Genomic knockout of FceRIa from RBL-2H3 cells was performed with the lentiCRISPR v2.0 (Addgene plasmid #52961) system (102), which was a gift from F. Zhang (Massachusetts Institute of Technology). The guide sequence 5′-ATTACACAATTGAGTATCGT-3′ was ligated into the vector using Bsm BI restriction sites. This sequence targets nucleotides 584 to 603 of rat FceR1a, corresponding to residues 196 to 201 of the translated protein, and has a fidelity score of 85 as determined by the CRISPR design tool ( Lentiviral particles containing this vector sequence were generated by cotransfection of HEK 293T cells with lentiCRISPR v2.0 and with pMD.G and p8.91 using GeneJuice (Novagen) as per the manufacturer’s instructions. Harvested lentivirus was incubated with RBL-2H3 cells in culture for 5 days before the cell surface expression of FcεRIα was assessed by flow cytometry analysis of cells stained with Alexa Fluor 647–conjugated mouse IgE. A pure FcεRI−ve population was then derived by fluorescence-activated cell sorting. To obtain an FcεRI-restored (FcεRI+ve) RBL-2H3 line, a CRISPR-resistant FcεRIα mutant was generated by site-directed mutagenesis of the target sequence to 5′-AcTAtACgATcGAaTAcCGg-3′ (mutations in lowercase), which translates to the same protein sequence as that encoded by the WT gene. The mutant FceRIa gene was inserted into the lentiviral pHR vector and used to transduce FcεRI−ve RBL-2H3 cells as described earlier. As before, FcεRIα cell surface expression was assessed by flow cytometry, and a pure FcεRI+ve RBL-2H3 population of cells was obtained by cell sorting.

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