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

Liang, M.; Liu, L.; Wang, W.; Zeng, X.; Liu, J.; Loganathan, K.; Zhu, G.; Bai, L.; Hou, C.; Chen, X.; Ouyang, L.; Liu, X.; Hu, B.; Xia, X.; Tong, Y.; Lou, C.; Alterovitz, G.; Tan, G.-Y.; Zhang, L.-X.

doi:10.1101/2020.06.25.170191

bioRxiv · 10.1101/2020.06.25.170191

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

Liang, M.·Liu, L.·Wang, W.·Zeng, X.·Liu, J.·Loganathan, K.·Zhu, G.·Bai, L.·Hou, C.·Chen, X.·Ouyang, L.·Liu, X.·Hu, B.·Xia, X.·Tong, Y.·Lou, C.·Alterovitz, G.·Tan, G.-Y.·Zhang, L.-X.

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Abstract

Directly cloning of biosynthetic gene clusters (BGCs) from microbial genomes has been revolutionizing the natural product-based drug discovery. However, it is still very challenging to efficiently clone, for example, large (> 80kb) and GC-rich (> 70%), streptomycete originating BGCs. In this study, we developed a simple, fast yet efficient and low-cost in vitro platform for direct cloning large BGCs from streptomycete genomic DNA, named as CAT-FISHING (CRISPR/Cas12a- and Agarose plug-based sysTem for Fast bIoSyntHetIc geNe cluster cloninG), by combining the advantages of CRISPR/Cas12a cleavage and bacterial artificial chromosome (BAC) library construction. CAT-FISHING was demonstrated by directly cloning large DNA fragments ranging from 47 to 139 kb with GC content of > 70% from the S. albus J1074 genome in a relatively efficient manner. Moreover, surugamides, encoded by a captured 87-kb BGC with GC content of 76%, was heterologously expressed in a Streptomyces chassis. These results indicate that CAT-FISHING is a powerful platform for BGCs batch cloning, which would be greatly beneficial to the natural products-based drug discovery. We believe that this system will lead a renaissance of interest in microorganisms as a source for drug development.

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Liang, M., Liu, L., Wang, W., Zeng, X., Liu, J., Loganathan, K., Zhu, G., Bai, L., Hou, C., Chen, X., Ouyang, L., Liu, X., Hu, B., Xia, X., Tong, Y., Lou, C., Alterovitz, G., Tan, G.-Y., Zhang, L.-X.. 2020-06-25. Simple cloning of large natural product biosynthetic gene cluster by CRISPR/Cas12a-mediated fast direct capturing strategy. https://doi.org/10.1101/2020.06.25.170191

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