Functional characterization of Pol III U6 promoters for gene knockdown and knockout in Plutella xylostella. (October 2017)

• Record Type: Journal Article
• Authors: Huang, Yuping; Wang, Yajun; Zeng, Baosheng; Liu, Zhaoxia; Xu, Xuejiao; Meng, Qian; Huang, Yongping; Yang, Guang; Vasseur, Liette; Gurr, Geoff M.; You, Minsheng
• Is Part Of: Insect biochemistry and molecular biology. Volume 89(2017)
• Page Start: 71
• Page End: 78
• Publication Date: 2017-10
• Subjects: CRISPR/Cas9 -- Diamondback moth -- Lepidoptera -- Pest control -- shRNA -- sgRNA; Insect biochemistry -- Periodicals; Insects -- Physiology -- Periodicals; Insects -- Molecular aspects -- Periodicals; Biochemistry -- Periodicals; Insectes -- Biochimie -- Périodiques; Insectes -- Composition -- Périodiques; Insectes -- Physiologie -- Périodiques; Insectes -- Aspect moléculaire -- Périodiques; Biochimie -- Périodiques; Biochemistry; Insect biochemistry; Insects -- Molecular aspects; Insects -- Physiology; Periodicals; 572.8157
• DOI: 10.1016/j.ibmb.2017.08.009
• Languages: English
• ISSNs: 0965-1748

Abstract

Abstract: 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. Graphical abstract: Highlights: Six candidate PxU6 promoters were identified and characterized from the genome of P. xylostella. EGFP specific shRNA expressed by different PxU6 promoters induced sufficient RNAi effect in P. xylostella cell line. PxU6:3 expressed sgRNAs combined with Cas9 induced effective disruption of EGFP expression in vitro. Pxabd-A sgRNA driven by PxU6:3 induced sequence specific mutagenesis and inherited defect phenotypes in vivo.