Zhao Xiangtong, Yang Lixian, Li Peng, Cheng Zijing, Jia Yongshi, Luo Limin, Bi Aihong, Xiong Hanchu, Zhang Haibo, Xu Hongen, Zhang Jinrui, Zhang Yaodong
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.
College of Public Health, Zhengzhou University, Zhengzhou, Henan, China.
Mol Ther Nucleic Acids. 2024 Dec 12;36(1):102428. doi: 10.1016/j.omtn.2024.102428. eCollection 2025 Mar 11.
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 and 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.
利用CRISPR系统同时靶向多个位点（一种称为多重CRISPR的工具），为操纵和阐明复杂细胞功能背后错综复杂且冗余的内源性网络提供了更大的可行性。由于不断涌现的Cas核酸酶的多功能性以及CRISPR阵列的使用，多重CRISPR已在众多研究中得到应用。然而，目前缺乏一种既方便又准确的简化实用的CRISPR阵列组装策略。在此，我们提出了一种新颖、高度准确、节省成本和时间的CRISPR阵列组装策略。使用该策略，我们在单个反应中高效组装了12个CRISPR RNA（crRNA）（用于AsCas12a）和15个crRNA（用于RfxCas13d）。与由Pol III启动子驱动的CRISPR阵列相比，由Pol II启动子驱动的CRISPR阵列表现出独特的表达模式，这可用于CRISPR强度的特定分布。随后设计并验证了用于表达长CRISPR阵列的改进方法。该研究为在DNA和RNA上方便地实施多重CRISPR提供了一种灵活而强大的工具，有助于剖析复杂的细胞网络以及未来实现多靶点基因治疗。
