Besides genome editing, CRISPR-Cas12a has recently been used for DNA detection applications with attomolar sensitivity but, to our knowledge, it has not been used for the detection of small molecules. Bacterial allosteric transcription factors (aTFs) have evolved to sense and respond sensitively to a variety of small molecules to benefit bacterial survival. By combining the single-stranded DNA cleavage ability of CRISPR-Cas12a and the competitive binding activities of aTFs for small molecules and double-stranded DNA, here we develop a simple, supersensitive, fast and high-throughput platform for the detection of small molecules, designated CaT-SMelor ( C RISPR-Cas12a- and aT F-mediated s mall m ol e cu l e detect or ). CaT-SMelor is successfully evaluated by detecting nanomolar levels of various small molecules, including uric acid and p -hydroxybenzoic acid among their structurally similar analogues. We also demonstrate that our CaT-SMelor directly measured the uric acid concentration in clinical human blood samples, indicating a great potential of CaT-SMelor in the detection of small molecules.

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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.

The versatile photosynthetic α-proteobacterium Rhodobacter sphaeroides , has recently been extensively engineered as a novel microbial cell factory (MCF) to produce pharmaceuticals, nutraceuticals, commodity chemicals and even hydrogen. However, there are no well-characterized high-activity promoters to modulate gene transcription during the engineering of R. sphaeroides . In this study, several native promoters from R. sphaeroides JDW-710 (JDW-710), an industrial strain producing high levels of co-enzyme Q 10 (Q 10 ) were selected on the basis of transcriptomic analysis. These candidate promoters were then characterized by using gusA as a reporter gene. Two native promoters, P rsp _ 7571 and P rsp _ 6124 , showed 620% and 800% higher activity, respectively, than the tac promoter, which has previously been used for gene overexpression in R. sphaeroides. In addition, a P rsp _ 7571 -derived synthetic promoter library with strengths ranging from 54% to 3200% of that of the tac promoter, was created on the basis of visualization of red fluorescent protein (RFP) expression in R. sphaeroides . Finally, as a demonstration, the synthetic pathway of Q 10 was modulated by the selected promoter T334* in JDW-710; the Q 10 yield in shake-flasks increased 28% and the production reached 226 mg/L . These well-characterized promoters should be highly useful in current synthetic biology platforms for refactoring the biosynthetic pathway in R. sphaeroides -derived MCFs.

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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.