CRISPR/CAS9

9 months ago

Strains, growth conditions and storage The S. cerevisiae strains used in this study (Table (Table1)1) share the CEN.PK genetic background (Entian and Kötter 2007; Nijkamp et al. 2012). Shake flask cultures were grown at 30 °C in 500 mL flasks containing 100 mL synthetic medium (SM) (Verduyn et al. 1992) with 20 g·L−1 gluc...

9 months ago

Cloning, expression and purification The S. pyogenes cas1 and cas9 genes were cloned into pET28a vectors with a C-terminal (His)6 tag (Supplementary Table S1). The S. pyogenes cas2 and csn2 genes were cloned into pET28a and pHMGWA vectors, respectively, each of which contain an N-terminal (His)6-maltose binding protein (MB...

11 months ago

Cell lines Human 293T/17 (ATCC No.: CRL-11268) cells were cultured in DMEM (Sigma-Aldrich) supplemented with 10% fetal calf serum (FCS; Sigma-Aldrich) and 2 mM L-glutamine (Sigma-Aldrich) at 37°C and 5% CO2. Human BlaER1 cells (a kind gift of Thomas Graf) (29) cells were grown in RPMI (Sigma-Aldrich) supplemented with 10% ...

11 months ago

Strains, plasmids, phage, and oligonucleotides Bacterial strains, plasmids, and phage used in this study, including descriptions and sources, are provided in Supplementary Table 1. Oligonucleotides used in this study are provided in Supplementary Table 2. Minimum inhibitory concentration (MIC) assay Cells were prepared b...

11 months ago

T cell editing Whole blood was obtained with donor consent from the Israeli Blood Bank (Magen David Adom, Sheiba Medical Center) in accordance with Tel Aviv University Review Board. PBMCs were extracted using Lymphocyte Separation Medium (mpbio) and cryopreserved until subsequent use. Following thawing, cells were activate...

11 months ago

Cells and viruses BSC-40 cells were obtained from American Type Culture Collection (ATCC, CRL-2761) and were cultured in Dulbecco’s Modified Eagle Medium (DMEM, Gibco 11965-092) supplemented with 10% (vol/vol) fetal bovine serum (FBS, Atlanta biologicals S11150H), 1% (vol/vol) glutamine (Gibco 35050-061), and 1% (vol/vol) ...

11 months ago

Plant materials Seeds of Nicotiana tabacum cv. ‘SR1’ were obtained from the Leaf Tobacco Research Center. Nicotiana benthamiana, N. tabacum ‘SR1’, and Torenia fournieri cv. ‘Blue and White’ plants were grown in soil in a green room at 25–30°C under long-day conditions (16 h light/8 h dark), and Solanum lycopersicum cv. ‘Mi...

11 months ago

Cell Lines and Antibodies U937 and HEK293 cell lines were obtained from American Type Culture Collection (ATCC). U937 cells were cultured in RPMI 1640 with HEPES (Gibco 22400-089) supplemented with 10% fetal bovine serum, defined (HyClone, GE Healthcare SH30070.03); 50 U/mL penicillin, 50 μg/mL streptomycin (Gibco 15070-06...

11 months ago

Reagents All chemicals were purchased from Sigma-Aldrich (St. Louis). All enzymes were purchased from New England Biolabs (Beverly, MA). All synthetic DNAs were purchased from Integrated DNA Technologies. C. elegans strains C. elegans strains were cultured using standard methods (Brenner, 1974) on nematode growth media (...

11 months ago

Animal husbandry and ethics Medaka (Oryzias latipes) (Iwamatsu, 2004, Naruse et al., 2004, Kasahara et al., 2007) were maintained as closed stocks in a fish facility built according to the European Union animal welfare standards and all animal experiments were performed in accordance with European Union animal welfare guid...

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