High-accuracy crRNA array assembly strategy for multiplex CRISPR
Simultaneous targeting of multiple loci with the CRISPR system, a tool known as multiplex CRISPR, offers greater feasibility for manipulating and elucidating the intricate and redundant endogenous networks underlying complex cellular functions. Owing to the versatility of continuously emerging Cas n...
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| Format: | Article |
| Language: | English |
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Elsevier
2025-03-01
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| Series: | Molecular Therapy: Nucleic Acids |
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| Online Access: | http://www.sciencedirect.com/science/article/pii/S2162253124003159 |
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| author | Xiangtong Zhao Lixian Yang Peng Li Zijing Cheng Yongshi Jia Limin Luo Aihong Bi Hanchu Xiong Haibo Zhang Hongen Xu Jinrui Zhang Yaodong Zhang |
| author_facet | Xiangtong Zhao Lixian Yang Peng Li Zijing Cheng Yongshi Jia Limin Luo Aihong Bi Hanchu Xiong Haibo Zhang Hongen Xu Jinrui Zhang Yaodong Zhang |
| author_sort | Xiangtong Zhao |
| collection | DOAJ |
| description | Simultaneous targeting of multiple loci with the CRISPR system, a tool known as multiplex CRISPR, offers greater feasibility for manipulating and elucidating the intricate and redundant endogenous networks underlying complex cellular functions. Owing to the versatility of continuously emerging Cas nucleases and the use of CRISPR arrays, multiplex CRISPR has been implemented in numerous in vitro and in vivo studies. However, a streamlined, practical strategy for CRISPR array assembly that is both convenient and accurate is lacking. Here, we present a novel, highly accurate, cost-, and time-saving strategy for CRISPR array assembly. Using this strategy, we efficiently assembled 12 CRISPR RNAs (crRNAs) (for AsCas12a) and 15 crRNAs (for RfxCas13d) in a single reaction. CRISPR arrays driven by Pol II promoters exhibited a distinct expression pattern compared with those driven by Pol III promoters, which could be exploited for specific distributions of CRISPR intensity. Improved approaches were subsequently designed and validated for expressing long CRISPR arrays. The study provides a flexible and powerful tool for the convenient implementation of multiplex CRISPR across DNA and RNA, facilitating the dissection of sophisticated cellular networks and the future realization of multi-target gene therapy. |
| format | Article |
| id | doaj-art-ead53da16f05458fa9380b0f5f088a0c |
| institution | Kabale University |
| issn | 2162-2531 |
| language | English |
| publishDate | 2025-03-01 |
| publisher | Elsevier |
| record_format | Article |
| series | Molecular Therapy: Nucleic Acids |
| spelling | doaj-art-ead53da16f05458fa9380b0f5f088a0c2025-01-18T05:04:24ZengElsevierMolecular Therapy: Nucleic Acids2162-25312025-03-01361102428High-accuracy crRNA array assembly strategy for multiplex CRISPRXiangtong Zhao0Lixian Yang1Peng Li2Zijing Cheng3Yongshi Jia4Limin Luo5Aihong Bi6Hanchu Xiong7Haibo Zhang8Hongen Xu9Jinrui Zhang10Yaodong Zhang11Henan Provincial Key Laboratory of Children’s Genetics and Metabolic Diseases, Children’s Hospital Affiliated to Zhengzhou University, Henan Children’s Hospital, Zhengzhou Children’s Hospital, Zhengzhou, Henan, China; College of Public Health, Zhengzhou University, Zhengzhou, Henan, China; Corresponding author: Xiangtong Zhao, Henan Provincial Key Laboratory of Children’s Genetics and Metabolic Diseases, Children’s Hospital Affiliated to Zhengzhou University, Henan Children’s Hospital, Zhengzhou Children’s Hospital, Zhengzhou, Henan, China.Cancer Center, Department of Radiation Oncology, Zhejiang Provincial People’s Hospital, Affiliated People’s Hospital, Hangzhou Medical College, Hangzhou, Zhejiang, China; Corresponding author: Lixian Yang, Cancer Center, Department of Radiation Oncology, Zhejiang Provincial People’s Hospital, Affiliated People’s Hospital, Hangzhou Medical College, Hangzhou, Zhejiang, China.Department of Gastroenterology, Zhejiang Provincial People’s Hospital, Affiliated People’s Hospital, Hangzhou Medical College, Hangzhou, Zhejiang, ChinaSchool of Anesthesiology, Xuzhou