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Issue:ISSN 2095-1353
           CN 11-6020/Q
Director:Chinese Academy of Sciences
Sponsored by:Chinese Society of Entomological;institute of zoology, chinese academy of sciences;
Address:Chaoyang District No. 1 Beichen West Road, No. 5 hospital,Beijing City,100101, China
Your Position :Home->Past Journals Catalog->2018年55 No.2

Construction of a CRISPR vector to edit the β-fructofuranosidase gene BmSuc1 and its incorporation in the silkworm Bombyx mori
Author of the article:ZHU Wen-Kai1** GAN Quan1, 2** HE Wei1 ZHANG Xin-Wei1, 2 ZHOU Yue1 WU Meng-Xue1 SUN Tong-Tong1
Author's Workplace:1.School of Life Sciences, Anhui Agricultural University, Hefei 230036, China; 2.Anhui International Joint Research and Development Center of Sericulture Resources Utilization, Hefei 230036, China
Key Words:CRISPR/Cas9, genome edition, sgRNA, Bombyx mori, β-fructofuranosidase

 [Objectives]  CRISPR/Cas9 is a novel genome-editing tool that has recently had broad application in many fields of research. CRISPR/Cas9 has the advantages of simplicity, efficiency and high specificity. BmSuc1 encodes β-fructofuranosidase (β-FFase, a fructosyl-hydrolytic sucrase) in the silkworm Bombyx mori (Lepidoptera). BmSuc1 is also the first β-FFase gene to have been cloned and identified in an animal. The physiological function of BmSUC1 in the silkworm is probably related to defense against mulberry alkaloids. However, further research is needed to confirm this. In order to clarify the role of BmSUC1 we used the CRISPR/Cas9 genome-editing system to construct a dual-sgRNA expressing vector. [Methods]  We first designed two sgRNAs against BmSuc1 ORF and inserted these into the Sal I and Nhe I restriction enzyme sites of a CRISPER edition vector using the homologous recombination method. We then microinjected the recombinant CRISPR plasmid into G0 silkworm eggs and created a G1 generation by G0 sib-crossing. [Results]  Both sgRNA insertions were verified by polymerase chain reaction and sequencing. DsRed2 fluorescence confirmed that we successfully obtained positive transgenic G1 individuals. [Conclusion]  This study provides a detailed description of the construction of a CRISPR edition vector to express dual sgRNAs. The resultant positive transgenic silkworms will help clarify the biological role of BmSuc1-coding β-FFase in the silkworm.

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