Using DIPA-CRISPR for simple and efficient endogenous protein tagging in insects.

CRISPR-Cas9 is rapidly expanding across diverse organisms. Among these advances, in-frame knockins of reporter genes have become essential for studying gene expression and protein localization. However, in hemimetabolan insects such as the German cockroach Blattella germanica, a phylogenetically basal and relevant pest species, functional fusion proteins have remained technically difficult to obtain. We present a streamlined gene-editing strategy to knock in a reporter gene in-frame with the distal-less gene, generating a functional fusion protein in B. germanica. By combining direct parental CRISPR with donor constructs designed for homology-directed repair carrying the mCherry gene, we successfully achieved targeted integration at the distal-less locus. The resulting fusion protein was functional and heritable and enabled live visualization of Distal-less protein distribution, showing fluorescence in developing appendages and the nervous system. This simple and robust methodology opens the door to generating fusion proteins in non-model insects, providing a valuable molecular tool for ecological, developmental, and pest-management research.