Riboregulated toehold-gated gRNA for programmable CRISPR–Cas9 function

Siu, Ka-Hei; Chen, Wilfred

doi:10.1038/s41589-018-0186-1

Cited by 127 publications

(126 citation statements)

References 26 publications

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“…Recently, Siu and Chen demonstrated a median ≈6.6-fold OFF→ON conditional response using toehold switch cgRNAs with subtly different structural details in the sequestration of the targetbinding region. 29 Unlike the terminator switch and splinted switch mechanisms for ON→OFF logic, toehold switch cgR-NAs for OFF→ON logic are not allosteric as the cgRNA initially down-regulates cgRNA:dCas9 function by sequestering the target-binding region (orange domain "u" in Figure 4a) with a portion of the trigger-binding region (orange domain "u*"). As a result, the toehold switch cgRNAs offer only partial sequence independence between the trigger X and the target gene Y ("u" is a subsequence of both X and Y).…”

Section: Resultsmentioning

confidence: 99%

Conditional Guide RNAs: Programmable Conditional Regulation of CRISPR/Cas Function in Bacteria via Dynamic RNA Nanotechnology

Hanewich-Hollatz

Chen

Huang

et al. 2019

Preprint

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Dynamic RNA Nanotechnology in Living Cells Programmable RNA trigger X dCas9 Constitutively inactive (OFF ON logic) cgRNA Y Programmable DNA target Constitutively active (ON OFF logic) X dCas9 Y cgRNAA guide RNA (gRNA) directs the function of a CRISPR protein effector to a target gene of choice, providing a versatile programmable platform for engineering diverse modes of synthetic regulation (edit, silence, induce, bind). However, the fact that gRNAs are constitutively active places limitations on the ability to confine gRNA activity to a desired location and time. To achieve programmable control over the scope of gRNA activity, here we apply principles from dynamic RNA nanotechnology to engineer conditional guide RNAs (cgRNAs) whose activity is dependent on the presence or absence of an RNA trigger. These cgRNAs are programmable at two levels, with the trigger-binding sequence controlling the scope of the effector activity and the target-binding sequence determining the subject of the effector activity. We demonstrate molecular mechanisms for both constitutively active cgRNAs that are conditionally inactivated by an RNA trigger (ON→OFF logic) and constitutively inactive cgRNAs that are conditionally activated by an RNA trigger (OFF→ON logic). For each mechanism, automated sequence design is performed using the reaction pathway designer within NUPACK to design an orthogonal library of three cgRNAs that respond to different RNA triggers. In E. coli expressing cgRNAs, triggers, and silencing dCas9 as the protein effector, we observe programmable conditional gene silencing with a median dynamic range of ≈6-fold for an ON→OFF "terminator switch" mechanism, ≈15-fold for an ON→OFF "splinted switch" mechanism, and ≈3.6-fold for an OFF→ON "toehold switch" mechanism; the median crosstalk within each cgRNA library is <2%, <2%, and ≈20% for the three mechanisms. By providing programmable control over both the scope and target of protein effector function, cgRNA regulators offer a promising platform for synthetic biology.

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Section: Resultsmentioning

confidence: 99%

Conditional Guide RNAs: Programmable Conditional Regulation of CRISPR/Cas Function in Bacteria via Dynamic RNA Nanotechnology

Hanewich-Hollatz

Chen

Huang

et al. 2019

Preprint

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“…The sgRNA that integrates the riboswitch properties allows the entire CRISPR system to acquire the ability to sense related ligands to regulate the "on-off" of the CRISPR system. Since the publication of our studies, there have been many studies attempting to modify sgRNAs in different ways to regulate the CRISPR system [40][41][42], depending on specific ligands. The interactions of ligands and the aptamers stabilize the structure of the aptamers, and this conformational change exposes the spacer of sgRNA blocked by the aptamer, which allows the sgRNA to restore the ability to guide the Cas protein to the targeted DNA sequence ( Figure 4A).…”

Section: Crispr-sgrna Based Signal Sensorsmentioning

confidence: 99%

Engineering Cellular Signal Sensors based on CRISPR-sgRNA Reconstruction Approaches

Zhan

Xiao

et al. 2020

Int. J. Biol. Sci.

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“…Multiplexing the precise transcriptional regulation through integration of both CRISPRa and CRISPRi is expected to reconstruct the metabolic network to enhance the precursor supply, reducing flux toward competing pathways or unwanted by-products, bypassing gene expression regulation, and expressing the SM-BGCs. Recently, toehold-gated gRNA was developed, which links endogenous signals to activation of the CRISPR/Cas system [133]. As the production of secondary metabolites requires sufficient accumulation of precursors and activation of biosynthetic genes, the combination of toehold-gated gRNA and CRISPRa and CRISPRi strategies will suggest a new metabolic engineering approach, linking the production of precursors to production of secondary metabolites.…”

Section: Future Perspectivementioning

confidence: 99%

Synthetic Biology Tools for Novel Secondary Metabolite Discovery in Streptomyces

Lee¹,

Hwang²,

Lee³

et al. 2019

Journal of Microbiology and Biotechnology

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Riboregulated toehold-gated gRNA for programmable CRISPR–Cas9 function

Cited by 127 publications

References 26 publications

Conditional Guide RNAs: Programmable Conditional Regulation of CRISPR/Cas Function in Bacteria via Dynamic RNA Nanotechnology

Conditional Guide RNAs: Programmable Conditional Regulation of CRISPR/Cas Function in Bacteria via Dynamic RNA Nanotechnology

Engineering Cellular Signal Sensors based on CRISPR-sgRNA Reconstruction Approaches

Synthetic Biology Tools for Novel Secondary Metabolite Discovery in Streptomyces

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