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

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 <1 h old, sixty-two successfully developed to adults, among which mutation in the vest loci was confirmed in 5 of the 18 wingless CPBs (29% phenotypic mutation efficiency). The mutation in vest resulted in a clear phenotype in the CPBs, which developed to adulthood with no hindwing and elytron formed. Altogether, this study provides for the first time a useful methodology involving the use of the CRISPR/Cas9 system for mutagenesis studies in one of the most important pest insects.