Orthogonal tuning of gene expression noise using CRISPR–Cas

Wu, Fan; Shim, Jiyoung; Gong, Ting; Tan, Cheemeng

doi:10.1093/nar/gkaa537

Cited by 5 publications

(5 citation statements)

References 34 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…It has been previously shown that dCas9:sgRNAs will compete for binding when their target regions overlap (Qi et al , 2013; Wu et al , 2020a). However, the distance constraints we measured showed that it would not be possible to obtain repression/derepression by targeting overlapping regions.…”

Section: Resultsmentioning

confidence: 99%

“…When multiple sgRNAs target overlapping regions, this leads to mutually exclusive binding (Qi et al , 2013). Tan and co‐workers harnessed this effect to control the strength and noise of an E. coli constitutive promoter by co‐transcribing different ratios of two sgRNAs that direct dCas9 (fused to an activating domain) to overlapping positions that either recruit or block RNAP (Wu et al , 2020a).…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Competitive dCas9 binding as a mechanism for transcriptional control

Anderson

Voigt

2021

Molecular Systems Biology

View full text Add to dashboard Cite

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.

show abstract

Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Competitive dCas9 binding as a mechanism for transcriptional control

Anderson

Voigt

2021

Molecular Systems Biology

View full text Add to dashboard Cite

show abstract

“…Rather than using repression by CRISPRi, one could also use CRISPR activation (CRISPRa) − based on dCas9-activator fusions, both as part of the oscillator circuit or for the control of secondary processes. The most exciting prospect would be to control bacterial gene expression of chromosomal genes with the oscillator, which could be achieved by targeting these genes with the corresponding sgRNAs.…”

Section: Discussionmentioning

confidence: 99%

Single Cell Characterization of a Synthetic Bacterial Clock with a Hybrid Feedback Loop Containing dCas9-sgRNA

et al. 2020

View full text Add to dashboard Cite

Genetic networks that generate oscillations in gene expression activity are found in a wide range of organisms throughout all kingdoms of life. Oscillatory dynamics facilitates the temporal orchestration of metabolic and growth processes inside cells and organisms, as well as the synchronization of such processes with periodically occurring changes in the environment. Synthetic oscillator gene circuits such as the “repressilator” can perform similar functions in bacteria. Until recently, such circuits were mainly based on a relatively small set of well-characterized transcriptional repressors and activators. A promising, sequence-programmable alternative for gene regulation is given by CRISPR interference (CRISPRi), which enables transcriptional repression of nearly arbitrary gene targets directed by short guide RNA molecules. In order to demonstrate the use of CRISPRi in the context of dynamic gene circuits, we here replaced one of the nodes of a repressilator circuit by the RNA-guided dCas9 protein. Using single cell experiments in microfluidic reactors we show that this system displays robust relaxation oscillations over multiple periods and over several days. With a period of ≈14 bacterial generations, our oscillator is similar in speed as previously reported oscillators. Using an information-theoretic approach for the analysis of the single cell data, the potential of the circuit to act as a synthetic pacemaker for cellular processes is evaluated. We also observe that the oscillator appears to affect cellular growth, leading to variations in growth rate with the oscillator’s frequency.

show abstract

“…Multiplexed CRISPRi can address non-coding element epistasis 30 but may be limited to elements that are contemporaneously active in the cell type being studied. More recently, methods for bidirectional perturbations of two loci simultaneously, including paired CRISPRa and CRISPRi, have been developed but have been applied only to non-mammalian cells, are transient or are targeted to only a few genes [31][32][33][34][35][36][37][38][39] . New tools are needed that are compatible with studying genetic interactions in human cells, pooled high-throughput single-cell readouts and multiplexed bidirectional control of non-coding elements and are highly scalable to hundreds or thousands of perturbations.…”

Section: Articlementioning

confidence: 99%

Bidirectional epigenetic editing reveals hierarchies in gene regulation

Pacalin,

Steinhart,

Shi

et al. 2024

Nat Biotechnol

View full text Add to dashboard Cite

CRISPR perturbation methods are limited in their ability to study non-coding elements and genetic interactions. In this study, we developed a system for bidirectional epigenetic editing, called CRISPRai, in which we apply activating (CRISPRa) and repressive (CRISPRi) perturbations to two loci simultaneously in the same cell. We developed CRISPRai Perturb-seq by coupling dual perturbation gRNA detection with single-cell RNA sequencing, enabling study of pooled perturbations in a mixed single-cell population. We applied this platform to study the genetic interaction between two hematopoietic lineage transcription factors, SPI1 and GATA1, and discovered novel characteristics of their co-regulation on downstream target genes, including differences in SPI1 and GATA1 occupancy at genes that are regulated through different modes. We also studied the regulatory landscape of IL2 (interleukin-2) in Jurkat T cells, primary T cells and chimeric antigen receptor (CAR) T cells and elucidated mechanisms of enhancer-mediated IL2 gene regulation. CRISPRai facilitates investigation of context-specific genetic interactions, provides new insights into gene regulation and will enable exploration of non-coding disease-associated variants.

show abstract

Orthogonal tuning of gene expression noise using CRISPR–Cas

Cited by 5 publications

References 34 publications

Competitive dCas9 binding as a mechanism for transcriptional control

Competitive dCas9 binding as a mechanism for transcriptional control

Single Cell Characterization of a Synthetic Bacterial Clock with a Hybrid Feedback Loop Containing dCas9-sgRNA

Bidirectional epigenetic editing reveals hierarchies in gene regulation

Contact Info

Product

Resources

About