Abstract
CRISPR-Cas9 has become one of the most prominent gene editing tools available and it has been utilized in various organisms from bacteria to fungi, plants, and animals. In this study, we developed a CRISPR-Cas9 protocol for the black garden ant Lasius niger, a common and easily available study species for lab and field experiments. To create indel mutations using CRISPR-Cas9 in L. niger, we targeted three different locations in a well-studied eye pigmentation gene cinnabar, generating several mutations that disrupt the ommochrome biosynthesis pathway and result in the lack of the pigment and therefore, abnormal eye coloration in adult workers. We also developed a protocol to collect L. niger eggs, inject them with CRISPR-Cas9 construct, and rear the eggs into mature adult workers with the assistance of nursing workers. We demonstrated for the first time in L. niger that CRISPR-Cas9 is an excellent tool to create targeted mutations for this species. Our protocol can be referred to when developing similar studies for other species of ants and eusocial insects.