Highly efficient DSB-free base editing for streptomycetes with CRISPR-BEST

Area of Science:

• Microbiology
• Molecular Biology
• Biotechnology

Background:

• Streptomycetes are crucial for producing valuable natural products.
• Existing CRISPR-Cas9 systems, while useful, can cause genome instability and Cas9 toxicity due to DNA double-strand breaks (DSBs).

Purpose of the Study:

• To develop a novel, DSB-free genome editing system for Streptomyces.
• To enhance genetic manipulation capabilities in Streptomyces species, including nonmodel organisms.

Main Methods:

• Development of CRISPR-BEST, a system utilizing cytidine (CRISPR-cBEST) and adenosine (CRISPR-aBEST) deaminases for base editing.
• Validation in various Streptomyces species, including inactivation of the kirN gene in Streptomyces collinus Tü365.
• Genome-wide off-target analysis to assess system fidelity.
• Implementation of a Csy4-based sgRNA processing machinery for multiplexed editing.
• Update of the CRISPy-web tool for designing CRISPR-BEST-specific sgRNAs.

Main Results:

• CRISPR-BEST enables precise C:G to T:A (CRISPR-cBEST) and A:T to G:C (CRISPR-aBEST) base conversions.
• Efficient simultaneous inactivation of duplicated kirN genes in Streptomyces collinus Tü365 was achieved, a feat not possible with DSB-based CRISPR-Cas9.
• High fidelity and broad applicability of CRISPR-BEST were confirmed through unbiased off-target evaluation.
• The system supports multiplexed editing and is supported by an updated web tool for sgRNA design.

Conclusions:

• CRISPR-BEST provides a robust, DSB-free alternative for precise genome editing in Streptomyces.
• This system overcomes key limitations of CRISPR-Cas9, enabling efficient genetic manipulation and the study of natural product biosynthesis.
• CRISPR-BEST expands the toolkit for genetic engineering in diverse Streptomyces species.