pRSFDuet™-1 DNA - Novagen

Protocol tips

Upstream tips	Protocol tips	Downstream tips
The combination of a “plain” T7 promoter pET plasmid [i.e., pET-3a-d, pET-20b(+), etc.] with a T7lac promoter plasmid is not recommended.

Upstream tips
The combination of a “plain” T7 promoter pET plasmid [i.e., pET-3a-d, pET-20b(+), etc.] with a T7lac promoter plasmid is not recommended.

Publication protocol

Plasmid Construction and sgRNA Target Design
The Cas9 sequence from the strain M1 GAS genome with a 3′ nuclear localization signal was codon-optimized for CHO cells, synthesized (for sequence, see Supplementary Materials and Methods) and subcloned into the mammalian expression vector pJ607-03 (DNA 2.0, Menlo Park, CA, A). The plasmid was then transformed into DH5α subcloning cells (Life Technologies, Paisley, U.K.). Transformant clones were selected on 100 µg/mL Ampicillin (Sigma–Aldrich, St. Louis, MO) LB plates. The chosen sgRNA target sequences are listed in Supplementary . The sgRNA expression constructs were designed by fusing tracrRNA and crRNA into a chimeric sgRNA (Jinek et al., ) and located immediately downstream of a U6 promoter (Chang et al., ). The sequences of the U6 promoter, scaffold and terminator are shown in Supplementary Materials and Methods. Initially, the sgRNA expression cassette (A) was synthesized as a gBlock (Integrated DNA Technologies, Leuven, Belgium) and subcloned into the pRSFDuet-1 vector (Novagen, Merck, Damstadt, Germany) using KpnI and HindIII restriction sites. This pRSFDuet-1/sgRNA expression vector was used as backbone in a PCR-based uracil-specific excision reagent (USER) cloning method (Hansen et al., ; Nour-Eldin et al., ). This method was designed to easily and rapidly change the 19 bp-long variable region (N) of the sgRNA in order to generate our sgRNA constructs. From the pRSFDuet-1/sgRNA expression vector, a 4,221 bp-long amplicon (expression vector backbone) was generated by PCR (1×: 98°C for 2 min; 30×: 98°C for 10 s, 57°C for 30 s, 72°C for 4 min 12 s; 1×: 72°C for 5 min) using two uracil-containing primers (sgRNA Backbone_fw and sgRNA Backbone_rv, Integrated DNA Technologies, Supplementary ) and the X7 DNA polymerase (Nørholm, ). Subsequent to Fastdigest DpnI (Thermo Fisher Scientific, Waltham, MA) treatment, the amplicon was purified from a 2% agarose TBE gel using the QIAEX II Gel Extraction Kit (Qiagen, Hilden, Germany). In parallel, 54 bp-long and 53 bp-long single stranded oligos (sense and antisense strand, respectively) comprising the variable region of the sgRNA were synthesized (TAG Copenhagen, Denmark, Supplementary ). The sense and antisense single stranded oligos (100 µM) were annealed in NEBuffer4 (New England Biolabs, Ipswich, MA) by incubating the oligo mix at 95°C for 5 min in a heating block and the oligo mix was subsequently allowed to slowly cool to RT by turning off the heating block. The annealed oligos were then mixed with the gel purified expression vector backbone and treated with USER enzyme (New England Biolabs) according to manufacturer's recommendations. After USER enzyme treatment, the reaction mixture was transformed into Mach1 competent cells (Life Technologies) according to standard procedures. Transformant clones were selected on 50 µg/mL Kanamycin (Sigma–Aldrich) LB plates. All constructs were verified by sequencing and purified by NucleoBond Xtra Midi EF (Macherey-Nagel, Düren, Germany) according to manufacturer's guidelines.

Cell Culture and Transfection
CHO-K1 adherent cells obtained from ATCC (#ATCC-CCL-61) were grown in CHO-K1 F-12K medium (ATCC) supplemented with 10% fetal calf serum (Life Technologies) and 1% Penicillin–Streptomycin (Sigma–Aldrich). Cells were expanded in T-75 cm vented cap tissue culture flasks (SARSTEDT, Nümbrecht, Germany) and experiments were performed in Advanced TC Cell Culture Multiwell plates (Greiner Bio-one, Frickenhausen, Germany). Cells were released from plastic ware using trypsin-EDTA (Sigma–Aldrich). Cells were transfected (Day 0) by the Nucleofector 2b device using the Amaxa Cell Line Nucleofector Kit V (Lonza, Basel, Switzerland) according to manufacturer's guidelines (program U-023). A total of 1 × 10 cells were transfected with 1 µg Cas9 plasmid and 1 µg sgRNA plasmid. Cells were incubated at 30°C, 5% CO from Day 1 to Day 2 (cold shock) and incubated at 37°C, 5% CO at all other times. Two days after transfection (Day 2), cells transfected with the pmaxGFP plasmid (Lonza) were used to estimate the transfection efficiency by analyzing GFP signal using a Celigo Imaging Cell Cytometer (Brooks Automation, Chelmsford, MA). The transfection efficiency was calculated as the percentage of GFP positive cells. Five days after transfection (Day 5), cells were trypsinized and pelleted (200 g, 5 min, RT). Genomic DNA was extracted from the cell pellets using QuickExtract DNA extraction solution (Epicentre, Illumina, Madison, WI) according to manufacturer's instructions and stored at −20°C.

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