Synthetic gRNA (sgRNA) was designed using benchling.com [28 ] to generate one double-strand break (DSB) in the FMR1 gene (ENSG00000102081) via Cas9 nuclease. DSB is followed by homology-directed repair (HDR) and stop tag insertion (Supplementary Table S1 ). CRISPR/Cas9 editing was achieved when CAS9 protein (1 μL; stock 61 μM; Alt-R® S.p. HiFi Cas9 Nuclease V3, IDT, Coralville, Ian USA)), FMR1 sgRNA (3 μL; stock 100 μM, Synthego, Redwood City, CA USA), and the ssODN (1 μL; stock 100 μM, IDT) in 20 μL of nucleofection buffer P3 (P3 Primary Cell NucleofectorTM Solution, Lonza, Montreal, QC, Canada) were nucleofected (program CA137, 4D-Nucleofector Device, Lonza) into 500,000 detached iPSCs [29 (link)]. Following nucleofection, iPSCs were evenly distributed into a flat-bottom 96-well plate in mTeSR media and 10 μM Y-27632. After limiting dilution, gene-edited clones were identified using a ddPCR (QX200™ Droplet Reader, BioRad, Hercules, CA, USA) [29 (link)] and Sanger sequencing. For more details, see our CRISPR editing [30 (link)], DNA sequencing [31 (link)] protocols and Supplementary Figure S1 .
P3 primary cell nucleofector solution
Manufactured by Lonza
Sourced in Switzerland, Canada
The P3 Primary Cell Nucleofector Solution is a specialized lab equipment designed for the efficient transfection of primary cells. It is a non-viral electroporation-based solution that facilitates the introduction of genetic material, such as DNA or RNA, into a variety of primary cell types.
Automatically generated - may contain errors
Lab products found in correlation
Amaxa 4d nucleofector, by Lonza (5 mentions)
4d nucleofector, by Lonza (4 mentions)
4d nucleofector device, by Lonza (3 mentions)
4d nucleofector x unit, by Lonza (3 mentions)
P3 primary cell 4d nucleofector x kit l, by Lonza (3 mentions)
Qx200 droplet reader, by Bio-Rad (2 mentions)
Nucleocuvette vessel, by Lonza (2 mentions)
Matrigel, by Corning (1 mentions)
Geneticin, by Thermo Fisher Scientific (1 mentions)
Y 27632, by Bio-Techne (1 mentions)
Accutase, by STEMCELL (1 mentions)
Accutase, by Thermo Fisher Scientific (1 mentions)
96 well shuttle device, by Lonza (1 mentions)
Y 27632 dihydrochloride, by Bio-Techne (1 mentions)
Supplement 1, by Lonza (1 mentions)
Chemically modified sgrna, by Synthego (1 mentions)
Cloner, by STEMCELL (1 mentions)
Dneasy blood and tissue kit, by Qiagen (1 mentions)
Amaxa 4d electroporator, by Lonza (1 mentions)
Pyrimethamine, by Merck Group (1 mentions)
23 protocols using p3 primary cell nucleofector solution
1
Efficient CRISPR/Cas9 Gene Editing in iPSCs
2
Efficient CRISPR/Cas9 Gene Editing in iPSCs
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Synthetic gRNA (sgRNA) was designed using benchling.com [28 ] to generate one double-strand break (DSB) in the FMR1 gene (ENSG00000102081) via Cas9 nuclease. DSB is followed by homology-directed repair (HDR) and stop tag insertion (Supplementary Table S1 ). CRISPR/Cas9 editing was achieved when CAS9 protein (1 μL; stock 61 μM; Alt-R® S.p. HiFi Cas9 Nuclease V3, IDT, Coralville, Ian USA)), FMR1 sgRNA (3 μL; stock 100 μM, Synthego, Redwood City, CA USA), and the ssODN (1 μL; stock 100 μM, IDT) in 20 μL of nucleofection buffer P3 (P3 Primary Cell NucleofectorTM Solution, Lonza, Montreal, QC, Canada) were nucleofected (program CA137, 4D-Nucleofector Device, Lonza) into 500,000 detached iPSCs [29 (link)]. Following nucleofection, iPSCs were evenly distributed into a flat-bottom 96-well plate in mTeSR media and 10 μM Y-27632. After limiting dilution, gene-edited clones were identified using a ddPCR (QX200™ Droplet Reader, BioRad, Hercules, CA, USA) [29 (link)] and Sanger sequencing. For more details, see our CRISPR editing [30 (link)], DNA sequencing [31 (link)] protocols and Supplementary Figure S1 .
