Amplicon-seq of CRISPR/Cas9-mediated genome editing to knockout Laccase2 gene in pea aphids

This study focuses on developing and optimizing CRISPR/Cas9-mediated gene knockout techniques for the pea aphid, Acyrthosiphon pisum, an insect with complex biology that includes cyclic parthenogenesis and diapause. Such traits present significant challenges to traditional genome-editing methods. The target gene, Laccase2 (Lac2), encodes phenol oxidases involved in cuticle hardening and pigmentation in other insects. We hypothesized that Lac2 plays a critical role in aphid egg pigmentation and survival during diapause. To test this, we used CRISPR/Cas9 ribonucleoprotein (RNP) microinjections and a new method called Directly Parental CRISPR (DIPA-CRISPR) to knock out Lac2. The resulting Lac2 knockout (KO) individuals (generation zero; G0) showed a high rate of pigment-less eggs, with pigmentation loss correlating with indel rates induced by the CRISPR system. Homozygous Lac2 KOs produced eggs with complete loss of melanization, which did not hatch, indicating that Lac2 is essential for melanization and viability during diapause. Diapause KO eggs occasionally contained late-stage embryos with developed eyes, suggesting that lethality may occur in late embryogenesis or during hatching. In G0 nymphs, mosaic knockout phenotypes were observed, showing partial pigmentation loss in the cuticle and compound eyes, suggesting that Lac2 may also contribute to postembryonic pigmentation. This study also enhanced the CRISPR/Cas9 gene knockout efficiency through egg microinjections, achieving stable G0 knockouts and enabling germline transmission of the KO allele to produce stable knockout lines. Furthermore, DIPA-CRISPR demonstrated effective indel mutation induction in fertilized eggs via body cavity injections in adult females. Overall, this research highlights Lac2's critical role in aphid development, pigmentation, and hatching, especially in overwintering eggs, and establishes an improved genome-editing protocol suitable for A. pisum, supporting broader applications in evolution, ecology, developmental biology, and agriculture research.