Anti-CRISPR RNAs: designing universal riboregulators with deep learning of Csy4-mediated RNA processing

Guo, Haotian; Song, Xiaohu; Lindner, Ariel B.

doi:10.1101/2020.11.15.384107

Cited by 3 publications

(7 citation statements)

References 47 publications

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“…The data suggest however a higher cleavage performance at 20 nM of DNA template compared to the fusion protein experiment. We are not able to directly explain this observation, but we hypothesize that the Csy4 binding hairpin could be heavily constrained by its flanking sequences, as shown by Guo et al 34 .…”

Section: Resultsmentioning

confidence: 73%

“…We anticipate this could have implications in the implementation of Csy4 derivatives in cell-free systems, such as the inactivated Csy4 40 or Csy4-mediated RNA switches. 34 The concentration of CRISPR-associated proteins can be tuned via the promoter strength of the expression cassette to achieve various processing efficiencies. The use of MGA aptamers enabled us to monitor the mRNA dynamics in diverse circuitry configurations, constituting novel examples of using a dual-reporter system.…”

Section: Resultsmentioning

confidence: 99%

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Functionalizing cell-free systems with CRISPR-associated proteins: Application to RNA-based circuit engineering

Lehr

Kuzembayeva²,

Kleindienst³

et al. 2021

Preprint

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Cell-free systems have become a compelling choice for the prototyping of synthetic circuits. Many robust protocols for preparing cell-free systems are now available along with toolboxes designed for a variety of applications. Thus far the production of cell-free extracts has often been decoupled from the production of functionalized proteins. Here, we leveraged the most recently published protocol for E. coli-based cell extracts with the endogenous production of two CRISPR-associated proteins, Csy4 and dCas9. We found pre-expression did not affect the resulting extract performance, and the final concentrations of the endonucleases matched the level required for synthetic circuit prototyping. We demonstrated the benefits and versatility of dCas9 and Csy4 through the use of RNA circuitry based on a combination of single-guide RNAs, small transcriptional activator RNAs, and toehold switches. For instance, we show that Csy4 processing increased fourfold the dynamic range of a previously published AND-logic gate. Additionally, blending the CRISPR-enhanced extracts enabled us to reduce leakage in a multiple inputs gate, and to extend the type of Boolean functions available for RNA-based circuits, such as NAND-logic. Finally, the use of dual transcriptional and translational reporters for the engineering of RNA-based circuits, allowed us to gain better insight into their underlying mechanisms. We hope this work will facilitate the adoption of advanced processing tools for RNA-based circuit prototyping in a cell-free environment.

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Section: Resultsmentioning

confidence: 73%

Section: Resultsmentioning

confidence: 99%

Functionalizing cell-free systems with CRISPR-associated proteins: Application to RNA-based circuit engineering

Lehr

Kuzembayeva²,

Kleindienst³

et al. 2021

Preprint

View full text Add to dashboard Cite

show abstract

“…The data suggest, however, a higher cleavage performance at 20 nM of DNA template compared to the fusion protein experiment. We are not able to directly explain this observation, but we hypothesize that the Csy4 binding hairpin could be heavily constrained by its flanking sequences, as shown by Guo et al Generating a control construct mimicking the cleaved product facilitated the analysis of otherwise hard to interpret UTR-related data. This could easily be applied to other experiments, e.g.…”

Section: Resultsmentioning

confidence: 90%

“…Csy4 cleavage activity displayed high efficiency for DNA concentrations traditionally used in cell-free circuit prototyping. We anticipate this could have implications in the implementation of Csy4 derivatives in cell-free systems, such as the inactivated Csy4 or Csy4-mediated RNA switches . The concentration of CRISPR-associated proteins can be tuned via the promoter strength of the expression cassette to achieve various processing efficiencies.…”

Section: Discussionmentioning

confidence: 99%

“…Taking advantage of the dual reporter system, we showed that Csy4 processing specifically enhances the translational activity of our multilevel regulated gate. This suggests that the Csy4 hairpin acts as en efficient insulator of the toehold switch component in our specific gate design, but future work would be needed to establish design rules in Csy4 hairpin positioning to maximize cleavage efficiency across multiple gates . Furthermore, this work could encourage the use of the short cleaving hairpin in RNA-based circuits or offer the possibility to take advantage of existing compatible DNA parts. , Future work could extend the one-pot strategy to include additional enhanced CFE extracts.…”

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

Functionalizing Cell-Free Systems with CRISPR-Associated Proteins: Application to RNA-Based Circuit Engineering

et al. 2021

View full text Add to dashboard Cite

Cell-free systems have become a compelling choice for the prototyping of synthetic circuits. Many robust protocols for preparing cell-free systems are now available along with toolboxes designed for a variety of applications. Thus far, the production of cell-free extracts has often been decoupled from the production of functionalized proteins. Here, we leveraged a recent protocol for producing an E. coli-based cell-free expression system with two CRISPR-associated proteins, Csy4 and dCas9, expressed prior to harvest. We found that pre-expression did not affect the resulting extract performance, and the final concentrations of the endonucleases matched the level required for synthetic circuit prototyping. We demonstrated the benefits and versatility of dCas9 and Csy4 through the use of RNA circuitry based on a combination of single guide RNAs, small transcriptional activator RNAs, and toehold switches. For instance, we show that Csy4 processing increased 4-fold the dynamic range of a previously published AND-logic gate. Additionally, blending the CRISPRenhanced extracts enabled us to reduce leakage in a multiple inputs gate, and to extend the type of Boolean functions available for RNA-based circuits, such as NAND-logic. Finally, we reported the use of simultaneous transcriptional and translational reporters in our RNA-based circuits. In particular, the AND-gate mRNA and protein levels were able to be independently monitored in response to transcriptional and translational activators. We hope this work will facilitate the adoption of advanced processing tools for RNAbased circuit prototyping in a cell-free environment.

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Engineering CRISPR guide RNAs for programmable RNA sensors

Liu,

Wang

2023

Biochemical Society Transactions

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As the most valuable feature of the CRISPR system, the programmability based on Watson–Crick base pairing has been widely exploited in engineering RNA sensors. The base pairing in these systems offers a connection between the RNA of interest and the CRISPR effector, providing a highly specific mechanism for RNA detection both in vivo and in vitro. In the last decade, despite the many successful RNA sensing approaches developed during the era of CRISPR explosion, a deeper understanding of the characteristics of CRISPR systems and the continuous expansion of the CRISPR family members indicates that the CRISPR-based RNA sensor remains a promising area from which a variety of new functions and applications can be engineered. Here, we present a systematic overview of the various strategies of engineering CRISPR gRNA for programmable RNA detection with an aim to clarify the role of gRNA's programmability among the present limitations and future development of CRISPR-enabled RNA sensors.

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Anti-CRISPR RNAs: designing universal riboregulators with deep learning of Csy4-mediated RNA processing

Cited by 3 publications

References 47 publications

Functionalizing cell-free systems with CRISPR-associated proteins: Application to RNA-based circuit engineering

Functionalizing cell-free systems with CRISPR-associated proteins: Application to RNA-based circuit engineering

Functionalizing Cell-Free Systems with CRISPR-Associated Proteins: Application to RNA-Based Circuit Engineering

Engineering CRISPR guide RNAs for programmable RNA sensors

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