3
CRISPR-mediated EGFP Knockin in hiPSCs
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A cell line overexpressing the EGFP transgene was generated using clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology. First, to target exon 1 of beta 2-microglobulin, the guide RNA sequence 5′-CGGAGCGAGACATCT-3′ was ligated to the pCAG-SpCas9-GFP-U6-gRNA plasmid (Addgene plasmid #79145) and a donor template containing the EF1a-EGFP and G418-resistance expression cassette flanked by homology arms. Next, we transfected the CRISPR/Cas9 and donor template plasmids into hiPSCs using 4D Nucleofector (Lonza, Basel, Switzerland) following the manufacturer’s instructions. Briefly, the cells were dissociated using accutase (Stemcell Technologies, Vancouver, BC, Canada), after which 1 × 106 cells were spun down at 600 rpm for 5 min, resuspended in 100 μL P3 Primary Cell NucleofectorTM Solution (Lonza, Basel, Switzerland), mixed with 1.5 μg CRISPR/Cas9 plasmid and 1.5 μg donor template, transferred to a NucleocuvetteTM vessel, and electroporated using program CM-130. The transfected cells were seeded in Matrigel (Corning, 354234)-coated 6-well plates containing E8 medium supplemented with 5 μm Y27632 (Tocris, Bristol, UK) and then selected using 500 μg/mL geneticin (Thermo Fisher Scientific, Waltham, MA, USA). Single-cell colonies were transferred to Matrigel-coated 24-well plates. Genotyping was performed by PCR and genomic DNA sequencing.
4
Efficient Gene Editing in Human iPSCs
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Codon-optimized CBE4max_SpRY mRNA from previous publication [5 (link)] was purchased from AmpTec (Hamburg, Germany). The sgRNAs were purchased from Synthego (California, CA, USA).
The iPSCs were detached with Accutase (Thermo Fisher Sci., Waltham, MA, USA). Total 300,000 cells per reaction were spun down and washed once with PBS. Cells were resuspended in 20 μL reaction with 18 μL P3 Primary Cell Nucleofector Solution (Lonza, Basel, Switzerland) premixed with 2 μL mRNA and sgRNA at desired concentration. The cells were electroporated by Amaxa 4D Nucleofector (Lonza, Basel, Switzerland) using the X Unit in 16-well nucleofection cuvettes with the program CB-150. Afterwards, 80 μL of pre-warmed mTeSR plus medium was added to each cuvette and the cells were transferred to a single well of a 6-well plate for standard iPSC culture. Fresh medium was changed regularly, and cells were analyzed 96 h after nucleofection.
The iPSCs were detached with Accutase (Thermo Fisher Sci., Waltham, MA, USA). Total 300,000 cells per reaction were spun down and washed once with PBS. Cells were resuspended in 20 μL reaction with 18 μL P3 Primary Cell Nucleofector Solution (Lonza, Basel, Switzerland) premixed with 2 μL mRNA and sgRNA at desired concentration. The cells were electroporated by Amaxa 4D Nucleofector (Lonza, Basel, Switzerland) using the X Unit in 16-well nucleofection cuvettes with the program CB-150. Afterwards, 80 μL of pre-warmed mTeSR plus medium was added to each cuvette and the cells were transferred to a single well of a 6-well plate for standard iPSC culture. Fresh medium was changed regularly, and cells were analyzed 96 h after nucleofection.
