Conditional control of RNA-guided nucleic acid cleavage and gene editing

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.

Page S3 with or without masking was incubated in the presence of 1.5 U RNase T1 in a reaction volume of 10 μL at 37 °C for various durations. Reaction was quenched by adding a 4.0-fold excess of quenching solution (0.1% SDS in formamide). RNA products were immediately separated on a denaturing 20% polyacrylamide gel (350 V, 1.0 hr).

Cell survival assay
Human HeLa-OC cells were grown in Gibco™ DMEM (Dulbecco's modification of Eagle medium), High Glucose medium (Thermo Fisher Scientific) containing 10% (v/v) Gibco™ fetal bovine serum (FBS, Thermo Fisher Scientific) and 1% penicillin/streptomycin (Invitrogen) at 37 °C , 5% CO2 humidified atmosphere. HeLa-OC cells (5 × 10 4 ) were seeded into wells of a 96well plate and incubated for 16 hr. Then cells were treated with different amounts of freshly dissolved DPBM or DMSO control for 24 hr. After centrifugation at 2000 rpm for 10 min, the medium was discarded and fresh medium was added. Subsequently, MTT (20 μL, 5 mg per mL in Page S4 DMSO) was added to each well and the mixture was incubated for an additional 4 hr. After centrifugation at 2000 rpm for 10 min, the medium was removed. Subsequently, DMSO (150 μL) was added to each well and the OD values at 570 nm were detected by a microplate reader (SpectraMax M5, Molecular Devices) to evaluate cell viability.
The BrdU (5-Bromo-2-deoxyUridine) assay was performed according to the manufacturer's protocol (Abcam, ab126556). HeLa-OC cells (5 × 10 4 ) were seeded into wells of a 96-well plate and incubated for 16 hr. Then cells were treated with different amounts of freshly dissolved DPBM or DMSO control for 24 hr. BrdU (10 μL per well, 10 μM) was added and incubated for 4 hr at 37 °C. The cells were then fixed with fixing solution (3.7% formaldehyde in 1 × PBS, 1 mM KH2PO4, 155 mM NaCl, 3 mM Na2HPO4 @ pH 7.4) at room temperature for 30 min. After the fixing solution was removed, anti-BrdU monoclonal antibody conjugated with peroxidase (100 µL per well) was added and incubated at room temperature for 1 hr. Then, the cells were washed with 1 × PBS, and peroxidase-conjugated goat anti-mouse immunoglobulin G (IgG) antibody (1:2,000) was added and incubated for 30 min. Subsequently, the cells were washed with 1 × PBS and incubated with the peroxidase substrate solution for 30 min at room temperature in the dark.
Finally, 100 µL of stop solution was added, and the absorbance was monitored at 450 nm by a microplate reader.
Light control of RNA-guided DNA cleavage 4 The Protector DNA containing PhotoCleavable linkers (PC linkers) were obtained from TaKaRa company (Dalian, China). Individual gRNA was annealed with Protector complement at a molar ratio of 1:2 in annealing buffer, which contained 50 mM Tris-HCl, 100 mM NaCl and 10 mM MgCl2 at pH 7.9 @ 25°C. Desired amount of gRNA/Protector in reaction mixture was irradiated for varied periods using a Spectroline UV source (UV-365EH model, 365 nm) at a distance of 4 cm from the source to test activity, followed by a further incubation at 37 °C for 24 hr (unless otherwise indicated). Reactions were quenched by adding SDS containing loading dye and loaded onto a 1.5% agarose gel containing 1.5 × Super GelRed for visualization (100 V, 1.5 hr).

Light control of gene editing
Human HeLa-OC cells (4 × 10 5 per well) were seeded into 6-well plates overnight before transfection and washed twice with DPBS, and 300 μL of pre-warmed DMEM was added to each well. Different gRNA/Protector complex (7.5 μg at a concentration of 1.5 μg per uL) were mixed in 120 μL of DMEM. The Lipofectamine 3000 transfection agent (5.0 μL, Thermo Fisher Scientific) in 120 μL of DMEM per well were added to the diluted gRNA, followed by an incubation (10 min). The complex was added to the cells, and the medium was changed to complete DMEM after an incubation (6 hr) at 37 °C in 5 % CO2. Cells were irradiated using a Spectroline UV source (UV-365EH model, 365 nm) and further cultured for 24 hr at 37 °C. gRNA- The forward primer for gRNA construct (gRNA-GFP+4) The forward primer for gRNA construct (gRNA-GFP+8) The forward primer for gRNA construct (gRNA-ANTXR1) For tracrRNA construct