Unfortunately, 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. As such, 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). Programmable transcriptional activation and repression in principle offers on-demand control of specific biological processes without the need to permanently alter the genome of a cell. A potential path for cellular engineering therefore is the rewiring of transcriptional factors to alter gene regulatory networks. In response to cellular or external signals, transcription factors (TFs) in the cell interact with specific DNA sequences to mediate gene activation or repression. Transcriptional regulation governs almost every cellular process fundamental to life. 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.
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. 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. The evolved dCas9-AsiA can simultaneously mediate activation and repression of bacterial regulons in E. Using this platform, we identified a novel CRISPR activator, dCas9-AsiA, that could activate gene expression by up to 200-fold across genomic and plasmid targets with diverse promoters after directed evolution. Here, we present a generalizable platform for screening and selection of functional bacterial CRISPR-Cas transcription activators.
While CRISPR-Cas-mediated gene activation have been extensively developed for eukaryotic systems, similar strategies have been difficult to implement in bacteria. Programmable gene activation enables fine-tuned regulation of endogenous and synthetic gene circuits to control cellular behavior.
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