Materials and Methods

Extensive CRISPR RNA modification reveals chemical compatibility and structure-activity relationships for Cas9 biochemical activity

Chemically modified oligonucleotide synthesis

Standard phosphoramidite solid-phase synthesis conditions were used for the synthesis of all modified and unmodified oligonucleotides. Syntheses were performed on an Applied Biosystems 3400 or Expedite DNA Synthesizer at a 1 micromole scale using Unylink CPG support (ChemGenes). All phosphoramidites were prepared as 0.15 M solutions in acetonitrile (ACN), except DNA, which was prepared as 0.1 M solutions. 5-Ethylthiotetrazole (0.25 M in ACN) was used to activate phosphoramidites for coupling. Detritylations were accomplished with 3% trichloroacetic acid in CH2Cl2 for 110 s. Capping of failure sequences was achieved with acetic anhydride in tetrahydrofuran (THF) and 16% N-methylimidazole in THF. Oxidation was done using 0.1M I2 in 1:2:10 pyridine:water:THF. Coupling times were 10–15 min for RNA, 2′F-ANA, 2′F-RNA, 2′F,4′_O_Me-RNA, 2′,4′-di_O_Me-RNA and LNA phosphoramidites. Mixed sugar modifications were prepared using premixed 1:1 equivalents of RNA 2′-amidite with RNA 3′-amidite, or 1:1 equivalents of RNA 2′-amidite or DNA 3′-amidite, or 1:1 equivalents of RNA 2′-amidite with 2′F-RNA 3′-amidite. This 1:1 ratio results in ∼0.77:1 incorporation of 2′-amidite to 3′-amidite. Deprotection and cleavage from the solid support was accomplished with either 3:1 NH4OH:EtOH for 48 h at room temperature (RT), or at 55°C for 16 h. Oligonucleotides containing RNA were synthesized with standard 2′-TBDMS phosphoramidites, and desilylation was achieved with either neat triethylamine trihydrofluoride for 48 h at RT, or with triethylamine trihydrofluoride/N-methyl pyrrolidone/triethylamine (1.5:0.75:1 by volume) for 2.5 h at 65°C. Crude oligonucleotides were purified by anion exchange HPLC on an Agilent 1200 Series Instrument using a Protein-Pak DEAE 5PW column (7.5 × 75 mm) at a flow rate of 1 ml/min. The gradient was 0–24% of 1 M LiClO4 over 30 min at 60°C. Samples were desalted on NAP-25 desalting columns according to manufacturer protocol. Modified crRNAs were prepared for RNP assembly by heating to 95°C then placing on ice to prevent formation of stable secondary structures.

RNA and RNA–DNA oligonucleotide synthesis

DNA oligonucleotides and DNA–RNA chimeric oligonucleotides were synthesized by Integrated DNA Technologies (IDT). Chimeric crRNAs were prepared for RNP assembly by heating to 95°C then placing on ice to prevent formation of stable secondary structures. Cas9 tracrRNAs were prepared by T7 RNA polymerase in vitro transcription with DNA templates synthesized by IDT. Single-stranded DNA templates were annealed to T7 promoter oligo to generate double-stranded promoter regions, which support in vitro transcription by T7 RNA polymerase. In vitro transcriptions were performed by standard protocols for 2 h. Briefly, reactions contained purified T7 RNA polymerase (4 μM), 30 mM Tris (at pH 7.9), 12.5 mM NaCl, 40 mM MgCl2, 2% PEG 8000, 0.05% Triton X-100, 2 mM spermidine and 2.5 μM T7-DNA template. Afterward, the DNA template was degraded by DNase I treatment. Reactions were phenol–chloroform extracted and gel-purified from denaturing polyacrylamide gels. Purified RNA was refolded by heating to 95°C for 5 min in a heating block followed by slow cooling the block to RT (∼40 min). RNA was quantified by measuring absorbance at 260 nm and calculated extinction coefficients using nearest neighbor approximations and Beer’s Law.

