First report on CRISPR/Cas9-targeted mutagenesis in the Colorado potato beetle, Leptinotarsa decemlineata

Abstract Leptinotarsa decemlineata (Say), commonly known as the Colorado potato beetle (CPB), is an agricultural important pest for potatoes and other solanaceous plants. The CRISPR/Cas system is an efficient genome editing technology, which could be exploited to study the biology of CPB and possibly also lead to the development of better environmentally friendly pest management strategies. However, the use of CRISPR/Cas9 has been limited to only a few model insects. Here, for the first time, a CRISPR/Cas9 protocol for mutagenesis studies in CPB was developed. A gene with a clear phenotype such as the vestigial gene (vest), known to be involved in wing development in other insect species, was selected as a good indicator for the knockout study. First, vest was functionally characterized in CPB by using RNAi technology for knockdown studies. Once the expected deformed wing phenotypes were observed, a CRISPR/Cas9 work flow was established for mutagenesis in CPB. By co-injecting the Cas9 protein and a vest-guide RNA into 539 CPB eggs of