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. 2022:2479:207-225.
doi: 10.1007/978-1-0716-2233-9_14.

CRISPR/Cas-Mediated Genome Editing of Streptomyces

Lee Ling Tan #  1 , Elena Heng #  1 , Nadiah Zulkarnain  1 , Wan-Chi Hsiao  2   3 , Fong Tian Wong  4   5 , Mingzi M Zhang  6
Affiliations

CRISPR/Cas-Mediated Genome Editing of Streptomyces

Lee Ling Tan et al. Methods Mol Biol. 2022.

Abstract

Streptomyces are an important source and reservoir of natural products with diverse applications in medicine, agriculture, and food. Engineered Streptomyces strains have also proven to be functional chassis for the discovery and production of bioactive compounds and enzymes. However, genetic engineering of Streptomyces is often laborious and time-consuming. Here we describe protocols for CRISPR/Cas-mediated genome editing of Streptomyces. Starting from the design and assembly of all-in-one CRISPR/Cas constructs for efficient double-strand break-mediated genome editing, we also present protocols for intergeneric conjugation, CRISPR/Cas plasmid curing, and validation of edited strains.

Keywords: CRISPR/Cas; Deletion; Double-strand break; Genetic engineering; Genome editing; Knock-in; Streptomyces; Substitution.

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