Supramolecular CRISPR-OFF switches with host–guest chemistry

Abstract CRISPR (Clustered Regularly Interspaced Short Palindromic Repeat) technology is a powerful tool in biology and medicine. However, the safety and application of this technology is hampered by excessive activity of CRISPR machinery. It is particularly important to develop methods for switching off CRISPR activity in human cells. The current study demonstrates the concept of supramolecular CRISPR-OFF switches by employing host-guest chemistry. We demonstrate that the CRISPR systems show considerable tolerance to adamantoylation on guide RNAs (gRNAs), whereas supramolecular complexation tremendously affects the function of adamantoyl gRNAs. Host–guest chemistry is demonstrated to be novel and effective tools to reduce unwanted excessive activities of CRISPR complexes in human cells. This work indicates considerable potential of supramolecular strategy for controlling and enhancing CRISPR systems.


Materials
Page S4 Chemical synthesis Pages S4-6 General methods of biological assay Pages S6-9 Table S1 DNA and RNA sequences used in the current study Pages S10-11 Figure S1 Structures of ADIs Page S12

Figure S2
Native PAGE analysis of adamantoyl R-32nt in the absence or presence of CB7 Page S13 Figure  The ADI-1 was synthesized according to a previous protocol 1 .
1-Adamantanemethanol (compound 1, 166 mg, 1.0 mmol) was dissolved in anhydrous CH2Cl2 (5 mL) in a flame-dried flask. Carbonyldiimidazole (CDI,194 mg,1.2 mmol) was added and the solution was stirred at room temperature for 2 hr. The mixture was diluted into CH2Cl2 (20 mL) and washed with water (2 × 25 mL). The organic phase was washed with brine (1 × 25 mL), dried over anhydrous MgSO4, filtered, and concentrated in vacuum to obtain the crude product. The crude product was purified by column chromatography (ethyl acetate: petroleum ether = 1:5

Molecular optimization study using Gaussian09
All optimizations were done at M062X/def2-SVP level with Grimme's D3 empirical dispersion correction 3 . The volume of the optimized geometries are computed, defined as the volume inside a contour of 0.001 electrons/Bohr 3 density. All the calculations are performed using Gaussian09 program 4 .

Denaturing polyacrylamide gel electrophoresis
The denaturing polyacrylamide gel electrophoresis was run in a temperature-controlled vertical electrophoretic apparatus (DYCZ-22A, Liuyi Instrument Factory, Beijing, China). The acrylamide concentration of the separating gel was 20 % (19 : 1 monomer to bis ratio). About one hundred nanograms of DNA were loaded on the gel. Electrophoresis was run at 10 °C for 12 hr at 400 V. The Super GelRed was used for post-electrophoresis gel staining. The target nucleic acids in the gel were visualized using a Pharos FX Molecular imager (Bio-Rad, USA) in the fluorescence mode (λex = 590 nm).

Supramolecular control of reverse transcription
For the M-MuLV RT, this reaction was performed in 1 × M-MuLV buffer, which contained 50 mM The following quenching and electrophoresis steps are similar to the progression steps of the procedure described above (the M-MuLV RT).

RNase degrading assay
Adamantoyl RNAs (R-21nt-FAM) were prepared using the general protocol for RNA adamantoylation.
Adamantoyl RNAs (100 ng) were treated with CB7 at varied concentrations for 20 min, and were subjected to RNase degradation.
For RNase I assay, this assay was performed in 1 × buffer in PCR grade water, which contained 20 mM Tris-HCl (pH 8.0), 100 mM NaCl, 0.1 mM EDTA and 0.01% Triton X-100. The fluorescently labeled RNA (100 ng) with or without adamantoylation was incubated in the presence of 1.5 U RNase I in a reaction volume of 10 μL at 37 °C for 10 min. 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).
For RNase T1 assay, this assay was performed in 1 × buffer in PCR grade water, which contained 50 mM Tris-HCl and 2 mM EDTA at pH 7.5 @ 25 °C. The fluorescently labeled RNA (100 ng) with or without adamantoylation was incubated in the presence of 1.5 U RNase T1 in a reaction volume of 10 μL at 37 °C for 30 min. The following quenching and electrophoresis steps are similar to the progression steps of the procedure described above (RNase I).

