Versatile single-step-assembly CRISPR/Cas9 vectors for dual gRNA expression

CRISPR/Cas9 technology enables efficient, rapid and cost-effective targeted genomic modification in a wide variety of cellular contexts including cultured cells. Some applications such as generation of double knock-outs, large deletions and paired-nickase cleavage require simultaneous expression of two gRNAs. Although single plasmids that enable multiplex expression of gRNAs have been developed, these require multiple rounds of cloning and/or PCR for generation of the desired construct. Here, we describe a series of vectors that enable generation of customized dual-gRNA expression constructs via an easy one-step golden gate cloning reaction using two annealed oligonucleotide inserts with different overhangs. Through nucleofection of mouse embryonic stem cells, we demonstrate highly efficient cleavage of the target loci using the dual-guide plasmids, which are available as Cas9-nuclease or Cas9-nickase expression constructs, with or without selection markers. These vectors are a valuable addition to the CRISPR/Cas9 toolbox and will be made available to all interested researchers via the Addgene plasmid repository.CRISPR/Cas9技术能够在包括培养细胞在内的多种细胞环境中，实现高效、快速且经济高效的靶向基因组修饰。部分应用场景，例如构建双敲除细胞系、引入大片段缺失以及采用双切口酶切割策略，均需要同时表达两条向导RNA（gRNA）。尽管目前已开发出可实现gRNA多重表达的单质粒载体，但这类载体在构建目标重组质粒时，需经历多轮克隆及/或PCR扩增步骤。本文报道了一系列载体，可通过简便的一步式Golden Gate克隆反应，利用两条带有不同粘性末端的退火寡核苷酸插入片段，快速构建定制化的双gRNA表达重组载体。通过对小鼠胚胎干细胞进行核转染实验，我们证实该双向导RNA质粒可高效切割靶位点；这类质粒可分为Cas9核酸酶型与Cas9切口酶型两类，且可搭载或不搭载筛选标记。该系列载体是CRISPR/Cas9工具库的重要补充，所有感兴趣的研究人员均可通过Addgene质粒资源库获取此类载体。