CRISPR/Cas9 Assisted Multiplex Genome Editing Technique in Escherichia coli

Area of Science:

• Synthetic Biology
• Molecular Biology
• Microbial Engineering

Background:

• Genome editing is crucial for biological research and microbial cell factory development.
• Conventional methods typically target single genomic loci, limiting efficiency for complex modifications.
• Simultaneous editing of multiple loci is essential for engineering advanced microbial systems.

Purpose of the Study:

• To develop a CRISPR/Cas9 assisted multiplex genome editing (CMGE) technique for simultaneous modification of multiple genomic loci in Escherichia coli.
• To improve the efficiency and applicability of multiplex genome editing in prokaryotic organisms.
• To provide a versatile and accessible tool for microbial genetic engineering.

Main Methods:

• Developed a CMGE technique utilizing CRISPR/Cas9 system in Escherichia coli.
• Assembled functional genetic parts onto replicable plasmids.
• Employed stringent inducible expression systems to control Cas9 gene expression, decoupling transformation from editing.
• Designed a modular assembly strategy for constructing complex multi-guide RNA (gRNA) plasmids.

Main Results:

• Achieved 100% efficiency for editing two loci and 88.3% efficiency for editing three loci in Escherichia coli.
• Demonstrated over 30% efficiency for editing four loci, representing state-of-the-art results.
• The CMGE technique showed high efficiency and adaptability for multiplex genome engineering.

Conclusions:

• The developed CMGE technique enables efficient simultaneous editing of multiple genomic loci in Escherichia coli.
• This method offers a robust and adaptable strategy for microbial genetic engineering, applicable to other prokaryotic systems.
• The CMGE technique is user-friendly and requires no special laboratory equipment, facilitating its adoption.