Reversible RNA acylation for control of CRISPR-Cas9 gene editing.
Maryam HabibianColin McKinlayTimothy R BlakeAnna M KietrysRobert M WaymouthPaul A WenderEric T KoolPublished in: Chemical science (2019)
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.
Keyphrases
- crispr cas
- genome editing
- cell cycle arrest
- induced apoptosis
- living cells
- single molecule
- circulating tumor
- cell death
- nucleic acid
- fluorescent probe
- transcription factor
- endoplasmic reticulum stress
- cell free
- fatty acid
- signaling pathway
- gene expression
- cell proliferation
- genome wide
- smoking cessation
- circulating tumor cells
- replacement therapy
- molecular dynamics simulations
- combination therapy