Originally published 28 June 2012; corrected 15 August 2012
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/cgi/content/full/science.1225829/DC1
Supplementary Materials for
A Programmable Dual-RNA–Guided DNA Endonuclea in Adaptive
Bacterial Immunity
Martin Jinek, Krzysztof Chylinski, Ines Fonfara, Michael Hauer, Jennifer A. Doudna,*
Emmanuelle Charpentier*
*To whom correspondence should be addresd. E-mail: doudna@berkeley.edu (J.A.D.);适合卷春饼的菜
双喜怎么剪emmanuelle.charpentier@mims.umu. (E.C.)
数值转换Published 28 June 2012 on Science Express
DOI: 10.1126/science.1225829
This PDF file includes:脑络通
Materials and Methods
Figs. S1 to S15
Tables S1 to S3
Full Reference List
Correction: Formatting errors and typos have been corrected. Additionally, the format of the tables has been revid, and a duplicate entry has been removed from table S2.
SUPPLEMENTARY MATERIALS AND METHODS
Bacterial strains and culture conditions. Table S1 lists the bacterial strains ud in the study. Streptococcus pyogenes, cultured in THY medium (Todd Hewitt Broth (THB, Bacto, Becton Dickinso
n) supplemented with 0.2% yeast extract (Oxoid)) or on TSA (tryptica soy agar, BBL, Becton Dickinson) supplemented with 3% sheep blood, was incubated at 37°C in an atmosphere supplemented with 5% CO2 without shaking. Escherichia coli, cultured in Luria-Bertani (LB) medium and agar, was incubated at 37°C with shaking. When required, suitable antibiotics were added to the medium at the following final concentrations: ampicillin, 100 µg/ml for E. coli; chloramphenicol, 33 µg/ml for E. coli; kanamycin, 25 µg/ml for E. coli and 300 µg/ml for S. pyogenes. Bacterial cell growth was monitored periodically by measuring the optical density of culture aliquots at 620 nm using a microplate reader (SLT Spectra Reader). Transformation of bacterial cells. Plasmid DNA transformation into E. coli cells was performed according to a standard heat shock protocol (39). Transformation of S. pyogenes was performed as previously described with some modifications (40). The transformation assay performed to monitor in vivo CRISPR/Cas activity on plasmid maintenance was esntially carried out as described previously (4). Briefly, electro-competent cells of S. pyogenes were equalized to the same cell density and electroporated with 500 ng of plasmid DNA. Every transformation was plated two to three times and the experiment was performed three times independently with different batches of competent cells for statistical analysis. Transformation efficiencies were calculated as CFU (colony forming units) per µg of DNA. Control transformations were performed with sterile water and backbone vector pEC85.
DNA manipulations. DNA manipulations including DNA preparation, amplification, digestion, ligation, purification, agaro gel electrophoresis were performed according to standard techniques (39)with minor modifications. Protospacer plasmids for the in vitro cleavage and S. pyogenes transformation assays were constructed as described previously (4). Additional pUC19-bad protospacer plasmids for in vitro cleavage assays were generated by ligating annealed oligonucleotides between digested EcoRI and BamHI sites in pUC19. The GFP gene-containing plasmid has been described previously (41). Kits (Qiagen) were ud for DNA purification and plasmid preparation. Plasmid mutagenesis was performed using QuikChange® II XL kit (Stratagene) or QuikChange site-directed mutagenesis kit (Agilent). All plasmids ud in this study were quenced at LGC Genomics or the UC Berkeley DNA Sequencing Facility and are listed in Table S2. VBC-Biotech Services, Sigma-Aldrich and Integrated DNA Technologies supplied the synthetic oligonucleotides and RNAs listed in Table S3.
In vitro transcription and purification of RNA. RNA was in vitro transcribed using T7 Flash in vitro Transcription Kit (Epicentre, Illumina company) and PCR-generated DNA templates carrying a T7 promoter quence. RNA was gel-purified and quality-checked prior to u. The primers ud for the preparation of RNA templates from S. pyogenes SF370, Listeria innocua Clip 11262 and Neisria meningitidis A Z2491 are listed in Table S3.
