MIBiG 4.0: advancing biosynthetic gene cluster curation through global collaboration.

Specialized or secondary metabolites are small molecules of biological origin, often showing potent biological activities with applications in agriculture, engineering and medicine. Usually, the biosynthesis of these natural products is governed by sets of co-regulated and physically clustered genes known as biosynthetic gene clusters (BGCs). To share information about BGCs in a standardized and machine-readable way, the Minimum Information about a Biosynthetic Gene cluster (MIBiG) data standard and repository was initiated in 2015.

An in vitro CRISPR-Cas12a-mediated protocol for direct cloning of large DNA fragments.

Large biosynthetic gene cluster (BGC) cloning is important for discovering natural product-based drugs and remains challenging in high GC content microorganisms (e.g., Actinobacteria).

Systems Analysis of Highly Multiplexed CRISPR-Base Editing in Streptomycetes.

CRISPR tools, especially Cas9n-sgRNA guided cytidine deaminase base editors such as CRISPR-BEST, have dramatically simplified genetic manipulation of streptomycetes. One major advantage of CRISPR base editing technology is the possibility to multiplex experiments in genomically instable species. Here, we demonstrate scaled up Csy4 based multiplexed genome editing using CRISPR-mcBEST in .

Synthetic Biology Facilitates Antimicrobials Discovery.

We are currently facing two big global challenges: antibiotics shortage and multidrug resistance [...

Optogenetic tools for microbial synthetic biology.

Chemical induction is one of the most common modalities used to manipulate gene expression in living systems. However, chemical induction can be toxic or expensive that compromise the economic feasibility when it comes to industrial-scale synthetic biology applications. These complications have driven the pursuit of better induction systems.

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.

Berberine reverses multidrug resistance in Candida albicans by hijacking the drug efflux pump Mdr1p.

Clinical use of antimicrobials faces great challenges from the emergence of multidrug-resistant pathogens. The overexpression of drug efflux pumps is one of the major contributors to multidrug resistance (MDR). Reversing the function of drug efflux pumps is a promising approach to overcome MDR.

Integrating PCR-free amplification and synergistic sensing for ultrasensitive and rapid CRISPR/Cas12a-based SARS-CoV-2 antigen detection.

Antigen detection provides particularly valuable information for medical diagnoses; however, the current detection methods are less sensitive and accurate than nucleic acid analysis. The combination of CRISPR/Cas12a and aptamers provides a new detection paradigm, but sensitive sensing and stable amplification in antigen detection remain challenging. Here, we present a PCR-free multiple trigger dsDNA tandem-based signal amplification strategy and a designed dual aptamer synergistic sensing strategy.

Hyper-Synergistic Antifungal Activity of Rapamycin and Peptide-Like Compounds against Orthogonally via Tor1 Kinase.

is a life-threatening, opportunistic fungal pathogen with a high mortality rate, especially within the immunocompromised populations. Multidrug resistance combined with limited antifungal drugs even worsens the situation. Given the facts that the current drug discovery strategies fail to deliver sufficient antifungals for the emerging multidrug resistance, we urgently need to develop novel approaches.

A versatile genetic engineering toolkit for E. coli based on CRISPR-prime editing.

CRISPR base editing is a powerful method to engineer bacterial genomes. However, it restricts editing to single-nucleotide substitutions. Here, to address this challenge, we adapt a CRISPR-Prime Editing-based, DSB-free, versatile, and single-nucleotide resolution genetic manipulation toolkit for prokaryotes.

A versatile biosensing platform coupling CRISPR-Cas12a and aptamers for detection of diverse analytes.

Rapid and sensitive detection of various analytes is in high demand. Apart from its application in genome editing, CRISPR-Cas also shows promises in nucleic acid detection applications. To further exploit the potential of CRISPR-Cas for detection of diverse analytes, we present a versatile biosensing platform that couples the excellent affinity of aptamers for broad-range analytes with the collateral single-strand DNA cleavage activity of CRISPR-Cas12a.

