Masonic Cancer Center, University of Minnesota, Minneapolis, MN, United States of America.
Center for Genome Engineering, University of Minnesota, Minneapolis, MN, United States of America.
PLoS One. 2018 Sep 17;13(9):e0198714. doi: 10.1371/journal.pone.0198714. eCollection 2018.
The CRISPR/Cas9 system is an RNA guided nuclease system that evolved as a mechanism of adaptive immunity in bacteria. This system has been adopted for numerous genome engineering applications in research and recently, therapeutics. The CRISPR/Cas9 system has been largely implemented by delivery of Cas9 as protein, RNA, or plasmid along with a chimeric crRNA-tracrRNA guide RNA (gRNA) under the expression of a pol III promoter, such as U6. Using this approach, multiplex genome engineering has been achieved by delivering several U6-gRNA plasmids targeting multiple loci. However, this approach is limited due to the efficiently of delivering multiple plasmids to a single cell at one time. To augment the capability and accessibility of multiplexed genome engineering, we developed an efficient golden gate based method to assemble gRNAs linked by optimal Csy4 ribonuclease sequences to deliver up to 10 gRNAs as a single gRNA array transcript. Here we report the optimal expression of our guide RNA array under a strong pol II promoter. This system can be implemented alongside the myriad of CRISPR applications, allowing users to model complex biological processes requiring numerous gRNAs.
CRISPR/Cas9 系统是一种 RNA 指导的核酸内切酶系统，作为细菌适应性免疫的一种机制而进化。该系统已被广泛应用于研究和最近的治疗领域的多种基因组工程应用。CRISPR/Cas9 系统主要通过 Cas9 蛋白、RNA 或质粒与嵌合 crRNA-tracrRNA 向导 RNA（gRNA）的传递来实现，该 gRNA 在 III 型聚合酶启动子（如 U6）的表达下进行。通过这种方法，可以通过递送多个靶向多个基因座的 U6-gRNA 质粒来实现多重基因组工程。然而，由于同时向单个细胞有效递送多个质粒的效率有限，因此该方法受到限制。为了增强多重基因组工程的能力和可及性，我们开发了一种高效的基于 Golden Gate 的方法，通过最佳 Csy4 核糖核酸酶序列将 gRNA 连接在一起，作为单个 gRNA 阵列转录本递送至多达 10 个 gRNA。在这里，我们报告了在强 II 型聚合酶启动子下我们的 gRNA 阵列的最佳表达。该系统可以与众多 CRISPR 应用程序一起实施，允许用户模拟需要多个 gRNA 的复杂生物过程。
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