CRISPR/Cas9 technology is a very powerful genome editing tool and has been used in many insect species for functional genomics studies through targeted gene mutagenesis. Here, we successfully established CRISPR/Cas9 research platform in Asian multi-colored ladybird beetle, Harmonia axyridis, an important natural enemy in biological control. In this study, one pivotal gene dopa decarboxylase (DDC) in melanin synthesis was targeted by CRISPR/Cas9 to generate mutants in H. axyridis by CRISPR/Cas9 technology. Our results showed that injection of single guide RNA of the DDC and Cas9 protein into preblastoderm eggs induced one insertion and four deletion (indels) mutant H. axyridis. Mutations of HaDDC gene generated 25% mutant rate with melanin missing phenotype in larva, pupa,l and adult stage. The predation ability of the fourth instar larvae has no significant difference between wild (control) and mutant H. axyridis (G0), while these mutant fourth instar larvae had longer developmental period than that of the wild type. Consequently, the total predation of the fourth instar larvae was significantly increased in H. axyridis mutants comparing with the wild type. These results indicated that the success of CRISPR/Cas9 gene editing in H. axyridis. The gene editing platform in H. axyridis would facilitate the gene function research and promote special strain of predatory ladybird beetle generation.

中文翻译：

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黑色素合成途径中断：CRISPR/Cas9 介导的异色瓢虫（鞘翅目：瓢虫科）中的多巴脱羧酶 (DDC) 敲除

CRISPR/Cas9 技术是一种非常强大的基因组编辑工具，已通过靶向基因诱变在许多昆虫物种中用于功能基因组学研究。在这里，我们成功建立了亚洲多色瓢虫异色瓢虫这一生物防治的重要天敌的CRISPR/Cas9研究平台。在这项研究中，CRISPR/Cas9 靶向黑色素合成中的一个关键基因多巴脱羧酶 (DDC)，通过 CRISPR/Cas9 技术在异色瓢虫中产生突变体。我们的研究结果表明，将 DDC 和 Cas9 蛋白的单向导 RNA 注射到前胚层卵中会诱导一个插入和四个缺失 (indels) 突变体异色瓢虫。HaDDC 基因的突变产生了 25% 的突变率，在幼虫、蛹、1 和成虫阶段具有黑色素缺失表型。四龄幼虫的捕食能力在野生型（对照）和突变型异色瓢虫（G0）之间没有显着差异，而这些突变型四龄幼虫的发育期比野生型长。因此，与野生型相比，异色瓢虫突变体中第四龄幼虫的总捕食量显着增加。这些结果表明CRISPR/Cas9基因编辑在异色瓢虫中的成功。异色瓢虫基因编辑平台将促进基因功能研究，促进捕食性瓢虫特殊品系的产生。异色瓢虫突变体4龄幼虫的总捕食量较野生型显着增加。这些结果表明CRISPR/Cas9基因编辑在异色瓢虫中的成功。异色瓢虫基因编辑平台将促进基因功能研究，促进捕食性瓢虫特殊品系的产生。异色瓢虫突变体4龄幼虫的总捕食量较野生型显着增加。这些结果表明CRISPR/Cas9基因编辑在异色瓢虫中的成功。异色瓢虫基因编辑平台将促进基因功能研究，促进捕食性瓢虫特殊品系的产生。