A New-Generation Base Editor with an Expanded Editing Window for Microbial Cell Evolution In Vivo Based on...

Area of Science:

• Molecular Biology
• Genetics
• Biotechnology

Background:

• Base editors (BEs) are crucial for genome manipulation and cell evolution studies.
• Current BEs have limited editing windows (5-6 bases), restricting variant library diversity.
• Expanding the editing window is essential for efficient screening of targeted individuals.

Purpose of the Study:

• To develop a new generation of base editors (BEs) with an expanded editing window.
• To engineer a dCas12b-based CRISPRi system for gene expression repression.
• To enhance microbial cell evolution and screening efficiency.

Main Methods:

• Engineering CRISPR-Cas12b to create a dCas12b-based CRISPRi system.
• Developing new BEs based on dCas12b with an expanded editing window.
• Validating the new BEs in Bacillus subtilis and Escherichia coli.

Main Results:

• Established a dCas12b-based CRISPRi system for gene transcription repression.
• Developed novel BEs with an expanded editing window (entire protospacer or more) due to reduced steric hindrance.
• Successfully created a spectinomycin-resistant E. coli strain and increased protein secretion efficiency by 6.49-fold in E. coli.

Conclusions:

• The new BEs with expanded editing windows exponentially increase variant library capacity.
• This advancement significantly improves screening efficiency for microbial cell evolution.
• The dCas12b-based system offers a versatile tool for genome editing and gene regulation.