Editing of all primary cells was carried out using a ribonucleic protein (RNP) system at a molar ratio of either 1:2.5 or 1:5 (Cas9: sgRNA), unless otherwise stated. Recombinant S. pyogenes Cas9 protein was purchased from IDT (Integrated DNA Technologies, Coralville, Iowa, USA). Nucleofection was performed in P3 nucleofection solution (Lonza) and Lonza Nucleofector 4d (program DZ-100). Cells were plated at a concentration of 1.0 × 105–2.5 × 105 cells/ml. For T cells editing, electroporation was performed using Lonza Nucleofector 4d (program EO-115) with an RNP composition as used for CD34+ HSPCs editing. INDEL frequencies were quantified using TIDE online software on genomic DNA extracted using Quick Extract (Epicentre, an Illumina Company, cat no. QE09050) according to manufacturing specifications.
P3 nucleofection solution
Manufactured by Lonza
The P3 nucleofection solution is a reagent used in the nucleofection process, a non-viral method for introducing nucleic acids, such as DNA or RNA, into cells. It is designed to facilitate the delivery and uptake of these molecules into the cell nucleus. The P3 solution is a key component of the nucleofection technology and is used in conjunction with Lonza's proprietary nucleofector devices and protocols.
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Lab products found in correlation
4d nucleofector, by Lonza (2 mentions)
4d nucleofector x unit, by Lonza (2 mentions)
Cas9 nuclease, by Aldevron (2 mentions)
Taq dna polymerase, by New England Biolabs (1 mentions)
4d nucleofector device, by Lonza (1 mentions)
Sh800s cell sorter, by Sony (1 mentions)
Standard taq buffer, by New England Biolabs (1 mentions)
Opti mem reduced serum medium, by Thermo Fisher Scientific (1 mentions)
Opti mem, by Thermo Fisher Scientific (1 mentions)
Pan monocyte isolation kit, by Miltenyi Biotec (1 mentions)
Dynabead, by Thermo Fisher Scientific (1 mentions)
Easysep human t cell isolation kit, by STEMCELL (1 mentions)
P3 primary cell 4d nucleofector x kit s, by Lonza (1 mentions)
X vivo 15 media, by Lonza (1 mentions)
6 protocols using p3 nucleofection solution
1
CRISPR-Cas9 Editing of Primary Cells
2
Transposon-free iPSC Generation
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iRhpb#4 in feeder-free conditions was nucleofected using 4D-nucleofector device (Lonza). 1 × 106 cells were transfected with pBase-tdTomato transposase vector (6 µg), using P3 nucleofection solution and program CA-137 (Lonza). UPPS medium was supplemented for 2 days with PSF (5 µM) after transfection. After 3 days Tomato positive clones were sorted using SH800S Cell Sorter (Sony Biotechnology), sorting 10 cells per well in a 24 well plate. Potential transgene-free colonies were picked and expanded for 2–5 passages. Colonies were then pelleted and gDNA was extracted. Detection PCR was performed using primers specifically designed to detect different regions of the transposon (Supplementary Table 1 ). PCR was performed using Taq DNA polymerase with Standard Taq Buffer (New England BioLabs) according to manufacturer instructions. Original gel electrophoresis pictures of PCR analysis can be found in Supplementary Fig. 14 . Transgene-free clones were checked for the absence of the reprogramming construct every 10 passages.
Transfection efficiency was evaluated 3 days after transfection by FACS sorting. The analysis was performed in triplicates for two iPSC lines in feeder-free conditions (iRhpb#2 and 4). 10 × 103 events were recorded for the analysis of each sample.
Transfection efficiency was evaluated 3 days after transfection by FACS sorting. The analysis was performed in triplicates for two iPSC lines in feeder-free conditions (iRhpb#2 and 4). 10 × 103 events were recorded for the analysis of each sample.
