Abstract

Programmable gene activation enables fine‐tuned regulation of endogenous and synthetic gene circuits to control cellular behavior. While CRISPR‐Cas‐mediated gene activation has been extensively developed for eukaryotic systems, similar strategies have been difficult to implement in bacteria. Here, we present a generalizable platform for screening and selection of functional bacterial CRISPR‐Cas transcription activators. Using this platform, we identified a novel CRISPR activator, dCas9‐AsiA, that could activate gene expression by more than 200‐fold across genomic and plasmid targets with diverse promoters after directed evolution. The evolved dCas9‐AsiA can simultaneously mediate activation and repression of bacterial regulons in E. coli. We further identified hundreds of promoters with varying basal expression that could be induced by dCas9‐AsiA, which provides a rich resource of genetic parts for inducible gene activation. Finally, we show that dCas9‐AsiA can be ported to other bacteria of clinical and bioindustrial relevance, thus enabling bacterial CRISPRa in more application areas. This work expands the toolbox for programmable gene regulation in bacteria and provides a useful resource for future engineering of other bacterial CRISPR‐based gene regulators.

Highlights

  • Transcriptional regulation governs almost every cellular process fundamental to life

  • To expedite the discovery of bacterial CRISPRa components, we developed a screening-selection platform in Escherichia coli to identify candidate dead Cas9 (dCas9)-mediated transcription activators

  • The screening-selection platform to identify active CRISPR-Cas mediated transcriptional activation established in this work provides a highly modular and portable system to engineering new bacterial CRISPRa systems for enhancing potency of dCas9-activators, modifying guide RNA (gRNA) designs, or screening for targetable promoters

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Summary

Introduction

Transcriptional regulation governs almost every cellular process fundamental to life. In response to cellular or external signals, transcription factors (TFs) in the cell interact with specific DNA sequences to mediate gene activation or repression. Significant past efforts have been devoted to developing synthetic transcription activators by fusing DNA-binding proteins with transcription effector domains to recruit the RNA polymerase (RNAP) complex (Dove & Hochschild, 1998; Joung et al, 2000). These past synthetic TFs generally recognize only predefined DNA sequences and are difficult to reprogram to target other sequences, which greatly limits their utility for transcriptional regulation of diverse endogenous and engineered gene regulatory networks

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