siDNA) to cleave complementary invading DNA. This activity can be repurposed for programmable DNA cleavage. However, currently characterized DNA-cleaving pAgos require elevated temperatures (≥65^◦C) for their activity, making them less suitable for applications that require moderate temperatures, such as genome editing. Here, we report the functional and structural characterization of the siDNA-guided DNA-targeting pAgo from the mesophilic bacterium Clostridium butyricum (CbAgo). CbAgo displays a preference for siDNAs that have a deoxyadenosine at the 5'-end and thymidines at nucleotides 2-4. Furthermore, CbAgo mediates DNA-guided DNA cleavage of AT-rich double stranded DNA at moderate temperatures (37^◦C). This study demonstrates that certain pAgos are capable of programmable DNA cleavage at moderate temperatures and thereby expands the scope of the potential pAgo-based applications.

Original language	English
Pages (from-to)	5809-5821
Number of pages	13
Journal	Nucleic Acids Research
Volume	47
Issue number	11
DOIs	https://doi.org/10.1093/nar/gkz306
State	Published - 20 Jun 2019

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© The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.

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title = "DNA-guided DNA cleavage at moderate temperatures by Clostridium butyricum Argonaute",

abstract = "Prokaryotic Argonaute proteins (pAgos) constitute a diverse group of endonucleases of which some mediate host defense by utilizing small interfering DNA guides (siDNA) to cleave complementary invading DNA. This activity can be repurposed for programmable DNA cleavage. However, currently characterized DNA-cleaving pAgos require elevated temperatures (≥65◦C) for their activity, making them less suitable for applications that require moderate temperatures, such as genome editing. Here, we report the functional and structural characterization of the siDNA-guided DNA-targeting pAgo from the mesophilic bacterium Clostridium butyricum (CbAgo). CbAgo displays a preference for siDNAs that have a deoxyadenosine at the 5'-end and thymidines at nucleotides 2-4. Furthermore, CbAgo mediates DNA-guided DNA cleavage of AT-rich double stranded DNA at moderate temperatures (37◦C). This study demonstrates that certain pAgos are capable of programmable DNA cleavage at moderate temperatures and thereby expands the scope of the potential pAgo-based applications.",

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AU - Hegge, Jorrit W.

AU - Swarts, Daan C.

AU - Chandradoss, Stanley D.

AU - Cui, Tao Ju

AU - Kneppers, Jeroen

AU - Jinek, Martin

AU - Joo, Chirlmin

AU - Van Der Oost, John

PY - 2019/6/20

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N2 - Prokaryotic Argonaute proteins (pAgos) constitute a diverse group of endonucleases of which some mediate host defense by utilizing small interfering DNA guides (siDNA) to cleave complementary invading DNA. This activity can be repurposed for programmable DNA cleavage. However, currently characterized DNA-cleaving pAgos require elevated temperatures (≥65◦C) for their activity, making them less suitable for applications that require moderate temperatures, such as genome editing. Here, we report the functional and structural characterization of the siDNA-guided DNA-targeting pAgo from the mesophilic bacterium Clostridium butyricum (CbAgo). CbAgo displays a preference for siDNAs that have a deoxyadenosine at the 5'-end and thymidines at nucleotides 2-4. Furthermore, CbAgo mediates DNA-guided DNA cleavage of AT-rich double stranded DNA at moderate temperatures (37◦C). This study demonstrates that certain pAgos are capable of programmable DNA cleavage at moderate temperatures and thereby expands the scope of the potential pAgo-based applications.

AB - Prokaryotic Argonaute proteins (pAgos) constitute a diverse group of endonucleases of which some mediate host defense by utilizing small interfering DNA guides (siDNA) to cleave complementary invading DNA. This activity can be repurposed for programmable DNA cleavage. However, currently characterized DNA-cleaving pAgos require elevated temperatures (≥65◦C) for their activity, making them less suitable for applications that require moderate temperatures, such as genome editing. Here, we report the functional and structural characterization of the siDNA-guided DNA-targeting pAgo from the mesophilic bacterium Clostridium butyricum (CbAgo). CbAgo displays a preference for siDNAs that have a deoxyadenosine at the 5'-end and thymidines at nucleotides 2-4. Furthermore, CbAgo mediates DNA-guided DNA cleavage of AT-rich double stranded DNA at moderate temperatures (37◦C). This study demonstrates that certain pAgos are capable of programmable DNA cleavage at moderate temperatures and thereby expands the scope of the potential pAgo-based applications.

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DNA-guided DNA cleavage at moderate temperatures by Clostridium butyricum Argonaute

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