H460 cells were obtained from ATCC® (NCI-H460; Gaithersburg, MD). p53 knockout H460 cells (H460crp53) were generated as described elsewhere (36 ). Briefly, cells were co-transfected (3 × 106 cells in 10-cm dish) with 1 μg CRISPR-Cas9 plasmid targeting the p53 loci and 1 μg of a homology-directed repair plasmid for p53 (both from Santa Cruz Biotechnology® Inc., Santa Cruz, CA). Cells were transfected using PolyJet™ reagent (SignaGen Laboratories, Rockville, MD) following the manufacturer’s guidelines. After 72 h, cells were exposed to 2.5 μg/ml puromycin with daily media exchanges to replenish the selection agent. After all cells transfected with 1 μg of a control CRISPR/Cas9 plasmid (Santa Cruz Biotechnology, Inc.) were killed (~96 h), the cells were allowed to recover and grow as individual colonies, which were then selected and examined for expression of p53 by Western blotting. shATG5 H460 cells were generated as described below. Mission shRNA bacterial stocks for ATG5 were purchased from Sigma-Aldrich (St. Louis, MO). Lentiviruses were produced in HEK 293TN cells co-transfected using EndoFectin™ Lenti Transfection Reagent (GeneCopoeia, Rockville, MD) with a packaging mixture of psPAX2 and pMD2.G constructs (Addgene, Cambridge, MA). Media containing the viruses was used to infect the H460 cells.
Crispr cas9 plasmid
Manufactured by Santa Cruz Biotechnology
Sourced in United States
The CRISPR/Cas9 plasmid is a molecular biology tool that enables targeted gene editing. It contains the CRISPR-associated protein Cas9 and a guide RNA, which together can recognize and cleave specific DNA sequences. This plasmid can be used to introduce precise modifications, such as insertions, deletions, or substitutions, into the genome of various cell types.
Automatically generated - may contain errors
Lab products found in correlation
Lipofectamine 2000, by Thermo Fisher Scientific (4 mentions)
Endofectin lenti transfection reagent, by GeneCopoeia (2 mentions)
Pmd2 g constructs, by Addgene (2 mentions)
Mission shrna bacterial stocks for atg5, by Merck Group (2 mentions)
Polyjet reagent, by SignaGen (2 mentions)
Pspax2, by Addgene (2 mentions)
Ultracruz reagent, by Santa Cruz Biotechnology (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Glutamax, by Thermo Fisher Scientific (1 mentions)
Uridine, by Merck Group (1 mentions)
Pyruvate, by Merck Group (1 mentions)
Sodium pyruvate, by Thermo Fisher Scientific (1 mentions)
Crispr cas9 knockout plasmid, by Santa Cruz Biotechnology (1 mentions)
Puromycin, by Merck Group (1 mentions)
6 well plate, by Corning (1 mentions)
96 well plate, by Corning (1 mentions)
Crispr cas9 and hdr plasmids, by Santa Cruz Biotechnology (1 mentions)
14 protocols using crispr cas9 plasmid
1
Generation and Validation of p53 Knockout and shATG5 H460 Cells
2
Generation of eIF4A1 Knockout Cells
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For generation of CRISPR/Cas9-mediated eIF4A1 knockout (KO), a set of CRISPR/Cas9 plasmids (Santa Cruz, Dallas, TX, Cat. #—sc-402623) were transfected into therapy-naïve and paclitaxel-resistant tumor cells using UltraCruz reagent (Santa Cruz, Dallas, TX, Cat. #—sc-395739) following the manufacturer's instructions. After 24 h, the supernatant was removed and replaced with regular media and cells were further cultured for a total of 72 h. eIF4A1 KO cells were sorted out based on the expression of green fluorescent protein (GFP). Single KO cells were isolated by limiting dilution in 96-well plates. The KO was verified by immunoblotting for eIF4A. Non-targeting CRISPR/Cas9 plasmids were employed to obtain the CRISPR-control cells (Santa Cruz, Dallas, TX, Cat. #—sc-418922).
3
Generation and Characterization of Tumor Cell Lines
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B16-F10 and B16-F10-gp3368 (link) cells were given by Dr. Julián Pardo (University of Zaragoza). LLC cells were given by Dr. David Sancho (Centro Nacional de Investigaciones Cardiovasculares, Madrid). LLC-OVA tumor cells were kindly provided by Dmitry Gabrilovich (University of Pennsylvania). TC-1 cells were given by Dr. T.C. Wu (Johns Hopkins Medicine)69 (link). B16-F10-ZsGreenLuc and LLC-ZsGreenLuc cells were made in the laboratory by transfection with a lentivirus encoding ZsGreen and firefly luciferase Luc2P (pHIV-Luc2-ZsGreen, from Addgene) and sorted based on high ZsGreen expression. For the generation of LLC-B2m−/− and B16-F10-B2m−/− cell lines, parental cells were transfected with CRISPR-Cas9 plasmids targeting the β2-microglobulin gene (purchased from SantaCruz Biotechnology) and cells were selected with puromycin and then sorted based on lack of MHC-I expression after staining with an antibody directed to H2Kb/Db (Miltenyi). Tumor cells were cultured with complete DMEM, containing 10% inactivated Fetal Bovine Serum (FBS, Gibco), Glutamax (Gibco) and penicillin/streptomycin (Gibco) and were always used with less than 8 passages from thawing. The B16-F10-gp33 and LLC-OVA clones were maintained in complete DMEM containing 500 μg ml−1 of G418 (Gibco).
