CRISPR-Cas systems provide versatile tools for programmable genome editing. Here, we developed a caged RNA strategy that allows Cas9 to bind DNA but not cleave until light-induced activation. This approach, referred to as very fast CRISPR (vfCRISPR), creates double-strand breaks (DSBs) at the submicrometer and second scales. Synchronized cleavage improved kinetic analysis of DNA repair, revealing that cells respond to Cas9-induced DSBs within minutes and can retain MRE11 after DNA ligation. Phosphorylation of H2AX after DNA damage propagated more than 100 kilobases per minute, reaching up to 30 megabases. Using single-cell fluorescence imaging, we characterized multiple cycles of 53BP1 repair foci formation and dissolution, with the first cycle taking longer than subsequent cycles and its duration modulated by inhibition of repair. Imaging-guided subcellular Cas9 activation further facilitated genomic manipulation with single-allele resolution. vfCRISPR enables DNA-repair studies at high resolution in space, time, and genomic coordinates.
Reversible RNA acylation for control of CRISPR–Cas9 gene editing
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-N 3 ) 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.
- Award ID(s):
- 1856414
- Publication Date:
- NSF-PAR ID:
- 10274591
- Journal Name:
- Chemical Science
- Volume:
- 11
- Issue:
- 4
- Page Range or eLocation-ID:
- 1011 to 1016
- ISSN:
- 2041-6520
- Sponsoring Org:
- National Science Foundation
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- Free Publicly Accessible Full Text
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Accepted Manuscript1.0
- Publisher's Version of Record
- https://doi.org/10.1039/C9SC03639C
