Cas12a-assisted precise targeted cloning using in vivo Cre-lox recombination

Direct cloning represents the most efficient strategy to access the vast number of uncharacterized natural product biosynthetic gene clusters (BGCs) for the discovery of novel bioactive compounds. However, due to their large size, repetitive nature, or high GC-content, large-scale cloning of these BGCs remains an overwhelming challenge. Here, we report a scalable direct cloning method named Cas12a-assisted precise targeted cloning using in vivo Cre-lox recombination (CAPTURE) which consists of Cas12a digestion, a DNA assembly approach termed T4 polymerase exo + fill-in DNA assembly, and Cre-lox in vivo DNA circularization. We apply this method to clone 47 BGCs ranging from 10 to 113 kb from both Actinomycetes and Bacilli with ~100% efficiency. Heterologous expression of cloned BGCs leads to the discovery of 15 previously uncharacterized natural products including six cyclic head-to-tail heterodimers with a unique 5/6/6/6/5 pentacyclic carbon skeleton, designated as bipentaromycins A–F. Four of the bipentaromycins show strong antimicrobial activity to both Gram-positive and Gram-negative bacteria such as methicillin-resistant Staphylococcus aureus, vancomycinresistant Enterococcus faecium, and bioweapon Bacillus anthracis. Due to its robustness and efficiency, our direct cloning method coupled with heterologous expression provides an effective strategy for large-scale discovery of novel natural products.

Simple cloning of large natural product biosynthetic gene cluster by CRISPR/Cas12a-mediated fast direct capturing strategy

ABSTRACTDirectly 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.TABLE OF CONTENTSSIGNIFICANCE 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.