Issue 4, 2020

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.

Graphical abstract: Reversible RNA acylation for control of CRISPR–Cas9 gene editing

Supplementary files

Article information

Article type
Edge Article
Submitted
23 7月 2019
Accepted
28 11月 2019
First published
02 12月 2019
This article is Open Access

All publication charges for this article have been paid for by the Royal Society of Chemistry
Creative Commons BY-NC license

Chem. Sci., 2020,11, 1011-1016

Reversible RNA acylation for control of CRISPR–Cas9 gene editing

M. Habibian, C. McKinlay, T. R. Blake, A. M. Kietrys, R. M. Waymouth, P. A. Wender and E. T. Kool, Chem. Sci., 2020, 11, 1011 DOI: 10.1039/C9SC03639C

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