Xu Li, Zhao Lixia, Gao Yandi, Xu Jing, Han Renzhi
Department of Surgery, Davis Heart and Lung Research Institute, Biomedical Sciences Graduate Program, Biophysics Graduate Program, The Ohio State University Wexner Medical Center, Columbus, OH 43210, USA.
Nucleic Acids Res. 2017 Mar 17;45(5):e28. doi: 10.1093/nar/gkw1048.
Clustered regularly interspaced short palindromic repeat/Cas9 (CRISPR/Cas9) system has emerged in recent years as a highly efficient RNA-guided gene manipulation platform. Simultaneous editing or transcriptional activation/suppression of different genes becomes feasible with the co-delivery of multiple guide RNAs (gRNAs). Here, we report that multiple gRNAs linked with self-cleaving ribozymes and/or tRNA could be simultaneously expressed from a single U6 promoter to exert genome editing of dystrophin and myosin binding protein C3 in human and mouse cells. Moreover, this strategy allows the expression of multiple gRNAs for synergistic transcription activation of follistatin when used with catalytically inactive dCas9-VP64 or dCas9-p300core fusions. Finally, the gRNAs linked by the self-cleaving ribozymes and tRNA could be expressed from RNA polymerase type II (pol II) promoters such as generic CMV and muscle/heart-specific MHCK7. This is particularly useful for in vivo applications when the packaging capacity of recombinant adeno-associated virus is limited while tissue-specific delivery of gRNAs and Cas9 is desired. Taken together, this study provides a novel strategy to enable tissue-specific expression of more than one gRNAs for multiplex gene editing from a single pol II promoter.
成簇规律间隔短回文重复序列/Cas9（CRISPR/Cas9）系统近年来已成为一种高效的RNA引导基因操作平台。通过共递送多个导向RNA（gRNA），同时编辑或转录激活/抑制不同基因变得可行。在此，我们报告与自我切割核酶和/或tRNA相连的多个gRNA可从单个U6启动子同时表达，以在人和小鼠细胞中对肌营养不良蛋白和肌球蛋白结合蛋白C3进行基因组编辑。此外，当与催化失活的dCas9-VP64或dCas9-p300core融合蛋白一起使用时，该策略允许表达多个gRNA以协同转录激活卵泡抑素。最后，由自我切割核酶和tRNA连接的gRNA可从II型RNA聚合酶（pol II）启动子如通用的CMV和肌肉/心脏特异性的MHCK7表达。当重组腺相关病毒的包装能力有限而又需要gRNA和Cas9的组织特异性递送时，这对于体内应用特别有用。综上所述，本研究提供了一种新策略，可从单个pol II启动子实现组织特异性表达多个gRNA用于多重基因编辑。
