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, 11 (1), 91

Conditional Control of RNA-guided Nucleic Acid Cleavage and Gene Editing

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Conditional Control of RNA-guided Nucleic Acid Cleavage and Gene Editing

Shao-Ru Wang et al. Nat Commun.

Abstract

Prokaryotes use repetitive genomic elements termed CRISPR (clustered regularly interspaced short palindromic repeats) to destroy invading genetic molecules. Although CRISPR systems have been widely used in DNA and RNA technology, certain adverse effects do occur. For example, constitutively active CRISPR systems may lead to a certain risk of off-target effects. Here, we introduce post-synthetic masking and chemical activation of guide RNA (gRNA) to controlling CRISPR systems. An RNA structure profiling probe (2-azidomethylnicotinic acid imidazolide) is used. Moreover, we accomplish conditional control of gene editing in live cells. This proof-of-concept study demonstrates promising potential of chemical activation of gRNAs as a versatile tool for chemical biology.

Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1. Schematic illustration of the design and workflow.
a The post-synthetic masking was used to block gRNA biochemical activity, whereas the Staudinger reduction restored gRNA function. b Chemical activation of gRNA to controlling CRISPR systems.
Fig. 2
Fig. 2. Conditional control of RNA-guided DNA cleavage.
Reactions were carried out as described in the Experimental section. All samples were tested in three biological replicates. Image of representative data is shown here. Uncleaved t-GFP1 DNA (702 bp) cut to shorter cleavage fragments (469 bp and 233 bp) were indicated. a, b The influence of chemical masking of gRNA on Cas9 cleavage of t-GFP1. The gRNA (gRNA-GFP) was synthesized by in vitro transcription with T7 RNA polymerase. c, d The influence of DPBM on Cas9 cleavage of t-GFP1. The CRISPR/Cas9 system with acylated gRNA-GFP (200 mM NAI-N3, 2 h) was treated with various concentrations of DPBM. Source data is available in the Source Data file.
Fig. 3
Fig. 3. Conditional control of Cas13a cleavage.
Reactions were carried out as described in the Experimental section. All samples were tested in three biological replicates. Image of representative data is shown here. The fluorescence of the sample versus time was shown. a, c, e The influence of chemical masking on Cas13a cleavage. The crRNA13a was masked with NAI-N3 (200 mM) for different durations. b, d, f The influence of DPBM on Cas13a cleavage. The CRISPR/Cas13a system with masked crRNA13a (200 mM NAI-N3, 2.0 h) was treated with various concentrations of DPBM. Source data is available in the Source Data file.
Fig. 4
Fig. 4. Chemical masking protects gRNA from RNase degradation.
Reactions were carried out as described in the Experimental section. All samples were tested in three biological replicates. Lanes 1, 5, 9, 13: no RNase I control; lanes 2, 6, 10, 14: RNase I cleavage for 2.5 min; lanes 3, 7, 11, 15: RNase I cleavage for 10 min; lanes 4, 8, 12, 16: RNase I cleavage for 30 min. Source data is available in the Source Data file.
Fig. 5
Fig. 5. Interactions of crRNA13a with both Cas13a and target RNA.
a Representative melting profiles of the crRNA/target RNA with different treatments were recorded in 10 mM Tris-HCl buffer (pH 7.0, 100 mM NaCl). Melting temperature of the NAI-N3-treated sample was statistically significantly different from that of the no treatment control (P < 0.05). b The dCas13a was incubated with 5′-Cy3-labeled crRNA probe (crRNA13a-Cy3 in Supplementary Table 1) with different treatments, and the samples were separated on native 6% PAGE gels. Lanes 1, 3, 5, 7, 9: no protein control; lanes 2, 4, 6, 8, 10: crRNA13a was incubated with a 5.0 molar excess of protein. Source data is available in the Source Data file.
Fig. 6
Fig. 6. Gene editing in live cells.
The T7E1 nuclease assay was performed 24 h post-transfection using Cas9-expressing HeLa-OC cells transfected with gRNA-HBEGF with different treatments. a Uncleaved HBEGF DNA (621 bp) cut to shorter cleavage fragments (311 bp and 310 bp) were indicated. Lane 1: target control; lane 2: no gRNA-HBEGF control; lane 3: original gRNA-HBEGF; lane 4: original gRNA-HBEGF, 300 μM DPBM; lanes 5–9: masked gRNA-HBEGF (200 mM, 1 h), different concentrations of DPBM; lane 10: DNA markers. The “Lanes and Bands” tool in Image Lab software version 5.1 (Bio-Rad) was used for image acquisition and differential densitometric analysis of the associated bands from the gels. Although long exposure times were necessary to visualize faint bands, the calculations of indel formation were not influenced by the extent of exposure. b The Cyan color represents the original gRNA-HBEGF group, while the Blue color represents the masked gRNA-HBEGF group. Error bars: ±SEM. Source data are provided as a Source Data file.

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