Medical University, Xuzhou, Jiangsu, ChinaCancer Center, Department of Radiation Oncology, Zhejiang Provincial People’s Hospital, Affiliated People’s Hospital, Hangzhou Medical College, Hangzhou, Zhejiang, ChinaCancer Center, Department of Radiation Oncology, Zhejiang Provincial People’s Hospital, Affiliated People’s Hospital, Hangzhou Medical College, Hangzhou, Zhejiang, ChinaCancer Center, Department of Radiation Oncology, Zhejiang Provincial People’s Hospital, Affiliated People’s Hospital, Hangzhou Medical College, Hangzhou, Zhejiang, ChinaCancer Center, Department of Radiation Oncology, Zhejiang Provincial People’s Hospital, Affiliated People’s Hospital, Hangzhou Medical College, Hangzhou, Zhejiang, ChinaCancer Center, Department of Radiation Oncology, Zhejiang Provincial People’s Hospital, Affiliated People’s Hospital, Hangzhou Medical College, Hangzhou, Zhejiang, ChinaCancer Center, Department of Radiation Oncology, Zhejiang Provincial People’s Hospital, Affiliated People’s Hospital, Hangzhou Medical College, Hangzhou, Zhejiang, ChinaHenan Provincial Key Laboratory of Children’s Genetics and Metabolic Diseases, Children’s Hospital Affiliated to Zhengzhou University, Henan Children’s Hospital, Zhengzhou Children’s Hospital, Zhengzhou, Henan, ChinaHenan Provincial Key Laboratory of Children’s Genetics and Metabolic Diseases, Children’s Hospital Affiliated to Zhengzhou University, Henan Children’s Hospital, Zhengzhou Children’s Hospital, Zhengzhou, Henan, China; Corresponding author: Yaodong Zhang, Henan Provincial Key Laboratory of Children’s Genetics and Metabolic Diseases, Children’s Hospital Affiliated to Zhengzhou University, Henan Children’s Hospital, Zhengzhou Children’s Hospital, Zhengzhou, Henan, China.Simultaneous targeting of multiple loci with the CRISPR system, a tool known as multiplex CRISPR, offers greater feasibility for manipulating and elucidating the intricate and redundant endogenous networks underlying complex cellular functions. Owing to the versatility of continuously emerging Cas nucleases and the use of CRISPR arrays, multiplex CRISPR has been implemented in numerous in vitro and in vivo studies. However, a streamlined, practical strategy for CRISPR array assembly that is both convenient and accurate is lacking. Here, we present a novel, highly accurate, cost-, and time-saving strategy for CRISPR array assembly. Using this strategy, we efficiently assembled 12 CRISPR RNAs (crRNAs) (for AsCas12a) and 15 crRNAs (for RfxCas13d) in a single reaction. CRISPR arrays driven by Pol II promoters exhibited a distinct expression pattern compared with those driven by Pol III promoters, which could be exploited for specific distributions of CRISPR intensity. Improved approaches were subsequently designed and validated for expressing long CRISPR arrays. The study provides a flexible and powerful tool for the convenient implementation of multiplex CRISPR across DNA and RNA, facilitating the dissection of sophisticated cellular networks and the future realization of multi-target gene therapy.http://www.sciencedirect.com/science/article/pii/S2162253124003159MT: RNA/DNA Editingmultiplex CRISPRcrRNACRISPR arrayGolden Gate AssemblyAsCas12a |
| spellingShingle | Xiangtong Zhao Lixian Yang Peng Li Zijing Cheng Yongshi Jia Limin Luo Aihong Bi Hanchu Xiong Haibo Zhang Hongen Xu Jinrui Zhang Yaodong Zhang High-accuracy crRNA array assembly strategy for multiplex CRISPR Molecular Therapy: Nucleic Acids MT: RNA/DNA Editing multiplex CRISPR crRNA CRISPR array Golden Gate Assembly AsCas12a |
| title | High-accuracy crRNA array assembly strategy for multiplex CRISPR |
| title_full | High-accuracy crRNA array assembly strategy for multiplex CRISPR |
| title_fullStr | High-accuracy crRNA array assembly strategy for multiplex CRISPR |
| title_full_unstemmed | High-accuracy crRNA array assembly strategy for multiplex CRISPR |
| title_short | High-accuracy crRNA array assembly strategy for multiplex CRISPR |
| title_sort | high accuracy crrna array assembly strategy for multiplex crispr |
| topic | MT: RNA/DNA Editing multiplex CRISPR crRNA CRISPR array Golden Gate Assembly AsCas12a |
| url | http://www.sciencedirect.com/science/article/pii/S2162253124003159 |
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