Sequence-independent RNA sensing and DNA targeting by a split domain CRISPR-Cas12a gRNA switch.
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Issue Date
2021-02-22Submitted date
2021-02-22
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Show full item recordAbstract
CRISPR 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.Citation
Nucleic Acids Res. 2021 Mar 18;49(5):2985-2999. doi: 10.1093/nar/gkab100.Affiliation
HIRI, Helmholtz-Institut für RNA-basierte Infektionsforschung, Josef-Shneider Strasse 2, 97080 Würzburg, Germany.Publisher
Oxgord Uiversity PressJournal
Nucleic acids researchPubMed ID
33619539Type
ArticleLanguage
enEISSN
1362-4962ae974a485f413a2113503eed53cd6c53
10.1093/nar/gkab100
Scopus Count
The following license files are associated with this item:
- Creative Commons
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