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

Area of Science:

• Genetics
• Molecular Biology
• Entomology

Background:

• The domestic silkworm (Bombyx mori) is a key model organism in lepidopteran genetics and significant for silk production.
• Mutant strains of Bombyx mori are crucial for advancing basic and applied research in biomedical and cosmetic applications.
• CRISPR/Cas9 technology has emerged as a highly efficient tool for generating targeted mutations in Bombyx mori.

Purpose of the Study:

• To establish and demonstrate a complete and efficient workflow for generating stable mutant Bombyx mori lines.
• To enable rapid screening, characterization, and generational tracking of CRISPR/Cas9-induced mutations.
• To facilitate the creation of Bombyx mori lines with stably inherited single gene mutations.

Main Methods:

• Utilized a sequential combination of molecular methods including heteroduplex assay, cloning with Sanger sequencing, and Amplification Refractory Mutation System (ARMS) PCR.
• Applied these methods to screen and characterize CRISPR/Cas9-induced mutations.
• Focused on identifying mutations in genes located on both autosomes and sex chromosomes.

Main Results:

• Successfully generated Bombyx mori lines with stably inherited single CRISPR/Cas9-induced mutations.
• Demonstrated the efficacy of the workflow in identifying mutations across different chromosome types.
• Validated the selection process for establishing stable mutant lines.

Conclusions:

• The developed workflow provides a robust and efficient approach for generating stable mutant silkworm lines.
• This protocol significantly aids in the study of gene function in Bombyx mori through precise genetic manipulation.
• The methodology holds potential for application in screening CRISPR/Cas9 mutations in other haploid insects.