CRISPR–Cas9, CRISPRi and CRISPR-BEST-mediated genetic manipulation in streptomycetes

Yaojun Tong, Christopher M. Whitford, Kai Blin, Tue S. Jørgensen, Tilmann Weber*, Sang Yup Lee*

*Corresponding author for this work

Research output: Contribution to journalJournal articleResearchpeer-review


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.

Original languageEnglish
JournalNature Protocols
Issue number8
Pages (from-to)2470-2502
Publication statusPublished - 2020


Dive into the research topics of 'CRISPR–Cas9, CRISPRi and CRISPR-BEST-mediated genetic manipulation in streptomycetes'. Together they form a unique fingerprint.

Cite this