CRISPR-Cas9, CRISPRi and CRISPR-BEST-mediated genetic manipulation in streptomycetes.

Streptomycetes are prominent sources of bioactive natural products, but metabolic engineering of the natural products of these organisms is greatly hindered by relatively inefficient genetic manipulation approaches. New advances in genome editing techniques, particularly CRISPR-based tools, have revolutionized genetic manipulation of many organisms, including actinomycetes. We have developed a comprehensive CRISPR toolkit that includes several variations of 'classic' CRISPR-Cas9 systems, along with CRISPRi and CRISPR-base editing systems (CRISPR-BEST) for streptomycetes.

Designing sgRNAs for CRISPR-BEST base editing applications with CRISPy-web 2.0.

CRISPR/Cas9 systems are an established tool in genome engineering. As double strand breaks caused by the standard Cas9-based knock-out techniques can be problematic in some organisms, new systems were developed that can efficiently create knock-outs without causing double strand breaks to elegantly sidestep these issues. The recently published CRISPR-BEST base editor system for actinobacteria is built around a C to T or A to G base exchange.

An aurora of natural products-based drug discovery is coming.

Natural products (NPs), a nature's reservoir possessing enormous structural and functional diversity far beyond the current ability of chemical synthesis, are now proving themselves as most wonderful gifts from mother nature for human beings. Many of them have been used successfully as medicines, as well as the most important sources of drug leads, food additives, and many industry relevant products for millennia. Most notably, more than half of the antibiotics and anti-cancer drugs currently in use are, or derived from, natural products.

Programmable polyketide biosynthesis platform for production of aromatic compounds in yeast.

To accelerate the shift to bio-based production and overcome complicated functional implementation of natural and artificial biosynthetic pathways to industry relevant organisms, development of new, versatile, bio-based production platforms is required. Here we present a novel yeast-based platform for biosynthesis of bacterial aromatic polyketides. The platform is based on a synthetic polyketide synthase system enabling a first demonstration of bacterial aromatic polyketide biosynthesis in a eukaryotic host.

Dual-function chromogenic screening-based CRISPR/Cas9 genome editing system for actinomycetes.

Actinobacteria are one of the most important sources of pharmaceutically valuable and industrially relevant secondary metabolites. Modern genome mining reveals that the potential for secondary metabolite production of actinomycetes has been underestimated. Recently, the establishment of CRISPR/Cas9-based genetic manipulation approaches in actinomycetes opened a new era for genome engineering of this type of organism.

Highly efficient DSB-free base editing for streptomycetes with CRISPR-BEST.

Streptomycetes serve as major producers of various pharmacologically and industrially important natural products. Although CRISPR-Cas9 systems have been developed for more robust genetic manipulations, concerns of genome instability caused by the DNA double-strand breaks (DSBs) and the toxicity of Cas9 remain. To overcome these limitations, here we report development of the DSB-free, single-nucleotide-resolution genome editing system CRISPR-BEST (CRISPR-Base Editing SysTem), which comprises a cytidine (CRISPR-cBEST) and an adenosine (CRISPR-aBEST) deaminase-based base editor.

A CRISPR-Cas12a-derived biosensing platform for the highly sensitive detection of diverse small molecules.

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 (CRISPR-Cas12a- and aTF-mediated small molecule detector).

Synthetic biology and metabolic engineering of actinomycetes for natural product discovery.

Actinomycetes are one of the most valuable sources of natural products with industrial and medicinal importance. After more than half a century of exploitation, it has become increasingly challenging to find novel natural products with useful properties as the same known compounds are often repeatedly re-discovered when using traditional approaches. Modern genome mining approaches have led to the discovery of new biosynthetic gene clusters, thus indicating that actinomycetes still harbor a huge unexploited potential to produce novel natural products.