College of Life Sciences, Shandong Normal University, No. 88 East Wenhua Road, Lixia District, Jinan 250014, Shandong, China.
Shandong Shunfeng Biotechnology Co. Ltd., 11 Floor, Main Building, QiLu Innovalley Incubator, High-tech Industry Development Zone, Jinan 250000, Shandong, China.
Gene. 2022 Jan 10;807:145919. doi: 10.1016/j.gene.2021.145919. Epub 2021 Aug 26.
The application of CRISPR/Cas9 system for gene editing, as a technical coup for biotechnology, is worldwide and encompasses multiple of species. The inactivation of catalytical site in Cas9 (dCas9) has been reprogrammed as an effective approach to regulate the transcriptional level of target genes, especially for the functionally essential genes and redundant genes. Here, we exploited the CRISPR/dCas9 system to manipulate the transcriptional level of target genes in common wheat. To improve target gene's expression, we generated transcriptional activator by fusing 6×TAL-VP128 activation domain to the C-terminus of dCas9 in frame. For target gene's repressing expression transcriptionally, 3×SRDX repression domain was conjugated to the C-terminus of dCas9 in frame. Our results showed that dCas9 fused activation or repression domain could increase or decrease the transcriptional level of target gene effectively in stable transgenic lines of wheat. The study on the tRNA-processing system in CRISPR/dCas9 based transcriptional regulation system demonstrated that this robust multiplex targeted tool can be incorporated to the CRISPR/dCas9 system to facilitate the target regulation of several genes' transcriptional level. Our data broaden the application of CRISPR/dCas9 based transcriptional regulation and provide great opportunities to investigate the function of essential genes in common wheat.
CRISPR/Cas9 系统在基因编辑中的应用作为生物技术的一项技术突破，已经在全球范围内得到广泛应用，并涵盖了多个物种。Cas9（dCas9）催化位点的失活已被重新编程为一种有效调节靶基因转录水平的方法，特别是对于功能必需基因和冗余基因。在这里，我们利用 CRISPR/dCas9 系统来操纵普通小麦中靶基因的转录水平。为了提高靶基因的表达，我们通过将 6×TAL-VP128 激活结构域融合到 dCas9 的 C 末端来生成转录激活剂。为了在转录水平上抑制靶基因的表达，我们将 3×SRDX 抑制结构域融合到 dCas9 的 C 末端。我们的结果表明，dCas9 融合激活或抑制结构域可以在小麦的稳定转基因系中有效增加或降低靶基因的转录水平。对 CRISPR/dCas9 基于 tRNA 加工系统的转录调控系统的研究表明，这种强大的多重靶向工具可以整合到 CRISPR/dCas9 系统中，以促进几个基因转录水平的靶标调控。我们的数据拓宽了基于 CRISPR/dCas9 的转录调控的应用，并为研究普通小麦中必需基因的功能提供了很好的机会。
