DIL-CRISPR: a practical approach to mitigate G₀ mosaic lethality in insect gene editing

Genome editing in insects is typically conducted by injecting genome editing reagents into early embryos, producing generation zero (G₀) individuals that develop as genetic mosaics. Targeting genes whose disruption induces mosaic lethality is therefore challenging, since most or all G₀ individuals frequently fail to survive to adulthood, preventing germline transmission of edited alleles. Here, we present a straightforward and practical approach, DIL-CRISPR, to mitigate G₀ mosaic lethality by systematically diluting the CRISPR/Cas9 injection mix. Using the tobacco cutworm Spodoptera litura and the juvenile hormone receptor gene Met1 as a benchmark, we demonstrate that dilution of the injection mix lessens the severity of larval-pupal mosaic phenotypes and increases G₀ survival in a dose-dependent manner. Amplicon sequencing further showed that somatic mutation frequencies decline with dilution, while germline mutation rates remain sufficient to establish mutant lines. Notably, we detected a substantial discrepancy between somatic and germline editing efficiencies, likely reflecting selective loss of highly edited, lethal mosaics before they reach adulthood. We conclude that DIL-CRISPR therefore offers a reliable means to balance G₀ survival with germline editing, converting an empirically used dilution practice into a generalizable strategy. Overall, this approach provides a practical solution for generating mutant lines of mosaic lethal genes and is broadly applicable across diverse insect species, facilitating functional genetic studies in non-model insects.