Abstract
Over the past few years, tools that make use of the Cas9 nuclease have led to many breakthroughs, including in the control of gene expression. The catalytically dead variant of Cas9 known as dCas9 can be guided by small RNAs to block transcription of target genes, in a strategy also known as CRISPRi. Here, we reveal that the level of complementarity between the guide RNA and the target controls the rate at which RNA polymerase “kicks out” dCas9 from the target and completes transcription. We use this mechanism to precisely and robustly reduce gene expression by defined relative amounts. Alternatively, tuning repression by changing dCas9 concentration is noisy and promoter‐strength dependent. We demonstrate broad applicability of this method to the study of genetic regulation and cellular physiology. First, we characterize feedback strength of a model auto‐repressor. Second, we study the impact of amount variations of cell‐wall synthesizing enzymes on cell morphology. Finally, we multiplex the system to obtain any combination of fractional repression of two genes.
Highlights
A powerful way to investigate genes and their regulation in bacteria is to vary their expression levels and investigate the response of the cell
We demonstrate the versatility of our approach using two examples: first, the accurate control of the rate at which the RNA polymerase (RNAP) kicks out dCas9 enables us to quantify the degree of feedback in a model auto-repressor by measuring how much actual gene expression differs from the controlled rate
To quantify how CRISPR-dCas9 modulates gene expression at the single-cell level, we integrated expression cassettes for two constitutively expressed reporters, sfgfp coding for the superfolder green fluorescent protein (GFP) and mCherry coding for a red fluorescent protein (RFP) at two different chromosomal loci of E. coli strain MG1655
Summary
A powerful way to investigate genes and their regulation in bacteria is to vary their expression levels and investigate the response of the cell. Different strategies have been devised to knock down gene expression by relative amounts from their native levels: antisense transcription can reduce gene expression in a defined manner (Brophy & Voigt, 2016). While this approach works well for moderate promoter strength, it becomes less efficient the stronger the promoter. If the target is chosen downstream of the promoter, dCas serves as a roadblock that blocks transcription elongation We characterize this system at the single-cell level, with interesting implications for the native CRISPR immune system. We develop a strategy to use this system for precise and noise-preserving relative gene repression that is independent of promoter strength
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