Brady Daniel, Saviane Alessio, Cappellozza Silvia, Sandrelli Federica
Department of Biology, University of Padova, via U. Bassi 58/B, 35131 Padova, Italy.
Council for Agricultural Research and Economics, Research Centre for Agriculture and Environment, Sericulture Laboratory, 35143 Padova, Italy.
Methods Protoc. 2020 Dec 29;4(1):4. doi: 10.3390/mps4010004.
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.
家蚕作为鳞翅目遗传学的模式生物被广泛研究，并且在丝绸生产中具有经济价值。家蚕在生物医学和化妆品行业也有应用，突变品系的产生显著促进了家蚕的基础研究和应用研究。近年来，CRISPR/Cas9技术正迅速成为生成携带目标基因突变的家蚕品系的最有效分子工具。在此，我们阐述了一个完整且高效的工作流程，用于筛选、快速鉴定并追踪多代突变，从而生成稳定遗传单个CRISPR/Cas9诱导突变的品系。该方法依赖于使用不同的分子方法，即异源双链分析、克隆后进行桑格测序以及扩增不应性突变系统PCR。依次组合使用这些方法能够鉴定出常染色体和性染色体上基因的CRISPR/Cas9诱导突变，并选择合适的个体来建立稳定的突变品系。该方案可进一步应用于筛选单倍体昆虫中的CRISPR/Cas9突变。
