A platform for CRISPRi-seq in Streptomyces albidoflavus

Area of Science:

• Microbiology
• Genomics
• Biotechnology

Background:

• Streptomyces species are vital sources of natural products for drug discovery.
• Genome sequencing has advanced microbial physiology understanding, but functional genomics tools for Streptomyces lag.
• Existing technologies hinder the full exploitation of genomic data in Streptomyces.

Purpose of the Study:

• To develop a high-throughput platform for genome-wide functional screening in Streptomyces.
• To establish a clustered regularly interspaced short palindromic repeats interference sequencing (CRISPRi-seq) system for Streptomyces species.
• To identify essential transporter operons contributing to fitness in Streptomyces albidoflavus.

Main Methods:

• Developed an en masse CRISPRi-seq platform for Streptomyces.
• Designed 2,160 unique sgRNAs targeting all 432 membrane transporter operons in S. albidoflavus.
• Performed CRISPRi-seq on 1.1Mb of the S. albidoflavus genome, followed by hit validation.

Main Results:

• Identified a small subset of 13 transporter operons (25 kb) essential for S. albidoflavus fitness.
• Demonstrated the platform's capability for large-scale functional genomics screens.
• Validated the efficiency of the CRISPRi-seq system in Streptomyces.

Conclusions:

• The developed CRISPRi-seq platform is a first-in-class tool for high-throughput functional genomics in Streptomyces.
• This scalable and generalizable approach accelerates the dissection of gene function in this genus.
• The findings provide a blueprint for future en masse screens in Streptomyces, advancing systems-level understanding and drug discovery.