USDA ARS, U.S. Arid Land Agricultural Research Center, Maricopa, AZ, 85138, USA.
Sci Rep. 2022 Mar 22;12(1):4917. doi: 10.1038/s41598-022-08908-4.
The western tarnished plant bug, Lygus hesperus, is a key hemipteran pest of numerous agricultural, horticultural, and industrial crops in the western United States and Mexico. A lack of genetic tools in L. hesperus hinders progress in functional genomics and in developing innovative pest control methods such as gene drive. Here, using RNA interference (RNAi) against cardinal (LhCd), cinnabar (LhCn), and white (LhW), we showed that knockdown of LhW was lethal to developing embryos, while knockdown of LhCd or LhCn produced bright red eye phenotypes, in contrast to wild-type brown eyes. We further used CRISPR/Cas9 (clustered regularly interspaced palindromic repeats/CRISPR-associated) genome editing to generate germline knockouts of both LhCd (Card) and LhCn (Cinn), producing separate strains of L. hesperus characterized by mutant eye phenotypes. Although the cardinal knockout strain Card exhibited a gradual darkening of the eyes to brown typical of the wild-type line later in nymphal development, we observed bright red eyes throughout all life stages in the cinnabar knockout strain Cinn, making it a viable marker for tracking gene editing in L. hesperus. These results provide evidence that CRISPR/Cas9 gene editing functions in L. hesperus and that eye pigmentation genes are useful for tracking the successful genetic manipulation of this insect.
西方玷污叶蝉，Lygus hesperus，是美国西部和墨西哥众多农业、园艺和工业作物的重要半翅目害虫。L. hesperus 缺乏遗传工具，这阻碍了功能基因组学的进展，并阻碍了基因驱动等创新害虫控制方法的发展。在这里，我们使用针对 cardinal（LhCd）、cinnabar（LhCn）和 white（LhW）的 RNA 干扰（RNAi），表明 LhW 的敲低对发育中的胚胎是致命的，而 LhCd 或 LhCn 的敲低产生了明亮的红眼表型，与野生型棕色眼睛形成对比。我们进一步使用 CRISPR/Cas9（成簇的规律间隔的短回文重复序列/CRISPR 相关）基因组编辑来生成 LhCd（Card）和 LhCn（Cinn）的种系敲除，产生了具有突变眼表型的 L. hesperus 分离株。尽管 cardinal 敲除株 Card 在若虫发育后期眼睛逐渐变暗至棕色，与野生型系典型的颜色相似，但我们在 cinnabar 敲除株 Cinn 中观察到所有生命阶段都有明亮的红眼，使其成为跟踪 L. hesperus 基因编辑的可行标记。这些结果表明 CRISPR/Cas9 基因编辑在 L. hesperus 中起作用，并且眼色素基因可用于跟踪该昆虫的成功遗传操作。
