Programmable Base Editing in Mycobacterium tuberculosis Using an Engineered CRISPR RNA-Guided Cytidine Deaminase

Multidrug-resistant Mycobacterium tuberculosis (Mtb) infection seriously endangers global human health, creating an urgent need for new treatment strategies. Efficient genome editing tools can facilitate identification of key genes and pathways involved in bacterial physiology, pathogenesis, and dru...

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Main Authors: Xin-Yuan Ding, Si-Shang Li, Yi-Man Geng, Mei-Yi Yan, Guo-Bao Li, Guo-Liang Zhang, Yi-Cheng Sun
Format: Article
Language:English
Published: Frontiers Media S.A. 2021-12-01
Series:Frontiers in Genome Editing
Subjects:
Online Access:https://www.frontiersin.org/articles/10.3389/fgeed.2021.734436/full
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author Xin-Yuan Ding
Si-Shang Li
Yi-Man Geng
Mei-Yi Yan
Guo-Bao Li
Guo-Liang Zhang
Yi-Cheng Sun
author_facet Xin-Yuan Ding
Si-Shang Li
Yi-Man Geng
Mei-Yi Yan
Guo-Bao Li
Guo-Liang Zhang
Yi-Cheng Sun
author_sort Xin-Yuan Ding
collection DOAJ
description Multidrug-resistant Mycobacterium tuberculosis (Mtb) infection seriously endangers global human health, creating an urgent need for new treatment strategies. Efficient genome editing tools can facilitate identification of key genes and pathways involved in bacterial physiology, pathogenesis, and drug resistance mechanisms, and thus contribute to the development of novel treatments for drug-resistant tuberculosis. Here, we report a two-plasmid system, MtbCBE, used to inactivate genes and introduce point mutations in Mtb. In this system, the assistant plasmid pRecX-NucSE107A expresses RecX and NucSE107A to repress RecA-dependent and NucS-dependent DNA repair systems, and the base editor plasmid pCBE expresses a fusion protein combining cytidine deaminase APOBEC1, Cas9 nickase (nCas9), and uracil DNA glycosylase inhibitor (UGI). Together, the two plasmids enabled efficient G:C to A:T base pair conversion at desired sites in the Mtb genome. The successful development of a base editing system will facilitate elucidation of the molecular mechanisms underlying Mtb pathogenesis and drug resistance and provide critical inspiration for the development of base editing tools in other microbes.
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spelling doaj.art-75994ec9eeba4a85a9eb8d6c2919e6002022-12-21T21:35:22ZengFrontiers Media S.A.Frontiers in Genome Editing2673-34392021-12-01310.3389/fgeed.2021.734436734436Programmable Base Editing in Mycobacterium tuberculosis Using an Engineered CRISPR RNA-Guided Cytidine DeaminaseXin-Yuan Ding0Si-Shang Li1Yi-Man Geng2Mei-Yi Yan3Guo-Bao Li4Guo-Liang Zhang5Yi-Cheng Sun6NHC Key Laboratory of Systems Biology of Pathogens, Institute of Pathogen Biology, Center for Tuberculosis Research, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, ChinaNHC Key Laboratory of Systems Biology of Pathogens, Institute of Pathogen Biology, Center for Tuberculosis Research, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, ChinaDepartment of Clinical Laboratory, Henan Provincial People’s Hospital, People’s Hospital of Zhengzhou University, Zhengzhou, ChinaNHC Key Laboratory of Systems Biology of Pathogens, Institute of Pathogen Biology, Center for Tuberculosis Research, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, ChinaNational Clinical Research Center for Infectious Diseases, Shenzhen Third People’s Hospital, Southern University of Science and Technology, Shenzhen, ChinaNational Clinical Research Center for Infectious Diseases, Shenzhen Third People’s Hospital, Southern University of Science and Technology, Shenzhen, ChinaNHC Key Laboratory of Systems Biology of Pathogens, Institute of Pathogen Biology, Center for Tuberculosis Research, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, ChinaMultidrug-resistant Mycobacterium tuberculosis (Mtb) infection seriously endangers global human health, creating an urgent need for new treatment strategies. Efficient genome editing tools can facilitate identification of key genes and pathways involved in bacterial physiology, pathogenesis, and drug resistance mechanisms, and thus contribute to the development of novel treatments for drug-resistant tuberculosis. Here, we report a two-plasmid system, MtbCBE, used to inactivate genes and introduce point mutations in Mtb. In this system, the assistant plasmid pRecX-NucSE107A expresses RecX and NucSE107A to repress RecA-dependent and NucS-dependent DNA repair systems, and the base editor plasmid pCBE expresses a fusion protein combining cytidine deaminase APOBEC1, Cas9 nickase (nCas9), and uracil DNA glycosylase inhibitor (UGI). Together, the two plasmids enabled efficient G:C to A:T base pair conversion at desired sites in the Mtb genome. The successful development of a base editing system will facilitate elucidation of the molecular mechanisms underlying Mtb pathogenesis and drug resistance and provide critical inspiration for the development of base editing tools in other microbes.https://www.frontiersin.org/articles/10.3389/fgeed.2021.734436/fullmycobacteriacytidine deaminasegenome editingCRISPRbase editing
spellingShingle Xin-Yuan Ding
Si-Shang Li
Yi-Man Geng
Mei-Yi Yan
Guo-Bao Li
Guo-Liang Zhang
Yi-Cheng Sun
Programmable Base Editing in Mycobacterium tuberculosis Using an Engineered CRISPR RNA-Guided Cytidine Deaminase
Frontiers in Genome Editing
mycobacteria
cytidine deaminase
genome editing
CRISPR
base editing
title Programmable Base Editing in Mycobacterium tuberculosis Using an Engineered CRISPR RNA-Guided Cytidine Deaminase
title_full Programmable Base Editing in Mycobacterium tuberculosis Using an Engineered CRISPR RNA-Guided Cytidine Deaminase
title_fullStr Programmable Base Editing in Mycobacterium tuberculosis Using an Engineered CRISPR RNA-Guided Cytidine Deaminase
title_full_unstemmed Programmable Base Editing in Mycobacterium tuberculosis Using an Engineered CRISPR RNA-Guided Cytidine Deaminase
title_short Programmable Base Editing in Mycobacterium tuberculosis Using an Engineered CRISPR RNA-Guided Cytidine Deaminase
title_sort programmable base editing in mycobacterium tuberculosis using an engineered crispr rna guided cytidine deaminase
topic mycobacteria
cytidine deaminase
genome editing
CRISPR
base editing
url https://www.frontiersin.org/articles/10.3389/fgeed.2021.734436/full
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