Cobb Ryan E, Wang Yajie, Zhao Huimin
†Department of Chemical and Biomolecular Engineering, ‡Institute for Genomic Biology, §Departments of Chemistry, Biochemistry and Bioengineering, Center for Biophysics and Computational Biology, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801, United States.
ACS Synth Biol. 2015 Jun 19;4(6):723-8. doi: 10.1021/sb500351f. Epub 2014 Dec 8.
Actinobacteria, particularly those of genus Streptomyces, remain invaluable hosts for the discovery and engineering of natural products and their cognate biosynthetic pathways. However, genetic manipulation of these bacteria is often labor and time intensive. Here, we present an engineered CRISPR/Cas system for rapid multiplex genome editing of Streptomyces strains, demonstrating targeted chromosomal deletions in three different Streptomyces species and of various sizes (ranging from 20 bp to 30 kb) with efficiency ranging from 70 to 100%. The designed pCRISPomyces plasmids are amenable to assembly of spacers and editing templates via Golden Gate assembly and isothermal assembly (or traditional digestion/ligation), respectively, allowing rapid plasmid construction to target any genomic locus of interest. As such, the pCRISPomyces system represents a powerful new tool for genome editing in Streptomyces.
放线菌，尤其是链霉菌属的放线菌，仍然是发现和改造天然产物及其相关生物合成途径的宝贵宿主。然而，对这些细菌进行基因操作往往既费力又耗时。在此，我们展示了一种用于链霉菌菌株快速多重基因组编辑的工程化CRISPR/Cas系统，证明了在三种不同链霉菌物种中可实现靶向染色体缺失，缺失大小各异（从20碱基对到30千碱基对），效率在70%至100%之间。所设计的pCRISPomyces质粒分别适用于通过金门组装和等温组装（或传统消化/连接）来组装间隔序列和编辑模板，从而能够快速构建质粒以靶向任何感兴趣的基因组位点。因此，pCRISPomyces系统是链霉菌基因组编辑的一种强大新工具。
