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      Editing streptomycete genomes in the CRISPR/Cas9 age

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          Abstract

          This article reviews CRISPR/Cas9-based toolkits available to investigate natural product biosynthesis and regulation in streptomycete bacteria.

          Abstract

          Covering: up to December 2018

          This article aims to highlight advantages, drawbacks and issues that users should consider when implementing the use of CRISPR/Cas9-tools for genome editing in streptomycetes, the most prolific source of antimicrobial natural products to date. Here, we examine four toolkits that have so far been made available for streptomycete in vivo-engineering and one for in vitro-editing, and review how they have been applied over the last three years. Our critical evaluation of these toolkits intends to support potential users in determining what they could achieve, what they should consider and what system they should select/optimise for their application.

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          Most cited references45

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          Efficient In Vivo Genome Editing Using RNA-Guided Nucleases

          Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) systems have evolved in bacteria and archaea as a defense mechanism to silence foreign nucleic acids of viruses and plasmids. Recent work has shown that bacterial type II CRISPR systems can be adapted to create guide RNAs (gRNAs) capable of directing site-specific DNA cleavage by the Cas9 nuclease in vitro. Here we show that this system can function in vivo to induce targeted genetic modifications in zebrafish embryos with efficiencies comparable to those obtained using ZFNs and TALENs for the same genes. RNA-guided nucleases robustly enabled genome editing at 9 of 11 different sites tested, including two for which TALENs previously failed to induce alterations. These results demonstrate that programmable CRISPR/Cas systems provide a simple, rapid, and highly scalable method for altering genes in vivo, opening the door to using RNA-guided nucleases for genome editing in a wide range of organisms.
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            CRISPR interference (CRISPRi) for sequence-specific control of gene expression.

            Sequence-specific control of gene expression on a genome-wide scale is an important approach for understanding gene functions and for engineering genetic regulatory systems. We have recently described an RNA-based method, CRISPR interference (CRISPRi), for targeted silencing of transcription in bacteria and human cells. The CRISPRi system is derived from the Streptococcus pyogenes CRISPR (clustered regularly interspaced palindromic repeats) pathway, requiring only the coexpression of a catalytically inactive Cas9 protein and a customizable single guide RNA (sgRNA). The Cas9-sgRNA complex binds to DNA elements complementary to the sgRNA and causes a steric block that halts transcript elongation by RNA polymerase, resulting in the repression of the target gene. Here we provide a protocol for the design, construction and expression of customized sgRNAs for transcriptional repression of any gene of interest. We also provide details for testing the repression activity of CRISPRi using quantitative fluorescence assays and native elongating transcript sequencing. CRISPRi provides a simplified approach for rapid gene repression within 1-2 weeks. The method can also be adapted for high-throughput interrogation of genome-wide gene functions and genetic interactions, thus providing a complementary approach to RNA interference, which can be used in a wider variety of organisms.
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              Discovery of microbial natural products by activation of silent biosynthetic gene clusters.

              Microorganisms produce a wealth of structurally diverse specialized metabolites with a remarkable range of biological activities and a wide variety of applications in medicine and agriculture, such as the treatment of infectious diseases and cancer, and the prevention of crop damage. Genomics has revealed that many microorganisms have far greater potential to produce specialized metabolites than was thought from classic bioactivity screens; however, realizing this potential has been hampered by the fact that many specialized metabolite biosynthetic gene clusters (BGCs) are not expressed in laboratory cultures. In this Review, we discuss the strategies that have been developed in bacteria and fungi to identify and induce the expression of such silent BGCs, and we briefly summarize methods for the isolation and structural characterization of their metabolic products.
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                Author and article information

                Journal
                NPRRDF
                Natural Product Reports
                Nat. Prod. Rep.
                Royal Society of Chemistry (RSC)
                0265-0568
                1460-4752
                2019
                2019
                Affiliations
                [1 ]School of Life Sciences
                [2 ]Department of Chemistry
                [3 ]University of Warwick
                [4 ]Coventry CV4 7AL
                [5 ]UK
                Article
                10.1039/C8NP00081F
                30680376
                2a6c6d8b-e0d2-4f75-8069-7c0e22f0e099
                © 2019

                http://creativecommons.org/licenses/by/3.0/

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