HUH-tagged Cas9 as a platform for efficient ssODN-mediated knock-in via embryo and adult injection in insects

Recent advances in adult injection-based insect genome editing have enabled genetic manipulation of a wide range of insect species, including those previously considered difficult or even impervious to genetic modification. However, achieving efficient knock-in remains a significant challenge with this approach. Here, we demonstrate that fusing a HUH endonuclease tag to Cas9 significantly enhances both non-homologous end joining (NHEJ)-mediated knockout and homology-directed repair (HDR)-mediated knock-in via adult injection. This fusion increased knockout efficiency by up to fivefold in the beetle Tribolium castaneum through adult injection, likely due to its previously unrecognized nuclear localization activity. It also improved single-stranded oligodeoxynucleotide (ssODN)-mediated knock-in efficiency, which we attribute to its characteristic ssDNA-tethering activity. To evaluate its versatility, we tested the HUH-tagged Cas9 in conventional embryo injection, which significantly enhanced HDR-mediated knock-in of an epitope tag in cricket and milkweed bug embryos. Our findings establish the HUH-tag as a versatile platform for improving both NHEJ- and HDR-based genome editing, providing a robust framework to advance genetic engineering across a broad spectrum of arthropods.