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.
Directly cloning of biosynthetic gene clusters (BGCs) from even unculturable microbial genomes revolutionized nature products-based drug discovery. However, it is still very challenging to efficiently cloning, for example, the large (e.g. > 80kb) BGCs, especially for samples with high GC content in Streptomyces. In this study, by combining the advantages of CRISPR/Cas12a cleavage and bacterial artificial chromosome (BAC) library construction, we developed a simple, fast yet efficient in vitro platform for direct cloning of large BGCs based on CRISPR/Cas12a, named CAT-FISHING (CRISPR/Cas12a-mediated fast direct biosynthetic gene cluster cloning). It was demonstrated by the efficient direct cloning of large DNA fragments from bacterial artificial chromosomes or high GC (>70%) Streptomyces genomic DNA. Moreover, surugamides, encoded by a captured 87-kb gene cluster, was expressed and identified in a cluster-free Streptomyces chassis. These results indicate that CAT-FISHING is now poised to revolutionize bioactive small molecules (BSMs) drug discovery and lead a renaissance of interest in microorganisms as a source of BSMs for drug development. SIGNIFICANCE STATEMENTNatural products (NPs) are one of the most important resources for drug leads. One bottleneck of NPs-based drug discovery is the inefficient cloning approach for BGCs. To address it, we established a simple, fast and efficient BGC directed cloning method CAT-FISHING by combining the advantages of CRISPR/Cas12a (e.g. high specificity) and bacterial artificial chromosome (BAC) library (e.g. large DNA fragment and high GC content). As demonstrations, a series of DNA fragments ranging from 49 kb to 139 kb were successfully cloned. After further optimization, our method was able to efficiently clone and express an 87-kb long, GC-rich (76%) surugamides BGC in a Streptomyces chassis with reduced time-cost. CAT-FISHING presented in this study would much facilitate the process of NPs discovery.cosmid, fosmid and BAC) construction, recombination-based or RecET/Redαβ-based cloning, and Gibson assembly etc (Table 1). Additionally, the emergence of the CRISPR/Cas9 technique has enabled several new DNA cloning methods such as ExoCET, and CATCH etc (3-5). However, it is relatively cumbersome and time-consuming of using the aforementioned methods. Simple, fast and efficient strategy for large BGCs, especially with high GC content, is in urgent need to make natural BSMs accessible and affordable.
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