KO cell lines were generated by CRISPR/Cas9-mediated genome editing. A CD47-KO variant of 3LL ΔNRAS cells was generated using Gene Knockout Kit version 2 targeting murine CD47 (Synthego). Knockout variants of PC9, NCI-H358, MGH119, and/or MGH134 were generated using Gene Knockout Kit version 2 targeting human B2M or human CD73 (Synthego). Gene KO was performed via ribonucleoprotein transfection with recombinant Cas9 (Synthego). Cells were then stained for surface antigen expression and sorted using a FACSAria II (BD Biosciences) to generate negative cell lines. For murine CD47, staining was performed using APC anti-murine CD47 clone miap301 (BioLegend) and sorting was used to generate a clonal population. For human lines, staining was performed with APC anti-human B2M clone 2M2 (Biolegend) or APC anti-human CD73 clone AD2 (Biolegend) and used to sort polyclonal lines that were negative for surface antigen expression.
Recombinant cas9
Manufactured by Synthego
Recombinant Cas9 is a protein essential for the CRISPR gene editing system. It functions as a molecular scissors, capable of precisely cutting targeted DNA sequences. This product provides the Cas9 enzyme in a purified, recombinant form for use in genetic engineering applications.
Automatically generated - may contain errors
Lab products found in correlation
Gene knockout kit version 2, by Synthego (2 mentions)
Apc anti murine cd47 clone miap301, by BioLegend (2 mentions)
Apc anti human b2m clone 2m2, by BioLegend (2 mentions)
Apc anti human cd73 clone ad2, by BioLegend (2 mentions)
Facsaria 2, by BD (2 mentions)
Alt r cas9 electroporation enhancer, by Synthego (2 mentions)
Neon transfection system, by Thermo Fisher Scientific (2 mentions)
5 protocols using recombinant cas9
1
CRISPR-mediated Knockout Cell Lines
2
CRISPR Genome Editing of Cell Lines
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HeLa TBK1 KO and RAB7 KO cells were created using px459-based Cas9 plasmids (listed in Supp. Table 3 ). The gRNA sequences to define each target gene are listed in Supp. Table 4 . 1 × 105 cells were plated in a 6-well dish per well and transfection mix, containing OPTiMEM, FuGENE HD and 1 μg gRNA plasmid, was added to the cells. RAW 264.7 STING KO and TBK1 + IKKε KO cells were created using the Synthego CRISPR Gene Knockout (Bentley-DeSousa and Ferguson, 2023 ). 2.5 × 105 cells were plated into a 6-well dish. The following day, cells were transfected with ribonucleoprotein particles using Lipofectamine CRISPRiMAX (Thermo Fisher Scientific), gene specific sgRNA and recombinant Cas9 (Synthego). After 48 h, the media was changed. After 72 h, single cells were plated into 96-well dishes to obtain monoclonal populations, that were further validated by immunoblotting.
3
CRISPR-Cas9 RNP Electroporation Protocol
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gRNAs (Synthego) were diluted to 100 µM (100 pMol/μL) in 1x TE buffer (Sythego). 1.2 uL (120 pMol) of gRNA, 0.9 uL of 100 μM Alt-R® Cas9 Electroporation Enhancer (IDT) and 3.9 uL water were added to a 1.5 mL tube per sample for a total of 6 μL. 1 μL of recombinant Cas9 (20 pMol) (Synthego) was added to 5 μL water in a separate 1.5 mL tube. 6 μL of diluted Cas9 was added to 6 μL of gRNA-enhancer mixture for a total of 12 μL cRNP complex at a 1:3 molar ratio. The cRNP complex was allowed to incubate for at least 10 minutes at room temperature (RT) and electroporated using the Neon Transfection system (Thermo-Fisher) as described previously34 (link), 54 . Cells were then incubated at 37°C for either 10 minutes before adoptive transfer or 90 minutes before centrifugation and resuspension in complete media supplemented with 50 ng rmIL-15 for culturing in vitro. Cells were cultured in vitro for 3 days following electroporation prior to reading out gene editing efficiency by flow cytometry or sanger sequencing.
4
CRISPR-Mediated Knockout Cell Lines
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Knockout cell lines were generated by CRISPR/Cas9-mediated genome editing. A CD47 knockout variant of 3LL ΔNRAS cells was generated using Gene Knockout Kit version 2 targeting murine CD47 (Synthego). Knockout variants of PC9, NCI-H358, MGH119, and/or MGH134 were generated using Gene Knockout Kit version 2 targeting human B2M or human CD73 (Synthego). Gene knockout was performed via ribonucleoprotein transfection with recombinant Cas9 (Synthego). Cells were then stained for surface antigen expression and sorted using a FACSAria II (BD Biosciences) to generate negative cell lines. For murine CD47, staining was performed using APC anti-murine CD47 clone miap301 (BioLegend) and sorting was used to generate a clonal population. For human lines, staining wass performed with APC anti-human B2M clone 2M2 (Biolegend) or APC anti-human CD73 clone AD2 (Biolegend) and used to sort polyclonal lines that were negative for surface antigen expression.
5
CRISPR-Cas9 RNP Electroporation Protocol
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