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Reversible RNA Acylation for Control of CRISPR-Cas9 Gene Editing 

Abstract

We report the development of post-transcriptional chemical methods that enable control over CRISPR-Cas9 gene editing activity both in in vitro assays and in living cells. We show that an azide-substituted acyl imidazole reagent (NAI-N3) efficiently acylates CRISPR single guide RNAs (sgRNAs) in 20 minutes in buffer. Poly-acylated (“cloaked”) sgRNA was completely inactive in DNA cleavage with Cas9 in vitro, and activity was quantitatively restored after phosphine treatment. Delivery of cloaked sgRNA and Cas9 mRNA into HeLa cells was enabled by the use of charge-altering releasable transporters (CARTs), which outperformed commercial transfection reagents in transfecting sgRNA co-complexed with Cas9 encoding functional mRNA. Genomic DNA cleavage in the cells by CRISPR-Cas9 was efficiently restored after treatment with phosphine to remove the blocking acyl groups. Our results highlight the utility of reversible RNA acylation as a novel method for temporal control of genome-editing function.

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Publication details

The article was received on 23 Jul 2019, accepted on 28 Nov 2019 and first published on 02 Dec 2019


Article type: Edge Article
DOI: 10.1039/C9SC03639C
Chem. Sci., 2020, Accepted Manuscript
  • Open access: Creative Commons BY-NC license
    All publication charges for this article have been paid for by the Royal Society of Chemistry

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    Reversible RNA Acylation for Control of CRISPR-Cas9 Gene Editing

    M. M. Habibian, C. J. Mckinlay, T. R. Blake, R. Waymouth, A. M. Kietrys, P. Wender and E. Kool, Chem. Sci., 2020, Accepted Manuscript , DOI: 10.1039/C9SC03639C

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