Protocol for genetic dissection of class switch recombination using genome-editing tools
Summary: 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. W...
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| Format: | Article |
| Language: | English |
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Elsevier
2025-06-01
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| Series: | STAR Protocols |
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| Online Access: | http://www.sciencedirect.com/science/article/pii/S2666166725002886 |
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| author | Pengfei Dai Tingting Liu Dingpeng Yang Yifeng Luo Liu Daisy Liu Xia Xie Yafang Shang Xiaojing Liu Fei-Long Meng |
| author_facet | Pengfei Dai Tingting Liu Dingpeng Yang Yifeng Luo Liu Daisy Liu Xia Xie Yafang Shang Xiaojing Liu Fei-Long Meng |
| author_sort | Pengfei Dai |
| collection | DOAJ |
| description | Summary: 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.1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. |
| format | Article |
| id | doaj-art-9c1f29eeb97f4424870fefd77642f7c5 |
| institution | Kabale University |
| issn | 2666-1667 |
| language | English |
| publishDate | 2025-06-01 |
| publisher | Elsevier |
| record_format | Article |
| series | STAR Protocols |
| spelling | doaj-art-9c1f29eeb97f4424870fefd77642f7c52025-08-20T03:25:11ZengElsevierSTAR Protocols2666-16672025-06-016210388210.1016/j.xpro.2025.103882Protocol for genetic dissection of class switch recombination using genome-editing toolsPengfei Dai0Tingting Liu1Dingpeng Yang2Yifeng Luo3Liu Daisy Liu4Xia Xie5Yafang Shang6Xiaojing Liu7Fei-Long Meng8Key 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; Corresponding authorKey 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, ChinaKey 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, ChinaKey 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, ChinaKey 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, ChinaKey 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, ChinaKey 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, ChinaKey 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, ChinaKey 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; Corresponding authorSummary: 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.1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.http://www.sciencedirect.com/science/article/pii/S2666166725002886cell-based assaysimmunologyCRISPR |
| spellingShingle | Pengfei Dai Tingting Liu Dingpeng Yang Yifeng Luo Liu Daisy Liu Xia Xie Yafang Shang Xiaojing Liu Fei-Long Meng Protocol for genetic dissection of class switch recombination using genome-editing tools STAR Protocols cell-based assays immunology CRISPR |
| title | Protocol for genetic dissection of class switch recombination using genome-editing tools |
| title_full | Protocol for genetic dissection of class switch recombination using genome-editing tools |
| title_fullStr | Protocol for genetic dissection of class switch recombination using genome-editing tools |
| title_full_unstemmed | Protocol for genetic dissection of class switch recombination using genome-editing tools |
| title_short | Protocol for genetic dissection of class switch recombination using genome-editing tools |
| title_sort | protocol for genetic dissection of class switch recombination using genome editing tools |
| topic | cell-based assays immunology CRISPR |
| url | http://www.sciencedirect.com/science/article/pii/S2666166725002886 |
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