LwCas13a expression
The wild type and mutated LbuCas13a genes were purchased from Addgene (Plasmid #83482 and #91861). The fusion proteins contained an N-terminal His6-MBP-TEV protease cleavage site. Target plasmids were transformed in E. coli Rosetta (DE3) (Novagen) cells. A 16 mL starter culture was grown overnight, which was used to inoculate 4 L expression culture for growth at 37 °C and 300 RPM until an OD600 of 0.6. At this time, protein expression was induced by supplementation with IPTG (isopropyl-β-D-thiogalactoside, Sigma) to a final concentration of 500 µM, and cells were cooled to 16 °C for 16 hr for protein expression. Cells were collected by centrifugation and were lysed by sonication in a buffer (20 mM Tris-HCl, 500 mM NaCl, pH 7.5). All subsequent steps of the protein purification were performed at 4 °C. After centrifugation, the supernatant was incubated with Ni Sepharose (GE Healthcare). The bound protein was eluted with buffer containing 100 mM imidazole. The His6-MBP tag of eluted proteins were removed by TEV protease digestion. Target proteins were purified on an SP column (GE Healthcare), eluting with a buffer (20 mM Tris-HCl, 1M NaCl, pH 7.5). Target The protein was further purified by size-exclusion chromatography (Superdex 200 Increase 10/300, GE Healthcare) in a buffer containing 50 mM HEPES, pH 7.5, 150 mM KCl, and then concentrated to a concentration of 10 mg/mL. This study was performed by using fluorescently labeled nuclease-deficient Cas9 (dCas9), which was expressed and purified according to a previous literature.
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/mL in 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 forward primer for R-102nt construct The forward primer for R-106nt construct The forward primer for R-110nt construct The reverse primer for The forward primer for sg-SLX4IP construct The forward primer for sg-HPRT1 construct The forward primer for sg-HBEGF construct The reverse primer for sgRNA construct

Figure S1: Structures of ADIs
ADI-1 and ADI-2 are designed and synthesized for the preparation of adamantoyl RNAs. All of these molecules were well characterized by spectroscopic techniques such as 1 H NMR, 13 C NMR and mass spectrometry.

Figure S2. Native PAGE analysis of adamantoyl R-32nt in the absence or presence of CB7
Reactions were carried out as described in the Experimental Section. R-32nt was used in this study. All samples were tested in three biological replicates. Image of representative data is shown here. In gel RNAs were seen with  Table S1); lanes 2, 8: unmodified RNAs; lanes 3-7, 9-13: adamantoyl RNA with different modification levels.

Figure S3. Supramolecular complexation in unstructured RNA contexts (R-21nt and R-21nt-c)
Reactions were carried out as described in the Experimental Section. R-21nt and R-21nt-c were used in this study.

Figure S4. Supramolecular complexation in unstructured RNA contexts (R-44nt)
Reactions were carried out as described in the Experimental Section. R-44nt was used in this study. All samples were tested in three biological replicates. Image of representative data is shown here. In gel RNAs were seen with  Table S1); lanes 2, 8: unmodified RNAs; lanes 3-7, 9-13: adamantoyl RNAs with different modification levels.

Figure S10. Supramolecular complexation on Cas13a gRNAs
Reactions were carried out as described in the Experimental Section. The gRNA for Cas13a was used in this study.
All samples were tested in three biological replicates. Image of representative data is shown here. In gel RNAs were seen with GelRed staining. The CB7-bound gRNA experiences greater resistance and moves more slowly  Table S1); lanes 2, 8: unmodified gRNAs; lanes 3-7, 9-13: adamantoyl gRNAs with different modification levels.

Figure S36. The tolerance of HeLa-OC cells to CB7
HeLa-OC cells were treated with CB7 at different concentrations and toxicity was measured using the the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cytotoxicity assay. Values were plotted relative to the mean of DMEM control set to 100% (= relative growth). All data were presented as the means ± SEM from three independent experiments. Error bars: ±SEM. The cytotoxicity threshold for CB7 was higher than 500 μM.