3
Efficient CRISPR-Cas9 Knockout of Regnase-1 and Roquin-1 in T Cells
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Single-guide RNA (sgRNA) sequences targeting Regnase-1 and Roquin-1 were designed using Benchling online software (https://www.benchling.com ) and cross-validated with Synthego’s guide design verification tool (https://design.synthego.com/#/validate ). sgRNA sequences were synthesized by Integrated DNA Technologies (IDT). After screening several (3+) sgRNAs targeting each gene (SI Appendix, Table S1 ), the top sgRNA was selected for downstream experiments. Gene disruption was performed following an optimized protocol previously described (33 ). Briefly, T cells were washed three times in Opti-MEM™ reduced serum medium (Gibco) and resuspended at 1 × 108 cells/mL in P3 nucleofection solution (Lonza). Ribonucleoprotein (RNP) complexes were generated by incubating each sgRNA (5 μg per 10 × 106 cells) individually with Cas9 nuclease (Aldevron, 10 μg per 10 × 106 cells) for 10 min at room temperature. For no knockout (Mock) groups, no sgRNA was used and for multiple knockout groups, RNPs targeting each gene were incubated separately before combining. Cells were electroporated in batches of 10 × 106 cells (100 µL) with a mixture of RNP complex and 16.8 pmol of electroporation enhancer (IDT) in electroporation cuvettes (electroporation code EH111) in a 4D-Nucleofector X-Unit (Lonza).
4
CRISPR Knockout of ZFP36 in T Cells
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Single guide RNA (sgRNA) sequences targeting the ZFP36 genomic locus were designed using Benchling online software (https://www.benchling.com ) and cross-validated with Synthego’s guide design verification tool (https://design.synthego.com/#/validate ). sgRNA sequences were synthesized by Integrated DNA Technologies (IDT). After screening three sgRNAs against ZFP36, the top sgRNA was selected for downstream experiments (sgRNA sequence: 5′-CGCTCCACCAGCCTAGTGGA-3′). Gene disruption was performed following an optimized protocol previously described19 (link). Briefly, T cells were washed three times in Opti-MEM™ reduced serum medium (Gibco) and resuspended at 1 × 108 cells/mL in P3 nucleofection solution (Lonza). Ribonucleoprotein (RNP) complexes were generated by incubating each sgRNA (5ug per 10 × 106 cells) individually with Cas9 nuclease (Aldevron, 10ug per 10 × 106 cells) for 10 min at room temperature. For mock groups, no sgRNA was used and for multiple knockout groups, RNPs targeting each gene were incubated separately before combining. Cells were electroporated in batches of 10 × 106 cells (100 µL) with a mixture of RNP complex and 16.8 pmol of electroporation enhancer (IDT) in electroporation cuvettes (electroporation code EH111) in a 4D-Nucleofector X-Unit (Lonza).
5
CRISPR-Mediated Monocyte Gene Editing
6
T Cell Isolation, Activation, and Genetic Modification
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T cells were isolated from donor leukopaks (STEMCELL Technologies) by negative selection with CD3 magnetic beads (EasySep Human T Cell Isolation Kit; STEMCELL Technologies) according to the manufacturer’s protocol and frozen in LN2 at 20x106 cells/mL for storage. Donor T cells were then thawed, washed twice in X-VIVO 15 media (Lonza) then resuspended at 1x106 cells/mL in T cell medium (X-VIVO 15 supplemented with 5% FBS, 55 μM 2-mercaptoethanol and 200 IU/mL recombinant human IL2) prior to the addition of CD3/CD28 conjugated magnetic activation beads (Dynabeads; Invitrogen) at a 1:1 bead-to-cell ratio. Forty-eight hours later the cells were washed 1x in PBS, resuspended in 20 μL of supplemented P3 nucleofection solution (Lonza) at 50x106 cells/mL, and immediately added to the RNPs and mixed by gentle pipetting. The cell/RNP mixture was placed in one well of a 16-well cuvette strip (P3 Primary Cell 4D-Nucleofector X Kit S; Lonza) and electroporated using program EH115 on a Lonza X-Unit nucleofector. Immediately after, 100 μL of warm X-VIVO+5% FBS was added to the well, and the cells were placed in a 5% CO2 incubator at 37°C for 15 min, then plated in 4 mL of T cell medium. Cells were monitored daily and split 1:1 when the density reached 2x106 cells/mL or media showed signs of acidification.
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