Journal article
CRISPR–Cas9, CRISPRi and CRISPR-BEST-mediated genetic manipulation in streptomycetes
Technical University of Denmark1
Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark2
New Bioactive Compounds, Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark3
Center for Microbial Secondary Metabolites, Centers, Technical University of Denmark4
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. Here, we provide step-by-step protocols for designing and constructing the CRISPR plasmids, transferring these plasmids to the target streptomycetes, and identifying correctly edited clones.
Our CRISPR toolkit can be used to generate random-sized deletion libraries, introduce small indels, generate in-frame deletions of specific target genes, reversibly suppress gene transcription, and substitute single base pairs in streptomycete genomes. Furthermore, the toolkit includes a Csy4-based multiplexing option to introduce multiple edits in a single experiment.
The toolkit can be easily extended to other actinomycetes. With our protocol, it takes <10 d to inactivate a target gene, which is much faster than alternative protocols.
Language: | English |
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Year: | 2020 |
Pages: | 2470-2502 |
ISSN: | 17502799 and 17542189 |
Types: | Journal article |
DOI: | 10.1038/s41596-020-0339-z |
ORCIDs: | Tong, Yaojun , Weber, Tilmann , 0000-0003-0599-3091 , Whitford, Christopher M. , Blin, Kai and Jørgensen, Tue S. |