The CRISPR/Cas9 system is an efficient genome editing method that can be used in functional genomics research. The fall armyworm, Spodoptera frugiperda, is a serious agricultural pest that has spread over most of the world. However, very little information is available on functional genomics for this insect. We performed CRISPR/Cas9-mediated site-specific mutagenesis of three target genes: two marker genes [Biogenesis of lysosome-related organelles complex 1 subunit 2 (BLOS2) and tryptophan 2, 3-dioxygenase (TO)], and a developmental gene, E93 (a key ecdysone-induced transcription factor that promotes adult development). The knockouts (KO) of BLOS2, TO and E93 induced translucent mosaic integument, olive eye color, and larval-pupal intermediate phenotypes, respectively. Sequencing RNA isolated from wild-type and E93 KO insects showed that E93 promotes adult development by influencing the expression of the genes coding for transcription factor, Krüppel homolog 1, the pupal specifier, Broad-Complex, serine proteases, and heat shock proteins. Often, gene-edited insects display mosaicism in which only a fraction of the cells are edited as intended, and establishing a homozygous line is both costly and time-consuming. To overcome these limitations, a method to completely KO the target gene in S. frugiperda by injecting the Cas9 protein and multiple sgRNAs targeting one exon of the E93 gene into embryos was developed. Ten percent of the G0 larvae exhibited larval-pupal intermediates. The mutations were confirmed by T7E1 assay, and the mutation frequency was determined as >80%. Complete KO of the E93 gene was achieved in one generation using the multiple sgRNA method, demonstrating a powerful approach to improve genome editing in lepidopteran and other non-model insects.

中文翻译：

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秋季粘虫Spodoptera frugiperda中的基因组编辑：一代鉴定敲除的多种sgRNA / Cas9方法。

CRISPR / Cas9系统是一种有效的基因组编辑方法，可用于功能基因组学研究。秋天的夜蛾夜蛾（Spodoptera frugiperda）是一种严重的农业害虫，已扩散到世界大部分地区。但是，关于这种昆虫的功能基因组学的信息很少。我们对三个靶基因进行了CRISPR / Cas9介导的位点特异性诱变：两个标记基因[溶酶体相关细胞器复合物1亚基2（BLOS2）和色氨酸2、3-二加氧酶（TO）的生物发生]，以及一个发育基因， E93（蜕皮激素诱导的关键转录因子，可促进成人发育）。BLOS2，TO和E93的敲除（KO）分别诱导了半透明的镶嵌外皮，橄榄眼色和幼虫-pu中间表型。从野生型和E93 KO昆虫分离的RNA测序表明，E93通过影响编码转录因子，Krüppel同系物1，p指定子，宽复合体，丝氨酸蛋白酶和热休克蛋白的基因的表达来促进成年发育。通常，经基因编辑的昆虫表现出镶嵌性，其中只有一部分细胞按预期进行编辑，建立纯合系既昂贵又费时。为了克服这些局限性，开发了一种通过将Cas9蛋白和靶向E93基因一个外显子的多个sgRNA注射到胚胎中来完全将节俭链球菌中的靶基因KO的方法。10％的G0幼虫表现出幼虫-中间体。通过T7E1分析证实了突变，并且确定突变频率为＞ 80％。