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《工程(英文)》 >> 2022年 第15卷 第8期 doi: 10.1016/j.eng.2022.02.013

用于分枝杆菌碱基编辑的PAM扩展型嗜热链球菌Cas9 C到T和C到G碱基编辑器

a School of Physical Science and Technology, ShanghaiTech University, Shanghai 201210, China
b University of Chinese Academy of Sciences, Beijing 100049, China
c Clinical Research Center, the Second Hospital of Nanjing, Nanjing University of Chinese Medicine, Nanjing 210003, China
d Department of Tuberculosis, the Second Hospital of Nanjing, Nanjing University of Chinese Medicine, Nanjing 210003, China
e Gene Editing Center, School of Life Science and Technology, ShanghaiTech University, Shanghai 201210, China
f Guangzhou Laboratory, Guangzhou 510120, China

收稿日期: 2021-11-08 修回日期: 2022-02-13 录用日期: 2022-02-15 发布日期: 2022-04-29

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摘要

结核分枝杆菌(Mycobacterium tuberculosis, MTB)耐药性是临床治疗面临的重大挑战,迫切需要开发治疗耐药结核病的新型策略,而简单易用的MTB遗传操作方法能够加速这一进程。依赖于规律成簇的间隔短回文重复序列(CRISPR)的碱基编辑器能够快速有效地进行碱基编辑和基因失活,然而,目前还没有开发出可用于MTB的碱基编辑器。通过筛选不同的碱基编辑器,发现广泛使用的酿脓链球菌CRISPR相关蛋白9(SpCas9)或毛螺科菌Cpf1(LbCpf1)胞嘧啶碱基编辑器在分枝杆菌中不具有活性,而嗜热链球菌Cas9(St1Cas9)胞嘧啶碱基编辑器活性较好。虽然使用St1Cas9 胞嘧啶碱基编辑器能够实现C到T的碱基编辑,但是,在碱基编辑过程中却产生了大量的副产物。将尿嘧啶-N-糖基化酶抑制剂或尿嘧啶-N-糖基化酶分别融合到St1Cas9 胞嘧啶碱基编辑器中,得到了两种新的碱基编辑器——CTBE和CGBE,能将C分别转化为T或G。将CTBE和CGBE用于耻垢分枝杆菌的基因编辑时,产物的纯度得到了提高,并且能够进行多位点编辑。此外,由于野生型St1Cas9 在靶向DNA序列时需要识别严格的前间隔序列邻近基序(PAM),因此,通过结构介导的蛋白质工程设计了PAM扩展的St1Cas9,扩大了碱基编辑器的靶向范围。首先在耻垢分枝杆菌中测试了CTBE和CGBE的编辑效率,随后测试了CTBE在MTB中的编辑效率。本文提出的方法显著减少了在MTB中进行精确遗传操作所需要的时间,并将推动分枝杆菌功能基因组学、抗生素-耐药性机制研究和药物-靶点研究的发展。

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