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<p>The CRISPR/Cas9 system is a powerful gene-editing method that has been successful applied in a wide variety of insects, including Lepidoptera, for gene function studies. However, the majority of gene-edited insects showed mosaicism and required further serial crossbreeding to establish homozygous strains for phenotypic analysis. Such a procedure is laborious and time-consuming and greatly restricts the progress of functional genomics research in lepidopterans. Here, we report a simple CRISPR/Cas9-based technique for rapid genetic screening in the lepidopteran pest <i>Helicoverpa armigera</i>. Redundant targeting of single genes with multiple sgRNAs at distinct sites consistently produced complete knockout phenotypes in 40–80% of the G<sub>0</sub> individuals. Simultaneous dual-gene disruption in G<sub>0</sub> was also feasible by redundantly targeting each gene. We developed a pooled multi-sgRNA synthesis method to reduce the cost and increase throughput of sgRNA production. Using the redundant targeting technique, we performed a reverse genetic screen in G<sub>0</sub> and uncovered a role for <i>chitin synthase 2</i> in the development of resistance to the <i>Bacillus thuringiensis</i> Vip3Aa toxin in <i>H. armigera</i>. This study developed a simple and efficient G<sub>0</sub> screening method in <i>H. armigera</i>. This method also provides a framework for large-scale screening of genes of interest in other lepidopterans.</p>

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Redundant targeting with multiple sgRNAs enables rapid genetic screening in the lepidopteran pest Helicoverpa armigera

  • 期刊论文

  • 发表日期：

    2025年12月9日

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  • Yonghui Song,
  • Jie Wang,
  • Xiaohan Yao,
  • Chang Liu,
  • Lu Gao,
  • Lisi Wang,
  • Yihua Yang,
  • Yidong Wu,
  • Ya-Zhou He

摘要

The CRISPR/Cas9 system is a powerful gene-editing method that has been successful applied in a wide variety of insects, including Lepidoptera, for gene function studies. However, the majority of gene-edited insects showed mosaicism and required further serial crossbreeding to establish homozygous strains for phenotypic analysis. Such a procedure is laborious and time-consuming and greatly restricts the progress of functional genomics research in lepidopterans. Here, we report a simple CRISPR/Cas9-based technique for rapid genetic screening in the lepidopteran pest Helicoverpa armigera. Redundant targeting of single genes with multiple sgRNAs at distinct sites consistently produced complete knockout phenotypes in 40–80% of the G0 individuals. Simultaneous dual-gene disruption in G0 was also feasible by redundantly targeting each gene. We developed a pooled multi-sgRNA synthesis method to reduce the cost and increase throughput of sgRNA production. Using the redundant targeting technique, we performed a reverse genetic screen in G0 and uncovered a role for chitin synthase 2 in the development of resistance to the Bacillus thuringiensis Vip3Aa toxin in H. armigera. This study developed a simple and efficient G0 screening method in H. armigera. This method also provides a framework for large-scale screening of genes of interest in other lepidopterans.