阅读说明：本技术 多重引导rna (Multiple guide RNAs ) 是由 S·特赛 K·J·乔昂格 于 2014-09-18 设计创作，主要内容包括：本申请涉及多重引导RNA。用于任选地在哺乳动物细胞中将高活性CRISPR引导RNA(gRNA)从RNA聚合酶II和III启动子进行多重表达的方法和构建体。本发明至少部分地基于Csy4的发现,Csy4是一种内切核糖核酸酶,该内切核糖核酸酶识别短RNA发夹序列,可以用于切下在单个较长RNA转录物上编码的多功能gRNA(产生自RNA pol II或III启动子),在该较长RNA转录物中的各个gRNA由Csy4切割位点隔开。(The present application relates to multiple guide RNAs. Methods and constructs for multiplexed expression of high activity CRISPR guide RNAs (grnas) from RNA polymerase II and III promoters, optionally in mammalian cells. The present invention is based, at least in part, on the discovery that Csy4, Csy4 is an endoribonuclease that recognizes short RNA hairpin sequences and can be used to excise multifunctional grnas (generated from the RNA pol II or III promoters) encoded on a single longer RNA transcript in which each gRNA is separated by a Csy4 cleavage site.)