Activating cryptic biosynthetic gene cluster through a CRISPR-Cas12a-mediated direct cloning approach

Area of Science:

• Molecular Biology
• Genomics
• Natural Product Chemistry

Background:

• Direct cloning of microbial biosynthetic gene clusters (BGCs) is crucial for natural product drug discovery.
• Existing methods face challenges in capturing large and complex BGCs.

Purpose of the Study:

• To develop a fast and efficient platform for direct cloning of large BGCs.
• To demonstrate the utility of this platform in identifying novel natural products.

Main Methods:

• Development of CAT-FISHING (CRISPR/Cas12a-mediated fast direct biosynthetic gene cluster cloning).
• Utilizing Cas12a and bacterial artificial chromosome library construction for in vitro BGC capture.
• Heterologous expression of cloned BGCs in a Streptomyces chassis.

Main Results:

• CAT-FISHING successfully captured large BGCs (up to 145 kb, 75% GC content) from actinomycetal genomes.
• A 110 kb polyketide BGC was cloned and expressed, leading to the discovery of marinolactam A.
• Marinolactam A exhibited promising anticancer activity.

Conclusions:

• CAT-FISHING is a powerful and efficient method for cloning complex BGCs.
• This platform significantly advances natural product-based drug discovery efforts.
• The method is expected to be a valuable asset for the scientific community.