Zhang Yingxiao, Zhang Yong, Qi Yiping
Department of Plant Science and Landscape Architecture, University of Maryland, College Park, MD, USA.
Department of Biotechnology, School of Life Science and Technology, Center for Informational Biology, University of Electronic Science and Technology of China, Chengdu, China.
Methods Mol Biol. 2019;1917:245-256. doi: 10.1007/978-1-4939-8991-1_18.
CRISPR-Cpf1 (Cas12a) is a class II type V endonuclease, which has been used as a genome editing tool in different biological systems. Here we describe a fast, efficient, and user-friendly system for CRISPR-Cpf1 expression vector assembly. In this system, the Pol II promoter is used to drive the expression of both Cpf1 and its crRNA, with the crRNA flanked by hammerhead (HH) and hepatitis delta virus (HDV) ribozyme RNAs for precise crRNA processing. All the components of this system can be modified depending on plant species and experimental goals. Using this system, nearly 100% editing efficiency and 90% gene expression decrease were achieved in rice and Arabidopsis, respectively.
CRISPR-Cpf1（Cas12a）是一种II类V型核酸内切酶，已被用作不同生物系统中的基因组编辑工具。在此，我们描述了一种用于CRISPR-Cpf1表达载体组装的快速、高效且用户友好的系统。在该系统中，Pol II启动子用于驱动Cpf1及其crRNA的表达，crRNA两侧分别为锤头状（HH）和丁型肝炎病毒（HDV）核酶RNA，用于精确的crRNA加工。该系统的所有组件均可根据植物物种和实验目标进行修改。使用该系统，在水稻和拟南芥中分别实现了近100%的编辑效率和90%的基因表达降低。