Preparation of Cas9 enzymes

Plasmid encoding an _Sp_Cas9 (simply referred to as Cas9) with a C-terminal fusion of a nuclear localization signal (NLS) and a 6x-Histidine tag (pET-Cas9-NLS-6xHis) was obtained from Addgene (62933). A dead Cas9 (dCas9) version was prepared by performing site-directed mutagenesis on this plasmid to generate H840A and D10A mutations (pET-dCas9-NLS-6xHis). Cas9 proteins were prepared similar to that previously described (35). Briefly, protein expression was induced in Rosetta (DE3) cells with 0.4 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) at 18°C for 16 h. Cell pellets were resuspended in 12 ml of chilled binding buffer (20 mM Tris–HCl, pH 8.0, 250 mM NaCl, 1 mM PMSF, 5 mM imidazole) per 0.5 l of culture pellet. Resuspended cells were sonicated and clarified by centrifugation. Supernatant was purified by His-Pur Cobalt-CMA resin (Thermo Scientific) by sequentially increasing concentrations of NaCl wash buffer (Tris–HCl, pH 8, 0.25/0.5/0.75/1.0 M NaCl, 10 mM imidazole). Protein was eluted with 130 mM imidazole wash buffer. The eluent was concentrated and exchanged into 2x Cas9 storage buffer (40 mM HEPES-KOH, pH 7.5, 300 mM KCl, 2 mM ethylenediaminetetraacetic acid (EDTA), 2 mM DTT) then one volume of glycerol added. Concentration was determined by UV absorbance at 280 nm using a calculated extinction coefficient (120 450 M−1 cm−1) and Beer’s law.

In vitro Cas9 cleavage activity assays

In vitro cleavage assays were performed as previously described (32). Linearized plasmid target DNA (100 ng) harboring the tetracycline receptor (TR) gene or EGFP (EG) were combined with the Cas9 RNP complex (0.75 μM Cas9, 0.25 μM tracrRNA, 0.3 μM crRNA final concentration) in a 1× cleavage buffer (20 mM Tris–HCl, pH 7.5, 100 mM KCl, 5% glycerol, 1 mM DTT, 0.5 mM EDTA, 2 mM MgCl2) supplemented with 0.1 mg/ml of purified yeast tRNA. The concentration of tracrRNA was purposely set as the limiting component of the RNP complex and used to predict final RNP concentration. Molar excess of Cas9 and crRNA will ensure complete assembly of tracrRNA into RNP complexes and does not result in aggregation at concentrations used in our assays (32). Standard reaction conditions were 37°C for 2 h in a final reaction volume of 40 μl. The mixture was treated with 10 μg of RNase A (Thermo Scientific) for 15 min followed by 20 μg of Proteinase K (Thermo Scientific) for 15 min at room temperature. The DNA products were precipitated in 10 volumes of acetone with 2% LiClO4 at −20°C for >1 h. DNA cleavage products were resolved by agarose electrophoresis and visualized using ethidium bromide staining. The fraction of target plasmid cleaved was quantified using ImageJ software. The band intensity for the cleavage product band was divided by the combined intensity of the largest cleavage product and uncut substrate plasmid bands and reported as fraction cleaved (i.e. ‘cut’/‘cut + uncut’). Error bars for all quantified data represent experimental replicates, not technical replicates. Sample size was selected based on the expectation that three or more replicates will be representative of typical in vitro assay conditions.

Radiolabeling of DNA target

A total of 100 pmols of antisense DNA target strand was radiolabeled with γ- 32P-ATP using T4 polynucleotide kinase following the manufacturer’s recommended enzyme protocol (Thermo Fisher). Reactions were phenol-chloroform extracted and radiolabeled DNA was gel-purified on 15% denaturing polyacrylamide gels by the crush-and-soak method. Gel-purified radiolabeled RNA and DNA was quantified by scintillation counting.

Dot-blot filter binding assays for duplex target binding

For target binding by Cas9 RNP complexes, radiolabeled duplex target DNA (1000 cpm/reaction) was combined with increasing concentrations of a pre-assembled dCas9–tracrRNA–crRNA complex in a final reaction of 20 μl 1× cleavage buffer and 0.1 mg/ml tRNA. After incubation at 37°C for 15 min, reactions were vacuum filtered over nitrocellulose membrane (Protran Premium NC, Amersham) using a 96-well dot blot apparatus. Wells were washed twice with 200 μl of 1× cleavage buffer. Membrane was then removed and washed with 1× PBS solution three times and air dried at RT. Binding of radiolabeled DNA was then visualized by GE Typhoon phosphorimager. Spots were quantified with ImageQuant software, values plotted in Prism (GraphPad) and data fit to a one-site binding hyperbola equation.