4
Generation of p53-Knockout and shATG5 NSCLC Cells
5
CRISPR-Mediated Modulation of ZC3H4 in Pulmonary Fibroblasts
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Pulmonary broblasts were quickly transfected with CRISPR/Cas9 plasmids according to the manufacturer's protocol (Santa Cruz®) to delete/upregulate ZC3H4 and observe its downstream effects.
The western blotting assay was used to determine the transfection e ciency. In brief, 24-well plates were used for cell seeding (2x10 5 cells /well), and the cells reached 40-80% con uency. The medium was changed to 200 µl fresh antibiotic-free growth medium, and solutions A and B were added as follows. For solution A, transfection reagent (1 μl) was poured into plasmid transfection medium (9 μl), and for solution B, plasmid DNA (1 μl) was poured into plasmid transfection medium (9 μl). After 5 min, solution A was poured dropwise directly into solution B; the sample was then immediately vortexed and incubated at room temperature for >20 min. The mixed solution was added dropwise to 200 μl of the medium in the 24-well plate, and the contents of the well were mixed by swirling the plate gently. The medium was added or replaced when necessary 12 hr after transfection. The pulmonary broblast cells were incubated for an additional 24-72 hr to conduct further experiments.
The western blotting assay was used to determine the transfection e ciency. In brief, 24-well plates were used for cell seeding (2x10 5 cells /well), and the cells reached 40-80% con uency. The medium was changed to 200 µl fresh antibiotic-free growth medium, and solutions A and B were added as follows. For solution A, transfection reagent (1 μl) was poured into plasmid transfection medium (9 μl), and for solution B, plasmid DNA (1 μl) was poured into plasmid transfection medium (9 μl). After 5 min, solution A was poured dropwise directly into solution B; the sample was then immediately vortexed and incubated at room temperature for >20 min. The mixed solution was added dropwise to 200 μl of the medium in the 24-well plate, and the contents of the well were mixed by swirling the plate gently. The medium was added or replaced when necessary 12 hr after transfection. The pulmonary broblast cells were incubated for an additional 24-72 hr to conduct further experiments.
6
Generating PKR-deficient HeLa Cells
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HeLa cells with an inactivated PKR gene were generated by using the CRISPR/Cas9 plasmid (Santa Cruz Biotechnology) according to the manufacturer’s instructions. After transfection, cells were treated with puromycin (2 μg/mL) for 48 h. The surviving cells were plated in 96-well plates with ~1–5 cells/well. Colonies were expanded and were selected according to their ability to support replication of vMyxM029KO virus.
7
SDHB Knockout in HCT-116 Cells
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HCT-116 cells were plated at 15000 cells/3 mL/well on 6-well plates (Corning) and cultured overnight. The next day, the cells were transfected with 1 μg of SDHB clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 knockout plasmid (Santa Cruz) and SDHB homology directed repair (HDR) plasmid (Santa Cruz) or a control CRISPR/Cas9 plasmid (Santa Cruz) using Lipofectamine 2000 (Thermo Fisher Scientific) as described by the manufacturer. After forty-eight hours post transfection, cells were selected with 2 μg/μL of puromycin (Sigma) for 1 week. Cells transfected with the SDHB knockout plasmid were collected as SDHB knockout polyclonal cells. Cells were cloned by limited dilution on 96-well plates (Corning), and cloned cells were collected for analysis of protein expression and genome copy number of SDHB. After transfection of SDHB knockout plasmid, 1 mM sodium pyruvate (Gibco) and 50 μg/ml of uridine (Sigma) were added to the culture medium described above because cells that lack a functional mitochondrial electron transport chain such as ρ0 cells fail to proliferate without supra-physiological levels of uridine and pyruvate in the culture medium [28 (link)].
8
CRISPR/Cas9-mediated SP110 knockout in Jurkat T cells
9
CRISPR Cas9 Plasmid Transfection and Clonal Selection
10
CRISPR-Cas9 for MGAT5 KO in GSCs
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MGAT5 KO GSCs were made using the CRISPR Cas 9 technique [31 (link)]. GSC transfection was carried out by electroporation with 1 μg of CRISPR Cas 9 plasmid (Santa Cruz) per 1 million cells in Amaxa mouse neural stem cell Nucleofector. Transfected plasmid contained GFP for Fluorescence-activated cell sorting and to deposit one cell per well in Corning® 96 wells round bottom ultra-low attachment microplates. After proliferation and the formation of a single neurosphere, KO efficiency was checked by western blot.
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