5
Efficient mESC Transfection Protocol
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mESCs were trypsinized and washed with 1× PBS (Nacalai Tesque) once. Cells (2 × 105) were transfected with 40 pmol of siRNA and 20 μl of the P3 Primary Cell Nucleofector Solution (Lonza; supplement 1 added) using program CG-104 in 96-well Shuttle Device (Lonza) according to the manufacturers’ instructions. Transfected cells were then seeded into iMatrix-511 silk–precoated culture dish for culture and further experiments.
6
CRISPR-Mediated Cardiac Differentiation of hiPSCs
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The control hiPSC line was kindly provided by Dr. Jianyi Zhang (University of Alabama at Birmingham), this hiPSC line was reprogrammed from cardiac fibroblasts of a healthy female donor and modified to overexpress cyclin D2 to enhance the yield of cardiac differentiation (Zhu et al., 2018 (link); Zhao et al., 2021 (link)). To generate mutant hiPSCs, 2 h prior to electroporation, hiPSCs were treated with 10 μM ROCK inhibitor (Y27632 dihydrochloride, Tocris). Cells were then passaged and 250 K cells were then spun down in PBS. Cells were resuspended in P3 Primary Cell Nucleofector™ Solution containing Supplement 1 (Lonza, Switzerland) and 1 μg of chemically modified sgRNA (Synthego, Menlo Park, CA) and 1.5 μg codon optimized BE4 mRNA (TriLink Biotechnologies, San Diego, CA) and placed in the 20 μL cuvette provided and CB-150 was the Amaxa protocol used. Cells were given 10 μM ROCKi for 48 h post electroporation followed by normal culturing conditions. For hiPSC single cell isolation ∼300 single cell suspended iPSC were cultured in a 10 cm dish with 10 μM ROCKi for 48 h. For the next 10 days cells were given daily media changes and then colonies were isolated using a 10 μL pipette to scrape half of the colony for isolated culture and half the colony for genomic DNA isolation.
7
Transfection of Toxoplasma with Plasmids
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Endogenous tagging plasmids and/or ectopic expression plasmids (with the gene of interest expressed off its native promoter) were transfected into Toxoplasma via electroporation using the Amaxa 4D Nucleofector (Lonza) system. Tachyzoites were mechanically released in phosphate-buffered saline (PBS), pelleted, and resuspended in 20 μl P3 primary cell Nucleofector solution (Lonza) with —to 25 μg DNA for transfection. After transfection, parasites were allowed to infect HFFs in DMEM in T25 flasks for 24 h, after which the medium was changed to complete DMEM supplemented with 50 μg/ml mycophenolic acid and 50 μg/ml xanthine for selection for the hypoxanthine-xanthine-guanine-phosphoribosyltransferae (HXGPRT or HPT) marker for 3 to 5 days.
8
Generating Knockout Monocytes using CRISPR-Cas9
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Nontargeting,
SLC46A2, and SLC46A3 sgRNAs were purchased from IDT and resuspended
to 100 μM in TE buffer. Cas9 RNPs were formed by adding 8 μL
of 61 μM Alt-R S.p. Cas9 Nuclease V3 (IDT) to 12 μL of
100 μM sgRNA and incubating for 10 min at room temperature.
Freshly purified CD14+ monocytes were washed once with
cold PBS, then resuspended in P3 Primary Cell nucleofector solution
(Lonza) to a density of 107 cells/100 μL. A volume
of 100 μL of resuspended monocytes was then added to the Cas9
RNPs, transferred to a nucleofection cuvette, and nucleofected using
program CM-137 on a Nucleofector 4D device (Lonza). Electroporated
cells were then transferred to a six-well plate containing 2 mL of
DMEM with 10% heat-inactivated FBS and 1% penicillin-streptomycin.
