CRISPR/Cas9-Mediated Knockout of the White Gene in Agasicles hygrophila

<i>Agasicles hygrophila</i> is the most effective natural enemy for the control of the invasive weed <i>Alternanthera philoxeroides</i> (Mart.) Griseb. However, research on the gene function and potential genetic improvement of <i>A. hygrophila</i> is limited due to a lack of effective genetic tools. In this study, we employed the <i>A. hygrophila white</i> (<i>AhW</i>) gene as a target gene to develop a CRISPR/Cas9-based gene editing method applicable to <i>A. hygrophila</i>. We showed that injection of Cas9/sgRNA ribonucleoprotein complexes (RNPs) of the <i>AhW</i> gene into pre-blastoderm eggs induced genetic insertion and deletion mutations, leading to white eyes. Our results demonstrate that CRISPR/Cas9-mediated gene editing is possible in <i>A. hygrophila</i>, offering a valuable tool for studies of functional genomics and genetic improvement of <i>A. hygrophila</i>, which could potentially lead to more effective control of invasive weeds through the development of improved strains of this biocontrol agent. In addition, the white-eyed mutant strain we developed could potentially be useful for other transgenic research studies on this species.