# A platform for CRISPRi-seq in Streptomyces albidoflavus

## TL;DR

This paper introduces a new CRISPRi-seq platform for studying gene function in Streptomyces albidoflavus, a bacteria known for producing important natural products.

## Contribution

The study develops the first high-throughput CRISPRi-seq platform for Streptomyces species, enabling large-scale functional genomic studies.

## Key findings

- Only 13 out of 432 transporter operons in S. albidoflavus are essential for fitness.
- The platform targets 1.1Mb of the genome using 2,160 unique sgRNAs.
- The method provides a scalable blueprint for functional screening in Streptomyces.

## Abstract

Streptomyces produce a multitude of secondary metabolites, which have been exploited in drug discovery campaigns for more than three-quarters of a century. Our understanding of microbial physiology has been revolutionized by genome sequencing and large-scale functional studies. Technology for genome-wide investigations in Streptomyces species, however, has lagged behind that for other bacterial systems, hindering exploitation of unprecedented quantities of genomic data. Here, we develop a platform for en masse clustered regularly interspaced short palindromic repeats interference sequencing (CRISPRi-seq) for Streptomyces spp. By performing CRISPRi-seq with 2,160 unique sgRNAs targeting all operons (432 operons) encoding membrane transporters (629 genes) representing 1.1Mb of the 6.8Mb genome for S. albidoflavus, combined with hit validation, we discovered that only a small proportion (13 of 432 operons, 25 kb) contribute positively to fitness. Our work provides both a first-in-class platform for high-throughput functional genomics and a generalized blueprint for en masse screens in Streptomyces species.

Streptomyces bacteria are prolific producers of clinically essential natural products, yet high-throughput tools to systematically interrogate their genomes remain underdeveloped. By establishing a robust CRISPRi-seq platform for en masse functional screening in Streptomyces albidoflavus, our work closes a critical technological gap in Streptomyces functional genomics. Our study not only identifies a small subset of transporter operons essential for fitness but also introduces a scalable, generalizable approach for dissecting gene function. This platform will accelerate systems-level understanding of an industrially and medically important genus.