Competitive dCas9 binding as a mechanism for transcriptional control.
Daniel A AndersonChristopher A VoigtPublished in: Molecular systems biology (2022)
Catalytically dead Cas9 (dCas9) is a programmable transcription factor that can be targeted to promoters through the design of small guide RNAs (sgRNAs), where it can function as an activator or repressor. Natural promoters use overlapping binding sites as a mechanism for signal integration, where the binding of one can block, displace, or augment the activity of the other. Here, we implemented this strategy in Escherichia coli using pairs of sgRNAs designed to repress and then derepress transcription through competitive binding. When designed to target a promoter, this led to 27-fold repression and complete derepression. This system was also capable of ratiometric input comparison over two orders of magnitude. Additionally, we used this mechanism for promoter sequence-independent control by adopting it for elongation control, achieving 8-fold repression and 4-fold derepression. This work demonstrates a new genetic control mechanism that could be used to build analog circuit or implement cis-regulatory logic on CRISPRi-targeted native genes.
Keyphrases
- transcription factor
- dna binding
- escherichia coli
- dna methylation
- gene expression
- genome wide
- crispr cas
- genome wide identification
- cancer therapy
- binding protein
- nitric oxide
- genome editing
- pseudomonas aeruginosa
- cystic fibrosis
- hydrogen peroxide
- copy number
- sensitive detection
- living cells
- single molecule
- fluorescent probe
- nuclear factor
- drug delivery
- toll like receptor
- heat shock protein