Thermal denaturation monitored by UV absorbance

Thermal denaturation was as previously described (32). Cas9 alone or complexed to tracrRNA and crRNA at 1 μM final concentration (equimolar concentrations of all components) was incubated at room temperature for 10 min in degassed 1× UV melt buffer (20 mM Cacodylate, pH 7.5, 150 mM KCl, 1 mM MgCl2). Samples were melted in a Cary 400 UV/Vis spectrophotometer at a ramp rate of 1°C/min while UV absorbance at 280 nm was collected every 1 min. Experiments were repeated in duplicate. Melting temperatures were determined using Van’t Hoff calculations and error determined by standard error of the mean using two experimental replicates for each sample. Melt data was plotted using Prism (GraphPad) software.

Cell-based editing measured by flow cytometry

HEK 293T cells expressing EGFP and _Sp_Cas9 were a kind gift from Wen Xue (UMass Medical Center) (29). Cells were grown in Dulbecco’s modified eagle’s medium (DMEM) with 1× non-essential amino acids (NEAA), 5% Cosmic calf serum (CCS) and 2.5% fetal bovine serum (FBS) without antibiotics. Cells were reverse transfected (40–50 000 cells) in four experimental replicates in 96-well plates with 20 pmols of crRNA:tracrRNA complex in a final of 200 μl using RNAiMAX (Invitrogen) following the manufacturer’s recommended protocol. After 12 h, one volume of media containing 15% FBS and 1× Penicillin-Streptomycin solution was added to the Opti-MEM and cells incubated for an additional 12 h. Media was then replaced with full media and cells grown for an additional 4 days.

For flow cytometry, cells were washed with PBS, trypsinized, washed again and then fixed with 1% paraformaldehyde in PBS for 8 min. Cells were washed again and counted in an Accuri C6 Flow Cytometer. EGFP was detected using the blue laser at excitation 488 nm; emission detection 530 ± 15 nm (FL1 channel). At least 20 000 events were collected and analyzed by Accuri CFlow Plus software. The cells were first gated based on forward and side scattering (FSC-A/SSC-A) to remove cell debris, then gated to select single cells (FSC-H/FSC-A). At last, cells were gated to select EGFP positive cells. The quadrant gate was established using the signal from non-EGFP expressing control cells. Untreated HEK 293T cells expressing EGFP and Cas9 contained ∼6% non-fluorescent cells. The average from four replicates was used for background subtraction to determine the extent of cell-based editing after treatment.

Article TitleExtensive CRISPR RNA modification reveals chemical compatibility and structure-activity relationships for Cas9 biochemical activity

Abstract

CRISPR (clustered regularly interspaced short palindromic repeat) endonucleases are at the forefront of biotechnology, synthetic biology and gene editing. Methods for controlling enzyme properties promise to improve existing applications and enable new technologies. CRISPR enzymes rely on RNA cofactors to guide catalysis. Therefore, chemical modification of the guide RNA can be used to characterize structure-activity relationships within CRISPR ribonucleoprotein (RNP) enzymes and identify compatible chemistries for controlling activity. Here, we introduce chemical modifications to the sugar–phosphate backbone ofStreptococcus pyogenesCas9 CRISPR RNA (crRNA) to probe chemical and structural requirements. Ribose sugars that promoted or accommodated A-form helical architecture in and around the crRNA ‘seed’ region were tolerated best. A wider range of modifications were acceptable outside of the seed, especiallyD-2′-deoxyribose, and we exploited this property to facilitate exploration of greater chemical diversity within the seed. 2′-fluoro was the most compatible modification whereas bulkierO-methyl sugar modifications were less tolerated. Activity trends could be rationalized for selected crRNAs using RNP stability and DNA target binding experiments. Cas9 activityin vitrotolerated most chemical modifications at predicted 2′-hydroxyl contact positions, whereas editing activity in cells was much less tolerant. The biochemical principles of chemical modification identified here will guide CRISPR-Cas9 engineering and enable new or improved applications.


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