A Golden Gate-Compatible CRISPR-Associated Transposon Tool for Multiplexed Bacterial Genome Editing

Area of Science:

• Synthetic Biology
• Bacterial Genetics
• CRISPR Technology

Background:

• Genome integration offers enhanced robustness and stability for large genetic circuits in bacteria.
• CRISPR-associated transposases (CASTs) facilitate RNA-guided DNA insertion without double-stranded breaks, functioning across diverse bacterial species.

Purpose of the Study:

• To present pSPIN-GG, an improved tool and protocol for simplified CAST-based bacterial genome engineering.
• To enhance the efficiency and accessibility of multiplexed genome engineering using CAST systems.

Main Methods:

• Development of pSPIN-GG with Golden Gate-compatible modules (promoter, guide, cargo) for easy assembly.
• Incorporation of a green fluorescent protein (GFP) dropout cassette for rapid verification of guide replacement.
• Identification and utilization of tested genomic sites in Escherichia coli BL21 for precise gene dosing.

Main Results:

• The pSPIN-GG system streamlines the assembly and screening process for genetic circuit insertion.
• Demonstrated accelerated library construction and reduced experimental burden.
• Expanded the accessibility of CAST systems for multiplexed genome engineering applications.

Conclusions:

• pSPIN-GG represents a significant refinement for CAST-based genome engineering, improving ease of use and efficiency.
• The system facilitates accelerated construction of genetic libraries and multiplexed genome engineering in bacteria.
• This tool enhances the accessibility of advanced synthetic biology techniques for researchers.