CRISPR/Cpf1 multiplex genome editing system increases silkworm tolerance to BmNPV

Area of Science:

• Genetics
• Molecular Biology
• Entomology

Background:

• CRISPR/Cas9 technology is valuable for insect studies but can be further optimized for genome engineering.
• Multiplex genome editing offers a powerful approach to modify multiple genes simultaneously.

Purpose of the Study:

• To establish a CRISPR/Cpf1 multiplex genome editing system targeting multiple genes in the BmNPV genome.
• To assess the efficacy of this system in modifying target genes and its impact on silkworm traits and antiviral responses.

Main Methods:

• Developed a transgenic silkworm line targeting BmNPV ie-1, gp64, and DNApoly genes.
• Created a binary hybrid expression system (FnCpf1 × gNPVM) to activate the FnCpf1 gene editing system.
• Applied multiplex genome editing to introduce mutations, deletions, and insertions at three target sites.

Main Results:

• Successfully introduced multiple genetic modifications at three target sites without negatively impacting economic traits of transgenic silkworms.
• Demonstrated a significant increase in the antiviral response of the multiplexed genome editing lines.
• Showed significant disruption of viral gene transcription and replication in the modified silkworm lines.

Conclusions:

• The CRISPR/Cpf1 multiplex genome editing system is effective for simultaneous modification of multiple genes in insects.
• This technology provides a simplified platform for efficient insect multi-genome engineering and genetic operations.
• The developed transgenic silkworm lines offer innovative resistance materials for silkworm breeding.