ARF1 KO was achieved by transient transfection of CRISPR-Cas9 KO plasmids as described (Khater et al., 2021a (link); Wei et al., 2021 (link)). CRISPR-Cas9 KO plasmids targeting human ARF1, as well as control plasmids, were purchased from Santa Cruz Biotechnology. The KO plasmid consists of a pool of three plasmids, each encoding the Cas9 nuclease and a target-specific 20 sgRNA. Cells were cultured on 6-well plates and transfected with KO plasmids (1 μg) using Lipofectamine 3000 for 24 h. The cells were transfected again for another 24 h. The cells were split at a ratio of 1:2, grown for additional 24 h and then starved for 48 h before stimulation with β-ionone at 100 μM for 5 min.
Crispr cas9 ko plasmid
Manufactured by Santa Cruz Biotechnology
Sourced in United States
The CRISPR/Cas9 KO plasmid is a tool used for gene editing. It contains the necessary components for the CRISPR/Cas9 system, which can be used to precisely modify target DNA sequences.
Automatically generated - may contain errors
Lab products found in correlation
Puromycin, by InvivoGen (2 mentions)
Dmem f12, by Thermo Fisher Scientific (2 mentions)
Rpmi 1640, by Thermo Fisher Scientific (1 mentions)
Puromycin, by Santa Cruz Biotechnology (1 mentions)
Polyethylenimine (pei), by Merck Group (1 mentions)
Gsk 3β, by Santa Cruz Biotechnology (1 mentions)
Pd l1, by Santa Cruz Biotechnology (1 mentions)
K646 100, by Abcam (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Hepg2 cell, by American Type Culture Collection (1 mentions)
Fugene hd, by Promega (1 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (1 mentions)
Iscove s modified dulbecco s medium, by Thermo Fisher Scientific (1 mentions)
Dulbecco modified eagle medium (dmem), by Merck Group (1 mentions)
Rpmi 1640, by Merck Group (1 mentions)
Riluzole, by Avantor (1 mentions)
Dspe peg2000, by Avanti Polar Lipids (1 mentions)
1 2 distearoyl sn glycero 3 phosphoethanolamine n, by Avanti Polar Lipids (1 mentions)
Atplite 1step, by PerkinElmer (1 mentions)
Trypsin edta, by American Type Culture Collection (1 mentions)
13 protocols using crispr cas9 ko plasmid
1
CRISPR-Mediated ARF1 Knockdown in Cell Culture
2
CRISPR-mediated NOD2-RIPK2 knockout
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U2OS/NOD2 RIPK2 KO cells were generated using CRISPR/Cas9 KO plasmids (containing gRNA, Cas9, and EGFP reporter; Santa Cruz Biotechnology) as described in Elliott et al (2016 ). Reconstitution with RIPK2 variants was performed by retroviral transduction using pBABE‐Puro plasmids and the retroviral packaging cell line Phoenix‐A. Cells were selected with 1 μg/ml puromycin (InvivoGen) for 1 week.
3
Cell Line Validation and Genetic Modification
4
Knocking Down PDL1 in HCT-116 Cells
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To knock down PDL1 in HCT-116 cells, they were co-transfected using polyethylenimine (PEI, Sigma) transfection reagent with pooled Pdcd-1L1 CRISPR/Cas9 knockout (KO) plasmids (sc-425636) and Pdcd-1L1 homology-directed DNA repair (HDR) plasmids (sc-425636-HDR) (Santa Cruz Biotechnology). After overnight incubation, the medium was replaced with a fresh medium, and 2 days later, transfected cells were selected using 2 μg/mL puromycin (InvivoGen).
Cell transfection was performed as described above using pooled CRISPR-Cas9 KO plasmids (Santa Cruz Biotechnology) and homology-directed DNA repair (HDR) plasmids, corresponding to the cut sites generated by the CRISPR-Cas9 KO plasmids, which are coded for a puromycin resistance cassette and red fluorescent protein (RFP; Santa Cruz Biotechnology).
Cell transfection was performed as described above using pooled CRISPR-Cas9 KO plasmids (Santa Cruz Biotechnology) and homology-directed DNA repair (HDR) plasmids, corresponding to the cut sites generated by the CRISPR-Cas9 KO plasmids, which are coded for a puromycin resistance cassette and red fluorescent protein (RFP; Santa Cruz Biotechnology).
