PAM-expanded <italic>Streptococcus thermophilus</italic> Cas9 C-to-T and C-to-G base editors for programmable base editing in mycobacteria

doi:10.26434/chemrxiv-2021-nt3ts

	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 ; Chen, Wei ; Ji, Quanjiang1
	2021-10-22
状态	已发表
摘要	New therapeutic strategies for rapid and effective treatment of tuberculosis are highly desirable, and their development can be drastically accelerated by facile genetic manipulation methods in Mycobacterium tuberculosis. 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 identified that only the unusual Streptococcus thermophilus Cas9 (St1Cas9) cytidine base editor (CBE), but not the widely used Streptococcus pyogenes Cas9 or Lachnospiraceae bacterium Cpf1 CBEs, are active in mycobacteria. Despite the notable C-to-T conversions, a high portion of undesired byproducts existed with St1Cas9 CBE. We thus engineered St1Cas9 CBE by 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. Because wild-type St1Cas9 recognizes a relatively strict PAM sequence for DNA targeting, we evolved a PAM-expanded St1Cas9 variant by structure-guided protein engineering for the base editors, substantially broadening the targeting scope. Our approaches significantly reduce the efforts and time for precise genetic manipulation and will facilitate functional genomics and drug-target exploration in M. tuberculosis and related organisms.
语种	英语
DOI	10.26434/chemrxiv-2021-nt3ts
相关网址	查看原文
出处	chemRxiv
收录类别	PPRN.PPRN
WOS记录号	PPRN:10703470
WOS类目	Chemistry, Multidisciplinary
资助项目	National Natural Science Foundation of China["M2020019","EKPG21-18","19QA1406000","21922705"] ; Shanghai Committee of Science and Technology, China[91753127] ; Emergency Key Program of Guangzhou Laboratory[2207783]
文献类型	预印本
条目标识符	https://kms.shanghaitech.edu.cn/handle/2MSLDSTB/348482
专题	物质科学与技术学院生命科学与技术学院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_PI研究组_季泉江组物质科学与技术学院_博士生
通讯作者	Chen, Wei; Ji, Quanjiang
作者单位	1.Shanghai Tech Univ, Sch Phys Sci & Technol, Shanghai 201210, Peoples R China 2.Univ Chinese Acad Sci, Beijing 100049, Peoples R China 3.Nanjing Univ Chinese Med, Hosp Nanjing 2, Clin Res Ctr, Nanjing 210003, Peoples R China 4.Nanjing Univ Chinese Med, Hosp Nanjing 2, Dept TB, Nanjing 210003, Peoples R China 5.Shanghai Tech Univ, Sch Life Sci & Technol, Shanghai 201210, Peoples R China
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. 2021.