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

Area of Science:

• Genetics
• Molecular Biology
• Entomology

Background:

• Genome editing in insects often involves injecting early embryos, resulting in mosaic G0 individuals.
• Disrupting essential genes can lead to mosaic lethality, preventing adult survival and germline transmission of edited genes.

Purpose of the Study:

• To develop a practical method, DIL-CRISPR, to overcome G0 mosaic lethality in insect genome editing.
• To establish a generalizable strategy for generating mutant lines of mosaic lethal genes in diverse insect species.

Main Methods:

• Systematically diluting CRISPR/Cas9 injection mixes.
• Utilizing the tobacco cutworm Spodoptera litura and the Met1 gene as a model.
• Assessing G0 survival, mosaic phenotypes, and somatic/germline mutation frequencies via amplicon sequencing.

Main Results:

• Dilution of the injection mix reduced mosaic lethality and increased G0 survival in a dose-dependent manner.
• Somatic mutation frequencies decreased with dilution, while germline mutation rates remained sufficient for establishing mutant lines.
• A discrepancy between somatic and germline editing efficiencies was observed, suggesting selective loss of lethal mosaics.

Conclusions:

• DIL-CRISPR provides a reliable strategy to balance G0 survival with germline editing efficiency.
• This approach facilitates the generation of mutant lines for mosaic lethal genes in insects.
• The method is broadly applicable to functional genetic studies in various insect species, including non-model organisms.