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Co-CRISPR: A valuable toolkit for mutation enrichment in the gene editing of Spodoptera frugiperda

文献类型： 外文期刊

作者： Han, Wei-Kang 1 ; Tang, Feng-Xian 1 ; Gao, Hao-Li 1 ; Wang, Yan 1 ; Yu, Na 1 ; Jiang, Jian-Jun 2 ; Liu, Ze-Wen 1 ;

作者机构： 1.Nanjing Agr Univ, Coll Plant Protect, Key Lab Integrated Management Crop Dis & Pests, Minist Educ, Nanjing, Peoples R China

2.Guangxi Acad Agr Sci, Plant Protect Res Inst, Guangxi Key Lab Biol Crop Dis & Insect Pests, Nanning, Peoples R China

关键词： co-CRISPR; kmo; mutation enrichment; Spodoptera frugiperda; Wnt1

期刊名称：INSECT SCIENCE （ 影响因子：3.605； 五年影响因子：3.323 ）

ISSN： 1672-9609

年卷期：

页码：

收录情况： SCI

摘要： The CRISPR/Cas9 system has been successfully applied in dozens of diverse species; although the screening of successful CRISPR/Cas9 editing events remains particularly laborious, especially for those that occur at relatively low frequency. Recently, a co-CRISPR strategy was proved to enrich the desired CRISPR events. Here, the co-CRISPR strategy was developed in the Fall armyworm, Spodoptera frugiperda, with kynurenine 3-monooxygenase gene (kmo) as a marker. The kmo mosaics induced by single-guide RNAs (sgRNAs)/Cas9 displayed the darker green color phenotype in larvae, compared with wild type (brown), and mosaic-eye adults were significantly acquired from the mosaic larvae group. In the kmo knockout strain, no significant difference was observed in larval development and adult reproduction. Acetylcholinesterase 2 (ace2) and Wnt1 were selected as target genes to construct the co-CRISPR strategy using kmo marker. By co-injection of kmo and ace2 sgRNAs, the mutant efficiency of ace2 was significantly increased in the kmo mosaic (larvae or adults) groups. Similarly, more malformed pupae with Wnt1 mutations were observed in the darker green larvae group. Taken together, these results demonstrated that kmo was a suitable visible marker gene for the application and extension of co-CRISPR strategy in Fall armyworm. Using darker green color in larvae or mosaic-eye in adults from kmo knockout as a marker, the mutant efficiency of a target gene could be enriched in a Fall armyworm group consisting of marked individuals. The co-CRISPR strategy is helpful for gene function studies by the knockout technique with no or lethal phenotypes.

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