A programmable dual-RNA-guided DNA endonuclease in adaptive

Originally published 28 June 2012; corrected 15 August 2012

http:///cgi/content/full/science.1225829/DC1

Supplementary Materials for

A Programmable Dual-RNA–Guided DNA Endonuclease in Adaptive

Bacterial Immunity

Martin Jinek, Krzysztof Chylinski, Ines Fonfara, Michael Hauer, Jennifer A. Doudna,*

Emmanuelle Charpentier*

*To whom correspondence should be addressed. E-mail: doudna@berkeley.edu (J.A.D.);

emmanuelle.charpentier@mims.umu.se (E.C.)

Published 28 June 2012 on Science Express

DOI: 10.1126/science.1225829

This PDF file includes:

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 revised, 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 used in the study. Streptococcus pyogenes, cultured in THY medium (Todd Hewitt Broth (THB, Bacto, Becton Dickinson) supplemented with 0.2% yeast extract (Oxoid)) or on TSA (trypticase 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 essentially 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, agarose 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-based 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 used 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 used in this study were sequenced 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 sequence. RNA was gel-purified and quality-checked prior to use. The primers used for the preparation of RNA templates from S. pyogenes SF370, Listeria innocua Clip 11262 and Neisseria meningitidis A Z2491 are listed in Table S3.

Protein purification. The sequence encoding Cas9 (residues 1-1368) was PCR-amplified from the genomic DNA of S. pyogenes SF370 and inserted into a custom pET-based expression vector using ligation-independent cloning (LIC). The resulting fusion construct contained an N-terminal hexahistidine-maltose binding protein (His6-MBP) tag, followed by a peptide sequence containing a tobacco etch virus (TEV) protease cleavage site. The protein was expressed in E. coli strain BL21 Rosetta 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 lysed in 20 mM Tris pH 8.0, 500 mM NaCl, 1 mM TCEP (supplemented with protease inhibitor cocktail (Roche)) in a homogenizer (Avestin). Clarified lysate was bound in batch to Ni-NTA agarose (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 protease, 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 separated from the fusion tag by purification on a 5 ml SP Sepharose 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 …… 此处隐藏：22279字，全部文档内容请下载后查看。喜欢就下载吧 ……