PAM-Expanded Streptococcus thermophilus Cas9 C-to-T and C-to-G Base Editors for Programmable Base Editing in Mycobacteria

doi:10.1016/j.eng.2022.02.013

	PAM-Expanded Streptococcus thermophilus Cas9 C-to-T and C-to-G Base Editors for Programmable Base Editing in Mycobacteria
	Zhang, Hongyuan1,2 ; Zhang, Yifei1,2 ; Wang, Wei-Xiao 3; Chen, Weizhong1 ; Zhang, Xia 4; Huang, Xingxu5,6 ; Chen, Wei 3; Ji, Quanjiang1,5,6
	2022-08
发表期刊	ENGINEERING (IF:10.1[JCR-2023],11.6[5-Year])
ISSN	2095-8099
DOI	10.1016/j.eng.2022.02.013
摘要	New therapeutic strategies for the rapid and effective treatment of drug-resistant tuberculosis are highly desirable, and their development can be drastically accelerated by facile genetic manipulation methods in Mycobacterium tuberculosis (M. tuberculosis). Clustered regularly interspaced short palindromic repeat (CRISPR) base editors allow for rapid, robust, and programmed single-base substitutions and gene inactivation, yet no such systems are currently available in M. tuberculosis. By screening distinct CRISPR base editors, we discovered that only the unusual Streptococcus thermophilus CRISPR associated protein 9 (St1Cas9) cytosine base editor (CBE)—but not the widely used Streptococcus pyogenes Cas9 (SpCas9) or Lachnospiraceae bacterium Cpf1 (LbCpf1) CBEs—is active in mycobacteria. Despite the notable C-to-T conversions, a high proportion of undesired byproducts exists with St1Cas9 CBE. We therefore engineered St1Cas9 CBE by means of uracil DNA glycosylase inhibitor (UGI) or uracil DNA glycosylase (UNG) fusion, yielding two new base editors (CTBE and CGBE) capable of C-to-T or C-to-G conversions with dramatically enhanced editing product purity and multiplexed editing capacity in Mycobacterium smegmatis (M. smegmatis). Because wild-type St1Cas9 recognizes a relatively strict protospacer adjacent motif (PAM) sequence for DNA targeting, we engineered a PAM-expanded St1Cas9 variant by means of structure-guided protein engineering for the base editors, substantially broadening the targeting scope. We first developed and characterized CTBE and CGBE in M. smegmatis, and then applied CTBE for genome editing in M. tuberculosis. Our approaches significantly reduce the efforts and time needed for precise genetic manipulation and will facilitate functional genomics, antibiotic-resistant mechanism study, and drug-target exploration in M. tuberculosis and related organisms. © 2022 THE AUTHORS
关键词	Aromatic compounds Bacteria C (programming language) Genes Genetic engineering Proteins Tubes (components) Base editing Cas9 Clustered regularly interspaced short palindromic repeat Genetic manipulations Genome editing Mycobacterium smegmatis Mycobacterium tuberculosis Palindromic Streptococcus thermophilus Uracil-DNA glycosylase
收录类别	EI ; SCIE
语种	英语
出版者	Elsevier Ltd
EI入藏号	20223112474090
EI主题词	DNA
EI分类号	461.2 Biological Materials and Tissue Engineering ; 461.8.1 Genetic Engineering ; 619.1 Pipe, Piping and Pipelines ; 723.1.1 Computer Programming Languages ; 804.1 Organic Compounds
原始文献类型	Article in Press
引用统计	正在获取...
文献类型	期刊论文
条目标识符	https://kms.shanghaitech.edu.cn/handle/2MSLDSTB/211757
专题	物质科学与技术学院_PI研究组_季泉江组生命科学与技术学院_PI研究组_黄行许组物质科学与技术学院_博士生
通讯作者	Chen, Weizhong; Ji, Quanjiang
作者单位	1.School of Physical Science and Technology, ShanghaiTech University, Shanghai; 201210, China; 2.University of Chinese Academy of Sciences, Beijing; 100049, China; 3.Clinical Research Center, the Second Hospital of Nanjing, Nanjing University of Chinese Medicine, Nanjing; 210003, China; 4.Department of Tuberculosis, the Second Hospital of Nanjing, Nanjing University of Chinese Medicine, Nanjing; 210003, China; 5.Gene Editing Center, School of Life Science and Technology, ShanghaiTech University, Shanghai; 201210, China; 6.Guangzhou Laboratory, Guangzhou; 510120, China
第一作者单位	物质科学与技术学院
通讯作者单位	物质科学与技术学院; 生命科学与技术学院
第一作者的第一单位	物质科学与技术学院
推荐引用方式 GB/T 7714	Zhang, Hongyuan,Zhang, Yifei,Wang, Wei-Xiao,et al. PAM-Expanded Streptococcus thermophilus Cas9 C-to-T and C-to-G Base Editors for Programmable Base Editing in Mycobacteria[J]. ENGINEERING,2022.
APA	Zhang, Hongyuan.,Zhang, Yifei.,Wang, Wei-Xiao.,Chen, Weizhong.,Zhang, Xia.,...&Ji, Quanjiang.(2022).PAM-Expanded Streptococcus thermophilus Cas9 C-to-T and C-to-G Base Editors for Programmable Base Editing in Mycobacteria.ENGINEERING.
MLA	Zhang, Hongyuan,et al."PAM-Expanded Streptococcus thermophilus Cas9 C-to-T and C-to-G Base Editors for Programmable Base Editing in Mycobacteria".ENGINEERING (2022).