Protein purification. The quence encoding Cas9 (residues 1-1368) was PCR-amplified from the genomic DNA of S. pyogenes SF370 and inrted into a custom pET-bad expression vector using ligation-independent cloning (LIC). The resulting fusion construct contained an N-terminal hexahistidine-malto binding protein (His6-MBP) tag, followed by a peptide quence containing a tobacco etch virus (TEV) protea cleavage site. The protein was expresd in E. coli strain BL21 Rotta 2 (DE3) (EMD Biosciences), grown in 2xTY medium at 18°C for 16 h following induction with 0.2 mM IPTG. The protein was purified by a combination of affinity, ion exchange and size exclusion chromatographic steps. Briefly, cells were lyd in 20 mM Tris pH 8.0, 500 mM NaCl, 1 mM TCEP (supplemented with protea inhibitor cocktail (Roche)) in a homogenizer (Avestin). Clarified lysate was bound in batch to Ni-NTA agaro (Qiagen). The resin was washed extensively with 20 mM Tris pH 8.0, 500 mM NaCl and the bound protein was eluted in 20 mM Tris pH 8.0, 250 mM NaCl, 10% glycerol. The His6-MBP affinity tag was removed by cleavage with TEV protea, while the protein was dialyzed overnight against 20 mM HEPES pH 7.5, 150 mM KCl, 1 mM TCEP, 10% glycerol. The cleaved Cas9 protein was parated from the fusion tag by purification on a 5 ml SP Sepharo HiTrap column (GE Life Sciences), eluting with a linear gradient of 100 mM – 1 M KCl. The protein was further purified by size exclusion chromatography on a Superdex 200 16/60 column in 20 mM HEPES pH 7.5, 150 mM KCl and 1 mM TCEP. Eluted protein was concentrated to ~8 mg.ml-1, flash-
frozen in liquid nitrogen and stored at -80°C. Cas9 D10A, H840A and D10A/H840A point mutants were generated using the QuikChange site-directed mutagenesis kit (Agilent) and confirmed by DNA quencing. The proteins were purified following the same procedure as for the wild-type Cas9 protein.
平准书Cas9 orthologs from Streptococcus thermophilus (LMD-9,YP_820832.1), L. innocua (Clip11262, NP_472073.1), Campylobacter jejuni (subsp. jejuni NCTC 11168, YP_002344900.1) and N. meningitidis (Z2491, YP_002342100.1) were expresd in BL21 Rotta (DE3) pLysS cells (Novagen) as His6-MBP (N. meningitidis and C. jejuni), His6-Thioredoxin (L. innocua) and His6-GST (S. thermophilus) fusion proteins, and purified esntially as for S. pyogenes Cas9 with the following modifications. Due to large amounts of co-purifying nucleic acids, all four Cas9 proteins were purified by an additional heparin pharo step prior to gel filtration, eluting the bound protein with a linear gradient of 100 mM – 2 M KCl. This successfully removed nucleic acid contamination from the C. jejuni, N. meningitidis and L. innocua proteins, but failed to remove co-purifying nucleic acids from the S. thermophilus Cas9 preparation. All proteins were concentrated to 1-8 mg.ml-1 in 20 mM HEPES pH 7.5, 150 mM KCl and 1 mM TCEP, flash-frozen in liquid N2 and stored at -80°C.
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Plasmid DNA cleavage assay. Synthetic or in vitro-transcribed tracrRNA and crRNA were pre-anneal
ed prior to the reaction by heating to 95°C and slowly cooling down to room temperature.Native or restriction digest-linearized plasmid DNA (300 ng (~8 nM)) was incubated for 60 min at 37°C with purified Cas9 protein (50-500 nM) and tracrRNA:crRNA duplex (50-500 nM, 1:1) in a Cas9 plasmid cleavage buffer (20 mM HEPES pH 7.5, 150 mM KCl, 0.5 mM DTT, 0.1 mM EDTA) with or without 10 mM MgCl2. The reactions were stopped with 5X DNA loading buffer containing 250 mM EDTA, resolved by 0.8 or 1% agaro gel electrophoresis and visualized by ethidium bromide
staining. For the Cas9 mutant cleavage assays, the reactions were stopped with 5X SDS loading buffer (30% glycerol, 1.2% SDS, 250 mM EDTA) prior to loading on the agaro gel.
Metal-dependent cleavage assay. Protospacer 2 plasmid DNA (5 nM) was incubated for 1 h at 37°C with Cas9 (50 nM) pre-incubated with 50 nM tracrRNA:crRNA-sp2 in cleavage buffer (20 mM HEPES pH 7.5, 150 mM KCl, 0.5 mM DTT, 0.1 mM EDTA) supplemented with 1, 5 or 10 mM MgCl2, 1 or 10 mM of MnCl2, CaCl2, ZnCl2, CoCl2, NiSO4 or CuSO4. The reaction was stopped by adding 5X SDS loading buffer (30% glycerol, 1.2% SDS, 250 mM EDTA), resolved by 1% agaro gel electrophoresis and visualized by ethidium bromide staining.
Single-turnover assay. Cas9 (25 nM) was pre-incubated 15 min at 37°C in cleavage buffer (20 mM H
EPES pH 7.5, 150 mM KCl, 10 mM MgCl2, 0.5 mM DTT, 0.1 mM EDTA) with duplexed tracrRNA:crRNA-sp2 (25 nM, 1:1) or both RNAs (25 nM) not pre-annealed and the reaction was started by adding protospacer 2 plasmid DNA (5 nM). The reaction mix was incubated at 37°C. At defined time intervals, samples were withdrawn from the reaction, 5X SDS loading buffer (30% glycerol, 1.2% SDS, 250 mM EDTA) was added to stop the reaction and the cleavage was monitored by 1% agaro gel electrophoresis and ethidium bromide staining. The same was done for the single turnover kinetics without pre-incubation of Cas9 and RNA, where protospacer 2 plasmid DNA (5 nM) was mixed in cleavage buffer with duplex tracrRNA:crRNA-sp2 (25 nM) or both RNAs (25 nM) not pre-annealed, and the reaction was started by addition of Cas9 (25 nM). Percentage of cleavage was analyzed by densitometry and the average of three independent experiments was plotted against time. The data were fit by nonlinear regression analysis and the cleavage rates (k obs [min-1]) were calculated.
