High-efficiency multiplex genome editing of Streptomyces species using an engineered CRISPR/Cas system.
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TLDR
The designed pCRISPomyces plasmids are amenable to assembly of spacers and editing templates via Golden Gate assembly and isothermal assembly, respectively, allowing rapid plasmid construction to target any genomic locus of interest.Abstract:
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.read more
Citations
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Multigene Editing in the Escherichia coli Genome via the CRISPR-Cas9 System
TL;DR: A targeted, continual multigene editing strategy that was applied to the Escherichia coli genome by using the Streptococcus pyogenes type II CRISPR-Cas9 system to realize a variety of precise genome modifications, including gene deletion and insertion, with the highest efficiency of 100%, is described.
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Systems strategies for developing industrial microbial strains.
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Synthetic biology to access and expand nature's chemical diversity.
TL;DR: How advances in synthetic biology — including novel DNA construction technologies, the use of genetic parts for the precise control of expression and for synthetic regulatory circuits — and multiplexed genome engineering can be used to optimize the design and synthesis of pathways that produce natural products are discussed.
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Metabolic engineering of Escherichia coli using CRISPR-Cas9 meditated genome editing.
TL;DR: A CRISPR-Cas9 based method for iterative genome editing and metabolic engineering of Escherichia coli is reported, which enables us to introduce various types of genomic modifications with near 100% editing efficiency and to introduce three mutations simultaneously.
Synthetic biology to access and expand nature's chemical diversity
TL;DR: The authors discuss how advances in synthetic biology, including novel DNA construction technologies, the use of genetic parts for the precise control of expression and for synthetic regulatory circuits, and multiplexed genome engineering can be used to optimize the design and synthesis of pathways that produce natural products.
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Le Cong,Le Cong,F. Ann Ran,F. Ann Ran,David M. Cox,David M. Cox,Shuailiang Lin,Shuailiang Lin,Robert P. J. Barretto,Naomi Habib,Patrick D. Hsu,Patrick D. Hsu,Xuebing Wu,Wenyan Jiang,Luciano A. Marraffini,Feng Zhang +15 more
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Multiplex Genome Engineering Using CRISPR/Cas Systems
Le Cong,F. A. Ran,David Benjamin Turitz Cox,Shuailiang Lin,Robert P. J. Barretto,Naomi Habib,Patrick D. Hsu,Xuebing Wu,Wenyan Jiang,Luciano A. Marraffini,Feng Zhang +10 more
TL;DR: Two different type II CRISPR/Cas systems are engineered and it is demonstrated that Cas9 nucleases can be directed by short RNAs to induce precise cleavage at endogenous genomic loci in human and mouse cells, demonstrating easy programmability and wide applicability of the RNA-guided nuclease technology.
Journal ArticleDOI
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TL;DR: An isothermal, single-reaction method for assembling multiple overlapping DNA molecules by the concerted action of a 5′ exonuclease, a DNA polymerase and a DNA ligase is described.