报告题目：CRISPR meets natural product discovery: the best is yet to come
Yaojun Tong his PhD from Prof. Lixin Zhang’s lab at Institute of Microbiology, CAS in 2014. And then he worked as a postdoc and later an assistant Professor with Prof. Sang Yup Lee, and Prof. Tilmann Weber at Novo Nordisk Foundation Center for Biosustainability (CfB), Technical University of Denmark (DTU).
Dr. Tong is on the path to being an independent junior group leader, he is currently leading the genomic manipulation toolbox development and the cell factory construction of actinomycetes at CfB. The research of Dr. Tong includes 1). developing and applying synthetic biology/system biology tools to understand the mechanisms of bacterial secondary metabolites synthesis, he has pioneered and is still developing CRISPR genome editing systems for actinomycetes, to facilitate the natural product discovery of actinomycetes; 2). studying the mode of action of bioactive natural products against microbial pathogens, he has revealed the synergistic antifungal mechanism of natural products beauvericin, berberine, rapamycin and so on; 3). studying how to overcome the bacterial multi-drug resistance.
Dr. Tong has published 17 peer reviewed articles (total citation > 600, data from Google Scholar), he is also involved in one PCT and four Chinese patent applications, with h-index of 12 (data from Google Scholar). He has been invited to give oral presentation in seven international conferences and he is the invited reviewer of more than ten peer reviewed journals, for example, Metabolic Engineering, mBio, ACS Synthetic Biology, Biotechnology and Bioengineering, and so on.
Actinomycetes are prolific resources of bioactive natural products. However, many of these strains are recalcitrant to genetic manipulation with traditional approaches, which is a severe bottleneck for systematic metabolic engineering. To facilitate the genetic manipulation of actinomycetes, we established a CRISPR toolkit (Figure 1), which includes an sgRNA designer, CRISPy-web; and three USER cloning compatible systems, pCRISPR-Cas9, pCRISPR-Cas9-ScaligD, and CRISPRi. Though, many sgRNA designers are available for the model organisms, only few tools that allow designing sgRNA for non-model organisms exist. To address this problem, we established the CRISPy-web (http://crispy.secondarymetabolites.org/), an easy to use web tool to design sgRNAs for any user-provided microbial genomes. Our CRISPR toolkit was validated in S. coelicolor  and many other streptomycetes. When no templates for homologous recombination were present, the DNA DSB introduced by Cas9 were repaired through a native, however only weakly active error-prone NHEJ pathway, resulting in a library of deletions with variable sizes around the DSB, from 1 bp to >30 kb. To the best of our knowledge, this is the first reported case of using NHEJ for DSB repair of genome editing in streptomycetes[4,5]. In a system with reconstituted NHEJ, the DSB was repaired with only small indels. When templates for homologous recombination were provided, in-frame deletion of the targeted gene were observed with close to 100% frequency. Moreover, we implemented a CRISPRi system to efficiently and reversibly control expression of the target genes by modulating their transcription level, based on a catalytically dead variant of Cas9 (dCas9). The described CRISPR-Cas9 toolkit comprises a powerful and broadly applicable set of tools to manipulate actinomycetal genomes, which opens new possibility of engineering this important group of organisms for fine natural product discovery.
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