Department of Applied Microbial Technology, Faculty of Biotechnology and Life Science, Sojo University, 4-22-1 Ikeda Nishi-ku, Kumamoto 860-0082, Japan.
Department of Applied Microbial Technology, Faculty of Biotechnology and Life Science, Sojo University, 4-22-1 Ikeda Nishi-ku, Kumamoto 860-0082, Japan.
J Biosci Bioeng. 2019 Sep;128(3):373-378. doi: 10.1016/j.jbiosc.2019.02.009. Epub 2019 Apr 19.
The clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 9 (CRISPR/Cas9) system is one of the most powerful tools for genome engineering. However, some of the steps are laborious, reducing its usability. In this study, we have developed a simplified method, called the guide RNA-transient expression system (gRNA-TES), to deliver gRNA in yeast. In gRNA-TES, a DNA fragment containing the promoter and gRNA is prepared by two simple PCR steps and co-transformed with a DNA module into the host strain; all steps including PCR steps and yeast transformation are completed within 5-6 h in a single day, in contrast to conventional plasmid-based gRNA delivery systems, which require at least 3-4 days to construct and verify the gRNA-expressing plasmids. The performance of gRNA-TES was evaluated by the replacement of 150-kb, 200-kb, 300-kb, 400-kb, and 500-kb regions of yeast chromosome 4 with a DNA module. Increased numbers of transformants with a high frequency of expected replacement of even the 500-kb region were obtained with gRNA-TES as compared with transformation without gRNA-TES. In addition, the integrity of the replaced region was verified in 67%-100% of transformants tested by colony PCR. We believe that gRNA-TES will vastly increase the accessibility of CRISPR/Cas9 technology to biologists and biotechnologists by offering a simple, fast, and cost-effective tool to deliver gRNA in genome engineering. Furthermore, it might be applied to plant and animal systems if appropriate gene promoters are incorporated in the technology.
簇状规律间隔短回文重复序列（CRISPR）和 CRISPR 相关蛋白 9（CRISPR/Cas9）系统是基因组工程中最强大的工具之一。然而，其中的一些步骤较为繁琐，降低了其可用性。在这项研究中，我们开发了一种简化的方法，称为引导 RNA 瞬时表达系统（gRNA-TES），用于在酵母中递送 gRNA。在 gRNA-TES 中，通过两步简单的 PCR 步骤制备包含启动子和 gRNA 的 DNA 片段，并与 DNA 模块共转化到宿主菌株中；包括 PCR 步骤和酵母转化在内的所有步骤都可以在一天内的 5-6 小时内完成，而传统的基于质粒的 gRNA 递送系统则需要至少 3-4 天来构建和验证表达 gRNA 的质粒。通过用 DNA 模块替换酵母染色体 4 上的 150-kb、200-kb、300-kb、400-kb 和 500-kb 区域，评估了 gRNA-TES 的性能。与没有 gRNA-TES 的转化相比，gRNA-TES 获得了更多的转化体，且这些转化体具有更高的预期替换频率，甚至可以替换 500-kb 区域。此外，通过菌落 PCR 验证了在 67%-100%的转化体中替换区域的完整性。我们相信，gRNA-TES 通过提供一种简单、快速且具有成本效益的工具来递送 gRNA，将极大地增加 CRISPR/Cas9 技术对生物学家和生物技术人员的可及性。此外，如果将适当的基因启动子纳入该技术，它可能会应用于植物和动物系统。
