Multiplexed sgRNA Expression Allows Versatile Single Nonrepetitive DNA Labeling and Endogenous Gene Regulation

Area of Science:

• Molecular Biology
• Synthetic Biology
• Genomics

Background:

• CRISPR/Cas9 technology is crucial for genome editing, gene regulation, and chromatin studies.
• Multiplexed biological investigations necessitate simultaneous expression of multiple single-guide RNAs (sgRNAs).
• Existing sgRNA coexpression methods (cotransfection, viral vectors) face challenges with efficiency, cost, and labor.

Purpose of the Study:

• To establish a novel, vector-independent method for efficient, scalable, and controllable coexpression of multiple sgRNAs.
• To demonstrate the utility of this system in live-cell applications like genomic locus imaging and coordinated gene regulation.

Main Methods:

• Developed a synthetic biology strategy to assemble multiple sgRNA expression cassettes in series within a single plasmid.
• Utilized this all-in-one sgRNA expression system for live-cell imaging of genomic loci.
• Applied the system for coordinated gene regulation studies in live cells.

Main Results:

• Successfully created a vector-independent method for assembling multiple sgRNA expression cassettes.
• Demonstrated the system's efficiency, controllability, and scalability.
• Showcased applications in genomic locus imaging and coordinated gene regulation in live cells.

Conclusions:

• The developed all-in-one sgRNA expression system offers a flexible and potent solution for multiplexed CRISPR applications.
• This method significantly advances research in understanding genome structure, function, and dynamics.
• Facilitates high-throughput and systematic investigations in complex biological systems.