Reversible RNA Acylation for Control of CRISPR-Cas9 Gene Editing

Overview

Journal Chem Sci

Publisher Royal Society of Chemistry

Specialty Chemistry

Date 2021 Jun 4

PMID 34084356

Citations 22

Authors

Maryam Habibian

Colin McKinlay

Timothy R Blake

Anna M Kietrys

Robert M Waymouth

Paul A Wender

Eric T Kool

Affiliations

Soon will be listed here.

Abstract

We report the development of post-transcriptional chemical methods that enable control over CRISPR-Cas9 gene editing activity both in assays and in living cells. We show that an azide-substituted acyl imidazole reagent (NAI-N) efficiently acylates CRISPR single guide RNAs (sgRNAs) in 20 minutes in buffer. Poly-acylated ("cloaked") sgRNA was completely inactive in DNA cleavage with Cas9 , 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.

Citing Articles

Chemical diversity of reagents that modify RNA 2'-OH in water: a review.

Shioi R, Kool E Chem Sci. 2024; .

PMID: 39309104 PMC: 11412305. DOI: 10.1039/d4sc05317f.

Reversible RNA Acylation Using Bio-Orthogonal Chemistry Enables Temporal Control of CRISPR-Cas9 Nuclease Activity.

Pandit B, Fang L, Kool E, Royzen M ACS Chem Biol. 2024; 19(8):1719-1724.

PMID: 39051564 PMC: 11334111. DOI: 10.1021/acschembio.4c00117.

Second-Generation Chiral Amino Acid Derivatives Afford High Stereoselectivity and Stability in Aqueous RNA Acylation.

Shioi R, Chatterjee S, Xiao L, Zhong W, Kool E J Org Chem. 2024; 89(11):8055-8063.

PMID: 38809698 PMC: 11898221. DOI: 10.1021/acs.joc.4c00686.

Selective Arylation of RNA 2'-OH Groups via SAr Reaction with Trialkylammonium Heterocycles.

Chatterjee S, Xiao L, Zhong W, Feng S, Kool E Angew Chem Int Ed Engl. 2024; 63(25):e202403496.

PMID: 38625814 PMC: 11905364. DOI: 10.1002/anie.202403496.

Efficient post-synthesis incorporation and conjugation of reactive ketones in RNA 2'-acylation.

Shioi R, Xiao L, Fang L, Kool E Chem Commun (Camb). 2023; 60(2):232-235.

PMID: 38054242 PMC: 10745195. DOI: 10.1039/d3cc05123d.

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