Huang Yuping, Wang Yajun, Zeng Baosheng, Liu Zhaoxia, Xu Xuejiao, Meng Qian, Huang Yongping, Yang Guang, Vasseur Liette, Gurr Geoff M, You Minsheng
State Key Laboratory for Ecological Pest Control of Fujian/Taiwan Crops and College of Life Science, Fujian Agriculture and Forestry University, Fuzhou 350002, China; Institute of Applied Ecology, Fujian Agriculture and Forestry University, Fuzhou 350002, China; Fujian-Taiwan Joint Innovation Centre for Ecological Control of Crop Pests, Fujian Agriculture and Forestry University, Fuzhou 350002, China; Key Laboratory of Integrated Pest Management for Fujian-Taiwan Crops, Ministry of Agriculture, Fuzhou 350002, China.
Key Laboratory of Insect Developmental and Evolutionary Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200032, China.
Insect Biochem Mol Biol. 2017 Oct;89:71-78. doi: 10.1016/j.ibmb.2017.08.009. Epub 2017 Sep 7.
RNA polymerase type III (Pol-III) promoters such as U6 are commonly used to express small RNAs, including short hairpin RNAs (shRNAs) and single guide RNAs (sgRNAs). Functional U6 promoters are widely used in CRISPR systems, and their characterization can facilitate genome editing of non-model organisms. In the present study, six U6 small nuclear RNA (snRNA) promoters containing two conserved elements of a proximal sequence element (PSEA) and a TATA box, were identified and characterized in the diamondback moth (Plutella xylostella) genome. Relative efficiency of the U6 promoters to express shRNA induced EGFP knockdown was tested in a P. xylostella cell line, revealing that the PxU6:3 promoter had the strongest expression effect. Further work with the PxU6:3 promoter showed its efficacy in EGFP knockout using CRISPR/Cas9 system in the cells. The expression plasmids with versatile Pxabd-A gene specific sgRNA driven by the PxU6:3 promoter, combined with Cas9 mRNA, could induce mutagenesis at specific genomic loci in vivo. The phenotypes induced by sgRNA expression plasmids were similar to those done in vitro transcription sgRNAs. A plasmid with two tandem arranged PxU6:3:sgRNA expression cassettes targeting Pxabd-A loci was generated, which caused a 28,856 bp fragment deletion, suggesting that the multi-sgRNA expression plasmid can be used for multi-targeting. Our work indicates that U6 snRNA promoters can be used for functional studies of genes with the approach of reverse genetics in P. xylostella. These essential promoters also provide valuable potential for CRISPR-derived gene drive as a tactic for population control in this globally significant pest.
III型RNA聚合酶（Pol-III）启动子，如U6，通常用于表达小RNA，包括短发夹RNA（shRNA）和单向导RNA（sgRNA）。功能性U6启动子广泛应用于CRISPR系统，其特性鉴定有助于非模式生物的基因组编辑。在本研究中，在小菜蛾（Plutella xylostella）基因组中鉴定并表征了六个包含近端序列元件（PSEA）和TATA框这两个保守元件的U6小核RNA（snRNA）启动子。在小菜蛾细胞系中测试了U6启动子表达诱导EGFP敲低的shRNA的相对效率，结果表明PxU6:3启动子具有最强的表达效果。对PxU6:3启动子的进一步研究表明，其在细胞中使用CRISPR/Cas9系统敲除EGFP方面具有功效。由PxU6:3启动子驱动的具有通用Pxabd-A基因特异性sgRNA的表达质粒，与Cas9 mRNA结合，可在体内特定基因组位点诱导诱变。sgRNA表达质粒诱导的表型与体外转录sgRNA诱导的表型相似。构建了一个具有两个串联排列的靶向Pxabd-A位点的PxU6:3:sgRNA表达盒的质粒，该质粒导致28,856 bp片段缺失，表明多sgRNA表达质粒可用于多靶点。我们的工作表明，U6 snRNA启动子可用于小菜蛾中采用反向遗传学方法进行基因功能研究。这些重要的启动子也为CRISPR衍生的基因驱动提供了宝贵的潜力，作为控制这种全球重要害虫种群的一种策略。