Multiple-turnover assay. Cas9 (1 nM) was pre-incubated for 15 min at 37°C in cleavage buffer (20 mM HEPES pH 7.5, 150 mM KCl, 10 mM MgCl2, 0.5 mM DTT, 0.1 mM EDTA) with pre-annealed tracrRNA:crRNA-sp2 (1 nM, 1:1). The reaction was started by addition of protospacer 2 plasmid DNA (5 nM). At defined time intervals, samples were withdrawn and the reaction was stopped by add
ing 5X SDS loading buffer (30% glycerol, 1.2% SDS, 250 mM EDTA). The cleavage reaction was resolved by 1% agaro gel electrophoresis, stained with ethidium bromide and the percentage of cleavage was analyzed by densitometry. The results of four independent experiments were plotted against time (min).
Oligonucleotide DNA cleavage assay. DNA oligonucleotides (10 pmol) were radiolabeled by incubating with 5 units T4 polynucleotide kina (New England Biolabs) and ~3–6 pmol (~20–40 mCi) [γ-32P]-ATP (Promega) in 1X T4 polynucleotide kina reaction buffer at 37°C for 30 min, in a 50 μL reaction. After heat inactivation (65°C for 20 min), reactions were purified through an Illustra MicroSpin G-25 column (GE Healthcare) to remove unincorporated label. Duplex substrates (100 nM) were generated by annealing labeled oligonucleotides with equimolar amounts of unlabeled complementary oligonucleotide at 95°C for 3 min, followed by slow cooling to room temperature. For cleavage assays, tracrRNA and crRNA were annealed by heating to 95°C for 30 s, followed by slow cooling to room temperature. Cas9 (500 nM final concentration) was
pre-incubated with the annealed tracrRNA:crRNA duplex (500 nM) in cleavage assay buffer (20 mM HEPES pH 7.5, 100 mM KCl, 5 mM MgCl2, 1 mM DTT, 5% glycerol) in a total volume of 9 μl. Reactions were initiated by the addition of 1 μl target DNA (10 nM) and incubated for 1 h at 37°C. Re
actions were quenched by the addition of 20 μl of loading dye (5 mM EDTA, 0.025% SDS, 5% glycerol in formamide) and heated to 95°C for 5 min. Cleavage products were resolved on 12% denaturing polyacrylamide gels containing 7 M urea and visualized by phosphorimaging (Storm, GE Life Sciences). Cleavage assays testing PAM requirements (Fig. 4B) were carried out using DNA duplex substrates that had been pre-annealed and purified on an 8% native acrylamide gel, and subquently radiolabeled at both 5’ ends. The reactions were t-up and analyzed as above.
Electrophoretic mobility shift assays. Target DNA duplexes were formed by mixing of each strand (10 nmol) in deionized water, heating to 95°C for 3 min and slow cooling to room temperature. All DNAs were purified on 8% native gels containing 1X TBE. DNA bands were visualized by UV shadowing, excid, and eluted by soaking gel pieces in DEPC-treated H2O. Eluted DNA was ethanol precipitated and dissolved in DEPC-treated H2O. DNA samples were 5’ end labeled with [γ-32P]-ATP using T4 polynucleotide kina (New England Biolabs) for 30 min at 37°C. PNK was heat denatured at 65°C for 20 min, and unincorporated radiolabel was removed using an Illustra MicroSpin G-25 column (GE Healthcare). Binding assays were performed in buffer containing 20 mM HEPES pH 7.5, 100 mM KCl, 5 mM MgCl2, 1 mM DTT and 10% glycerol in a total volume of 10 μl. Cas9 D10A/H840A double mutant was programmed with equimolar amounts of pre-annealed trac
rRNA:crRNA duplex and titrated from 100 pM to 1 μM. Radiolabeled DNA was added to a final concentration of 20 pM. Samples were incubated for 1 h at 37°C and resolved at 4°C on an 8% native polyacrylamide gel containing 1X TBE and 5 mM MgCl2. Gels were dried and DNA visualized by phosphorimaging.
In silico analysis of DNA and protein quences. Vector NTI package (Invitrogen) was ud for DNA quence analysis (Vector NTI) and comparative quence analysis of proteins (AlignX).凤仙花花期
In silico modeling of RNA structure and co-folding. In silico predictions were performed using the Vienna RNA package algorithms (42, 43). RNA condary structures and co-folding models were predicted with RNAfold and RNAcofold, respectively and visualized with VARNA (44).
