使用基因组编辑工具对类别转换重组进行遗传剖析的方案。
Protocol for genetic dissection of class switch recombination using genome-editing tools.
作者信息
Dai Pengfei, Liu Tingting, Yang Dingpeng, Luo Yifeng, Liu Liu Daisy, Xie Xia, Shang Yafang, Liu Xiaojing, Meng Fei-Long
机构信息
Key Laboratory of RNA Innovation, Science and Engineering, Shanghai Academy of Natural Sciences (SANS), Shanghai Institute of Biochemistry and Cell Biology, Center for Excellence in Molecular Cell Science, Chinese Academy of Sciences, University of Chinese Academy of Sciences, Shanghai 200031, China; International Peace Maternity and Child Health Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai Key Laboratory of Embryo Original Diseases, Shanghai 200030, China.
Key Laboratory of RNA Innovation, Science and Engineering, Shanghai Academy of Natural Sciences (SANS), Shanghai Institute of Biochemistry and Cell Biology, Center for Excellence in Molecular Cell Science, Chinese Academy of Sciences, University of Chinese Academy of Sciences, Shanghai 200031, China.
出版信息
STAR Protoc. 2025 Jun 20;6(2):103882. doi: 10.1016/j.xpro.2025.103882. Epub 2025 Jun 6.
Antibody class switch recombination is achieved through programmed DNA damage, and the processing of programmed DNA lesions requires the coordinated action of many DNA metabolic factors. Here, we present a protocol for inducing class switch recombination using base editors or CRISPR-Cas9. We provide optimized guide RNA (gRNA) sequences and describe steps for cytokine activation, electroporation, surface immunoglobulin detection, and data analysis. This method allows researchers to investigate the involvement of specific factors in antibody diversification and elucidate their functional roles. For complete details on the use and execution of this protocol, please refer to Dai et al..
抗体类别转换重组是通过程序性DNA损伤实现的，而程序性DNA损伤的处理需要许多DNA代谢因子的协同作用。在这里，我们介绍一种使用碱基编辑器或CRISPR-Cas9诱导类别转换重组的方案。我们提供了优化的引导RNA（gRNA）序列，并描述了细胞因子激活、电穿孔、表面免疫球蛋白检测和数据分析的步骤。该方法使研究人员能够研究特定因子在抗体多样化中的作用，并阐明其功能。有关本方案使用和执行的完整详细信息，请参阅戴等人的文章。
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