5
CRISPR/Cas9-mediated Knockout Cell Lines
6
HepG2 Cell Glycogen Quantification
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HepG2 cells (ATCC) were cultivated in Dulbecco’s modified Eagle’s medium with 25 mM of glucose, supplemented with 4 mM glutamine, 10% FBS, and 500 U/ml penicillin-streptomycin (GIBCO) as previously (52 ). Glycogen was quantified using a kit (K646-100, BioVision). HepG2 p53 knockout was generated using CRISPR/Cas9 KO plasmids (sc-416469, SantaCruz) as described elsewhere (52 ).
7
Generation of MANF-Knockout N2A Cells
8
CRISPR/Cas9-Mediated Ataxin 3 Knockout in Cell Lines
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Commercially available CRISPR/Cas9 KO plasmid containing three guide RNAs targeting Ataxin 3, and a homology‐directed repair plasmid containing a puromycin resistance cassette were purchased from Santa Cruz Biotechnology. Plasmids (2.5 μg each) were transfected into early‐passage HEK293, HeLa and U2OS cells using FuGENE HD (Promega Corporation). After 72 h of transfection, a medium supplemented with puromycin was added to the cells. The puromycin concentrations (1.25 μg/ml for HEK293 cells, 0.6 μg/ml for HeLa cells and 1 μg/ml for U2OS cells) required to kill wild‐type cells were determined by puromycin kill curve. After 72 h, the puromycin‐containing medium was removed and cells were sorted using a cell sorter into single‐cell populations on a 96‐well plate. ATX3 expression was analysed by Western blotting. Three HEK293, three HeLa and three U2OS clones showing loss of all detectable ATX3 were selected for subsequent analysis.
9
Generating LRP5 KO/Activation Clones
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The CRISPR/Cas9 KO plasmid or CRISPR activation plasmid that disrupts or activates the endogenous LRP5 gene was purchased (Santa Cruz, USA). HCT‐116 cells were plated at a density of 3 × 105 cells per well of a 6‐well plate 24 h before transfection. LRP5‐CRISPR/Cas9 KO plasmid (LRP5‐KO), LRP5‐CRISPR activation plasmid (LRP5‐ACT) or corresponding controls were transfected into cells separately by using lipofectamine 3000 (Invitrogen, USA) in serum‐free medium; then, cells were cultured in medium containing 5 μg/ml puromycin for two weeks, and single cell was selectively by using flow cytometer followed by transferred to 96‐well plate for expansion. Cell clones exhibiting the most efficient downregulation or upregulation of LRP5 compared with corresponding controls were verified by qRT‐PCR and immunoblot analysis and selected for subsequent analysis.
10
Characterization of Cell Lines for Immunotherapy
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We used osteosarcoma cell lines (OS2000 and KIKU), a myxofibrosarcoma cell line (MFH03), retrovirus packaging cell lines (PLAT‐A and PG13), an HLA class I‐ and II‐deficient CML cell line (K562), a human embryonic kidney cell line (293T), and a mutant TAP‐deficient cell line T2 transduced with HLA‐A*2402 (T2‐A24). T2‐A24 cells were obtained from K. Kuzushima (Aichi Cancer Research Institute). OS2000, KIKU, and MFH03 were established in our laboratory.
5 (link),
9 (link),
10 (link) 293T cells in which human β2M (beta 2 microglobulin) was knocked out using CRISPR/Cas9 KO Plasmid (#sc‐417704‐KO‐2; Santa Cruz Biotechnology) were transduced with HLA‐A*24:02 (293T‐A24). K562 and T2‐A24 cells were maintained in RPMI 1640 (Sigma‐Aldrich) supplemented with 10% FBS. OS2000 and MFH03 cells were cultured in Iscove's Modified Dulbecco's Medium (Gibco BRL) containing 10% FBS. KIKU and 293T cells were cultured in DMEM (Sigma‐Aldrich) containing 10% FBS in a 5% CO2 incubator. For T2‐A24 cells, G418 (0.8 mg/mL) was continuously added to the culture medium.
5 (link),
9 (link),
10 (link) 293T cells in which human β2M (beta 2 microglobulin) was knocked out using CRISPR/Cas9 KO Plasmid (#sc‐417704‐KO‐2; Santa Cruz Biotechnology) were transduced with HLA‐A*24:02 (293T‐A24). K562 and T2‐A24 cells were maintained in RPMI 1640 (Sigma‐Aldrich) supplemented with 10% FBS. OS2000 and MFH03 cells were cultured in Iscove's Modified Dulbecco's Medium (Gibco BRL) containing 10% FBS. KIKU and 293T cells were cultured in DMEM (Sigma‐Aldrich) containing 10% FBS in a 5% CO2 incubator. For T2‐A24 cells, G418 (0.8 mg/mL) was continuously added to the culture medium.
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