Functional analysis of Bombyx Wnt1 during embryogenesis using the CRISPR/Cas9 system

Area of Science:

• Molecular Biology
• Developmental Biology
• Genetics

Background:

• CRISPR-associated (CRISPR) systems offer advanced genome editing capabilities.
• CRISPR/Cas9 has enabled targeted gene mutagenesis in some insect species, but its application is limited outside of model organisms like Drosophila and Bombyx mori due to a lack of effective tools.

Purpose of the Study:

• To develop a simple and effective method for gene knockout analysis in insects, specifically for studying gene function during embryogenesis.
• To functionally analyze the Bombyx Wnt1 (BmWnt1) gene using CRISPR/Cas9-mediated mutagenesis in the silkworm, Bombyx mori.

Main Methods:

• CRISPR/Cas9 system utilizing Cas9 mRNA and BmWnt1-specific single-guide RNA (sgRNA).
• Direct injection of CRISPR/Cas9 components into Bombyx mori embryos.
• Phenotypic analysis of injected embryos, including hatching rates, body segmentation, and pigmentation.
• Quantitative real-time PCR (qRT-PCR) to assess gene expression changes (Hox genes).
• Analysis of generated DNA deletions.

Main Results:

• Direct injection of Cas9 mRNA and BmWnt1 sgRNA into Bombyx embryos resulted in a Wnt1-deficient phenotype.
• Injected embryos exhibited dose-dependent defects in hatching, body segmentation, and pigmentation.
• Quantitative real-time PCR revealed down-regulation of Hox genes following BmWnt1 depletion.
• CRISPR/Cas9 mediated large genomic deletions, up to 18Kb, at the target locus.

Conclusions:

• The CRISPR/Cas9 system is a viable and promising tool for targeted gene mutagenesis in insect embryogenesis.
• This approach facilitates the functional analysis of genes, such as BmWnt1, crucial for embryonic development in insects.
• The study highlights the utility of CRISPR/Cas9 for dissecting gene function in non-model insect species.