At 24 h after nucleofection, cells were pelleted and resuspended in
2 mL of fresh media. At 72 h after transfection, cells were used for
CDN stimulation assays and genomic DNA was isolated to measure the
knockout efficiency. The knockout efficiency was determined by amplifying
the region of genomic DNA surrounding sgRNA target sites (using the
primers listed inTable S1 ), performing
Sanger sequencing, and using the sequencing trace to estimate knockout
efficiency through TIDE analysis.69 (link)
SLC46A2, and SLC46A3 sgRNAs were purchased from IDT and resuspended
to 100 μM in TE buffer. Cas9 RNPs were formed by adding 8 μL
of 61 μM Alt-R S.p. Cas9 Nuclease V3 (IDT) to 12 μL of
100 μM sgRNA and incubating for 10 min at room temperature.
Freshly purified CD14+ monocytes were washed once with
cold PBS, then resuspended in P3 Primary Cell nucleofector solution
(Lonza) to a density of 107 cells/100 μL. A volume
of 100 μL of resuspended monocytes was then added to the Cas9
RNPs, transferred to a nucleofection cuvette, and nucleofected using
program CM-137 on a Nucleofector 4D device (Lonza). Electroporated
cells were then transferred to a six-well plate containing 2 mL of
DMEM with 10% heat-inactivated FBS and 1% penicillin-streptomycin.
At 24 h after nucleofection, cells were pelleted and resuspended in
2 mL of fresh media. At 72 h after transfection, cells were used for
CDN stimulation assays and genomic DNA was isolated to measure the
knockout efficiency. The knockout efficiency was determined by amplifying
the region of genomic DNA surrounding sgRNA target sites (using the
primers listed in
Sanger sequencing, and using the sequencing trace to estimate knockout
efficiency through TIDE analysis.69 (link)
9
Nucleofection of iPSCs and hESCs
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For nucleofection of iPSCs and hESCs, cells were detached by using Accutase. For each reaction, 1.0 × 106 cells were resuspended in 82 μl of P3 Primary Cell Nucleofector Solution and 18 μl of supplement 1 using the P3 Primary Cell 4D-Nucleofector X Kit L (Lonza). Three micrograms of base editor P2A-PuroR expression plasmid and 1 μg of sgRNA plasmid were added in the single-cell suspension and mixed well. The single-cell suspension was then transferred into a Nucleocuvette. Nucleofection was carried out in 4D-Nucleofector X Unit (Lonza) using code CB200, and cells were immediately plated on a Matrigel-coated 35-mm dish in mTeSR supplemented with 1× CloneR (STEMCELL Technologies). After 24 hours, puromycin (1.0 μg/ml−1) was supplemented into the medium for 1 day selection, and the surviving colonies were expanded for 10 to 14 days until extraction of the genome using the DNeasy Blood and Tissue Kit (Qiagen). The target region was PCR amplified using 30 cycles and sent for Sanger sequencing. EditR (baseeditr.com ) was used to quantify the mutation peaks of Sanger chromatograms for analyzing the base conversion.
10
CRISPR-based Genetic Modification of P. knowlesi
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The tightly synchronized mature schizont-stage parasites of P. knowlesi were transfected using the Amaxa 4D electroporator (Lonza, Basel, Switzerland) and the P3 Primary cell 4D Nucleofector X Kit L (Lonza) following previous reports (Moon et al., 2016 (link)). Briefly, a 20-μg repair template and 20 μg pCas9/sg (Mohring et al., 2019 (link)) containing sgRNA sequences for pkmsp1p were mixed with P3 Primary Cell nucleofector solution, including supplement 1 (Lonza), and transferred to a Nucleocuvette™ Vessel (Lonza), followed by nucleofection with program FP158. Transfected parasites were immediately transferred to complete media with RBCs and incubated at 550 rpm for approximately 30 min at 37°C to allow invasion before transferring to standard culture conditions. After 24 h, transfected parasites were selected by drug pressure with 100 nM pyrimethamine (Sigma-Aldrich), and the medium, including pyrimethamine, was replaced at daily intervals for 5 days. The transfected parasites were cloned out by limiting dilution and confirmed by genotyping with diagnostic primers of extracted genomic DNA (Supplementary Tables 1 2
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