An Efficient Workflow for Screening and Stabilizing CRISPR/Cas9-Mediated Mutant Lines in .

The domestic silkworm is extensively studied as a model organism for lepidopteran genetics and has an economic value in silk production. Silkworms also have applications in biomedical and cosmetic industries, and the production of mutant strains significantly enhances basic and applied silkworm research. In recent years, CRISPR/Cas9 technology is being rapidly adopted as the most efficient molecular tool for generating silkworm lines carrying mutations in target genes. Here we illustrate a complete and efficient workflow to screen, characterize rapidly and follow mutations through generations, allowing the generation of lines, stably inheriting single CRISPR/Cas9-induced mutations. This approach relies on the use of different molecular methods, the heteroduplex assay, cloning followed by Sanger sequencing, and the amplification refractory mutation system PCR. The use of these methodologies in a sequential combination allows the identification of CRISPR/Cas9-induced mutations in genes mapping on both autosomes and sex chromosomes, and the selection of appropriate individuals to found stable mutant lines. This protocol could be further applied to screen CRISPR/Cas9 mutations in haploid insects.