Synthetic CRISPR-Cas gene activators for transcriptional reprogramming in bacteria

Chen, Dong; Fontana, Jason; Patel, Anika; Carothers, James M.; Zalatan, Jesse G.

doi:10.1038/s41467-018-04901-6

Cited by 172 publications

(265 citation statements)

References 64 publications

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“…The grey line represents the baseline activity of the J3-J23117-mRFP1 reporter strain containing an empty vector instead of the CRISPRa component plasmid. For comparison, previous CRISPRa data at -81 and -91 are shown on the schematic above the plot 1 . C) The sharp positioning requirements of CRISPRa are observed when targeting a different minimal promoter.…”

Section: S6mentioning

confidence: 99%

Effective CRISPRa-mediated control of gene expression in bacteria must overcome strict target site requirements

et al. 2020

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Supplementary Figure 1: CRISPRa at the endogenous gene target ldhA does not follow predicted trends.Eight scRNA target sites (L1-L8) upstream of the ldhA promoter were selected. Three of the target sites (L5-L7) were within the 40 bp window where CRISPRa is effective (-100 to -60). While L1, L4, and L5 resulted in weak increases in gene expression, there was no apparent relationship between the position of the sites and ldhA expression levels. Gene expression was measured using RT-qPCR. Fold activation represents expression levels relative to an off-target control (hAAVS1). Bars indicate the average values between three technical replicates and black dots indicate the values of individual replicates. Error bars indicate the standard error of the mean between three technical replicates. S3 Supplementary Figure 2: CRISPRa activity depends on the target sequence on the scRNA. Reporter cassettes that differ only by the sequence of the 20 base scRNA target site give a broad range of gene expression levels, demonstrating that the sequence of the scRNA target site can have a substantial effect on CRISPRa. Three new reporter plasmids were constructed where the J306 target site, located at -81 from the TSS, on the J3-J23117-mRFP1 reporter was replaced by the J104, J106, and J108 sequence. Activation at each promoter was tested when CRISPRa was targeted to their cognate scRNA site. The off-target negative control (OT) represents a strain expressing the original reporter with the J306 site and the CRISPRa components to target an offtarget site (J206). Fluorescence/OD 600 values were measured using a plate reader. Bars indicate the average values between three biological replicates. Black dots indicate the values of individual biological replicates. Error bars indicate the standard deviation between biological replicates. S4 S5 Supplementary Figure 3: The sharp positioning dependence of CRISPRa is observed across multiple promoters. A) The sharp positioning requirements of CRISPRa are not significantly affected by the location or composition of the inserted sequence. Reporters were based on the J1-J23117-mRFP1 (Supplementary Figure 3A) with base shifts introduced in different ways. In the J1 reporter A, bases were inserted upstream of the -35 region. In the J1 reporter B, a different sequence was

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Section: S6mentioning

confidence: 99%

Effective CRISPRa-mediated control of gene expression in bacteria must overcome strict target site requirements

et al. 2020

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“…To enable these goals, we recently developed a new CRISPR-Cas transcriptional activation (CRISPRa) system that is effective in E. coli. Our system can be combined with CRISPRi gene repression to programmably and flexibly target multiple genes for simultaneous activation and repression 3 . While our CRISPRa system can be used with heterologous genes, an outstanding challenge is to understand the rules that define effective target sites at arbitrary promoters in the genome.…”

Section: Introductionmentioning

confidence: 99%

“…Targeting this complex to a promoter or an open reading frame (ORF) results in gene repression (CRISPRi) 4 . To enable simultaneous activation, we use modified guide RNAs, termed scaffold RNAs (scRNAs), that include a 3' MS2 hairpin to recruit a transcriptional activator fused to the MS2 coat protein (MCP) 3 . We can express multiple gRNAs and scRNAs to inhibit and activate genes simultaneously; gRNAs targeted to a promoter or ORF result in CRISPRi and scRNAs targeted to an appropriate site upstream of a minimal promoter result in CRISPRa.…”

Section: Introductionmentioning

confidence: 99%

“…We demonstrate here that the rules for targeting CRISPRa to effective sites in E. coli are surprisingly stringent. In prior work, we found that CRISPRa in E. coli was effective at target sites located in a narrow 40 base window between 60 and 100 bases upstream of the transcriptional start site (TSS) 3 . Here, we show that multiple factors combine to make the requirements for effective sites even more strict.…”

Section: Introductionmentioning

confidence: 99%

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Effective CRISPRa-Mediated Control of Gene Expression in Bacteria Must Overcome Strict Target Site Requirements

Fontana

Chen

Kiattisewee

et al. 2019

Preprint

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In bacterial systems, CRISPR-Cas transcriptional activation (CRISPRa) has the potential to dramatically expand our ability to regulate gene expression, but we currently lack a complete understanding of the rules for designing effective guide RNA target sites. We have identified multiple features of bacterial promoters that impose stringent requirements on bacterial CRISPRa target sites. Most importantly, we found that shifting a gRNA target site by 2-4 bases along the DNA target can cause a nearly complete loss in activity. The loss in activity can be rescued by shifting the target site 10-11 bases, corresponding to one full helical turn. Practically, our results suggest that it will be challenging to find a gRNA target site with an appropriate PAM sequence at precisely the right position at arbitrary genes of interest. To overcome this limitation, we demonstrate that a dCas9 variant with expanded PAM specificity allows activation of promoters that cannot be activated by S. pyogenes dCas9. These results provide a roadmap for future engineering efforts to further expand and generalize the scope of bacterial CRISPRa.

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“…Nevertheless, eukaryotic CRISPRa is more versatile than bacterial CRISPRa, since more transcriptional activators are known, and their spatial constraints are more relaxed than in bacteria. Bacterial CRISPRa works efficiently in a narrow region of about 90 bp upstream of the transcription start site limiting applicability [164] in addition to the rare occurrence of PAM sequences in this region [157].…”

Section: Limitations Of Crispri and Future Directionsmentioning

confidence: 99%

Impact of CRISPR interference on strain development in biotechnology

Schultenkämper

Brito

Wendisch

2020

Biotech and App Biochem

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Genetic perturbation systems are of great interest to redirect metabolic fluxes for value‐added production, as well as genetic screening for the development of new drugs, or to identify new targets for biotechnological applications. Here, we review CRISPR interference (CRISPRi), a method for gene expression using a catalytically inactive version of the CRISPR‐associated protein 9 (dCas9) of the widely applied CRISPR‐Cas9 genome editing system. In combination with the appropriate sgRNA, dCas9 binds to specific DNA sequences without causing double‐stranded DNA breakage but interfering with transcription initiation or elongation. Besides manifold uses to interrogate the physiology of a bacterial cell, CRISPRi is used in applications for metabolic engineering and strain development in industrial biotechnology. Albeit in its infancy, CRISPRi has already delivered the first success stories; however, we also analyze limitations of the CRISPRi system and give future perspectives.

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Synthetic CRISPR-Cas gene activators for transcriptional reprogramming in bacteria

Cited by 172 publications

References 64 publications

Effective CRISPRa-mediated control of gene expression in bacteria must overcome strict target site requirements

Effective CRISPRa-mediated control of gene expression in bacteria must overcome strict target site requirements

Effective CRISPRa-Mediated Control of Gene Expression in Bacteria Must Overcome Strict Target Site Requirements

Impact of CRISPR interference on strain development in biotechnology

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