Sequence-independent RNA sensing and DNA targeting by a split domain CRISPR-Cas12a gRNA switch.
Cast your vote
You can rate an item by clicking the amount of stars they wish to award to this item.
When enough users have cast their vote on this item, the average rating will also be shown.
Your vote was cast
Thank you for your feedback
Thank you for your feedback
MetadataShow full item record
AbstractCRISPR technologies increasingly require spatiotemporal and dosage control of nuclease activity. One promising strategy involves linking nuclease activity to a cell's transcriptional state by engineering guide RNAs (gRNAs) to function only after complexing with a 'trigger' RNA. However, standard gRNA switch designs do not allow independent selection of trigger and guide sequences, limiting gRNA switch application. Here, we demonstrate the modular design of Cas12a gRNA switches that decouples selection of these sequences. The 5' end of the Cas12a gRNA is fused to two distinct and non-overlapping domains: one base pairs with the gRNA repeat, blocking formation of a hairpin required for Cas12a recognition; the other hybridizes to the RNA trigger, stimulating refolding of the gRNA repeat and subsequent gRNA-dependent Cas12a activity. Using a cell-free transcription-translation system and Escherichia coli, we show that designed gRNA switches can respond to different triggers and target different DNA sequences. Modulating the length and composition of the sensory domain altered gRNA switch performance. Finally, gRNA switches could be designed to sense endogenous RNAs expressed only under specific growth conditions, rendering Cas12a targeting activity dependent on cellular metabolism and stress. Our design framework thus further enables tethering of CRISPR activities to cellular states.
CitationNucleic Acids Res. 2021 Mar 18;49(5):2985-2999. doi: 10.1093/nar/gkab100.
AffiliationHIRI, Helmholtz-Institut für RNA-basierte Infektionsforschung, Josef-Shneider Strasse 2, 97080 Würzburg, Germany.
PublisherOxgord Uiversity Press
JournalNucleic acids research
The following license files are associated with this item:
- Creative Commons
- Extinction of all infectious HIV in cell culture by the CRISPR-Cas12a system with only a single crRNA.
- Authors: Gao Z, Fan M, Das AT, Herrera-Carrillo E, Berkhout B
- Issue date: 2020 Jun 4
- CRISPR-Cas12a has widespread off-target and dsDNA-nicking effects.
- Authors: Murugan K, Seetharam AS, Severin AJ, Sashital DG
- Issue date: 2020 Apr 24
- Self-Supplying Guide RNA-Mediated CRISPR/Cas12a Fluorescence System for Sensitive Detection of T4 PNKP.
- Authors: Yuan X, Yuan H, Liu B, Liu Y
- Issue date: 2022 Dec 17
- Multiplexed Genome Engineering with Cas12a.
- Authors: Weisbach NR, Meijs A, Platt RJ
- Issue date: 2021
- Creating CRISPR-responsive smart materials for diagnostics and programmable cargo release.
- Authors: Gayet RV, de Puig H, English MA, Soenksen LR, Nguyen PQ, Mao AS, Angenent-Mari NM, Collins JJ
- Issue date: 2020 Sep