Programmable Single and Multiplex Base-Editing in Bombyx mori Using RNA-Guided Cytidine Deaminases

Area of Science:

• Genetics
• Molecular Biology
• Biotechnology

Background:

• Standard genome editing tools like CRISPR/Cas9 rely on DNA double-strand breaks.
• Recent advancements include CRISPR-based base editors that enable precise base conversions without double-strand breaks.
• These base editors have shown promise in various organisms.

Purpose of the Study:

• To demonstrate the efficacy of CRISPR/Cas9-dependent base editor 3 (BE3) for C-to-T base editing in the invertebrate Bombyx mori (silkworm).
• To evaluate BE3 as a tool for gene inactivation through base-editing-induced nonsense mutations.
• To assess the targetability and editing window of BE3 in the silkworm genome.

Main Methods:

• Utilized the CRISPR/Cas9-dependent base editor 3 (BE3) system in Bombyx mori.
• Applied BE3 for gene inactivation via base-editing-induced nonsense mutations.
• Performed genome-scale analysis to identify targetable sites and assessed editing efficiency and window.
• Co-transfected multiple guide RNAs (gRNAs) to evaluate multiplex editing capabilities.

Main Results:

• BE3 efficiently converted C to T in Bombyx mori with high frequency.
• Gene inactivation efficiency reached up to 66.2% using BE3-induced nonsense mutations.
• 96.5% of B. mori genes possess targetable sites for BE3-mediated inactivation, with a median of 11 sites per gene.
• The editing window extended up to 13 bases, and simultaneous base substitutions in up to 14 bases were observed with low indel frequency (0.6%) when 32 gRNAs were co-transfected.

Conclusions:

• RNA-guided cytidine deaminases (base editors) are capable of programmable single and multiplex base editing in an invertebrate model (Bombyx mori).
• BE3 is a powerful tool for gene knockout and precise genome modification in silkworms.
• This study expands the application of base editing technology to invertebrates, opening new avenues for genetic research and manipulation.