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

Direct cloning of biosynthetic gene clusters (BGCs) from microbial genomes facilitates natural product-based drug discovery. Here, by combining Cas12a and the advanced features of bacterial artificial chromosome library construction, we developed a fast yet efficient in vitro platform for directly capturing large BGCs, named CAT-FISHING (CRISPR/Cas12a-mediated fast direct biosynthetic gene cluster cloning). As demonstrations, several large BGCs from different actinomycetal genomic DNA samples were efficiently captured by CAT-FISHING, the largest of which was 145 kb with 75% GC content. Furthermore, the directly cloned, 110 kb long, cryptic polyketide encoding BGC from Micromonospora sp. 181 was then heterologously expressed in a Streptomyces chassis. It turned out to be a new macrolactam compound, marinolactam A, which showed promising anticancer activity. Our results indicate that CAT-FISHING is a powerful method for complicated BGC cloning, and we believe that it would be an important asset to the entire community of natural product-based drug discovery.

Automated summary

In this study, a method named CAT-FISHING was developed to directly capture large biosynthetic gene clusters (BGCs) by combining Cas12a and the advanced features of bacterial artificial chromosome library construction. Several large BGCs from different actinomycetal genomic DNA samples were efficiently captured using CAT-FISHING, and a new macrolactam compound with promising anticancer activity was discovered.