Empower multiplex cell and tissue-specific CRISPR-mediated gene manipulation with self-cleaving ribozymes and tRNA.
Empower multiplex cell and tissue-specific CRISPR-mediated gene manipulation with self-cleaving ribozymes and tRNA.
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DOI：
10.1093/nar/gkw1048
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发表时间：
2017-03-17
影响因子：
14.9
通讯作者：
Han R
中科院分区：
文献类型：
--
作者：
Xu L;Zhao L;Gao Y;Xu J;Han R
CRISPR/Cas9系统是近年来出现的一种高效的RNA引导的基因操作平台。不同基因的同时编辑或转录激活/抑制随着多个向导RNA（gRNA）的共递送而变得可行。在这里，我们报告了与自切割核酶和/或tRNA连接的多个gRNA可以从单个U6启动子同时表达，以在人类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，当与无催化活性的dCas 9-VP 64或dCas 9-p300核心融合体一起使用时，该策略允许表达用于卵泡抑素的协同转录激活的多种gRNA。最后，由自切割核酶和tRNA连接的gRNA可以从RNA聚合酶II型（pol II）启动子（如通用CMV和肌肉/心脏特异性MHCK 7）表达。这对于当重组腺相关病毒的包装能力有限而需要gRNA和Cas9的组织特异性递送时的体内应用特别有用。总之，本研究提供了一种新的策略，以使多于一种gRNA的组织特异性表达能够从单个pol II启动子进行多重基因编辑。
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.