Partially complementary oligonucleotides

The FcεRIγ-KO cell line was generated in RBL-2H3 cells by CRISPR-Cas9–mediated gene editing, resulting in insertion of a premature stop codon. RNA (5′-GCAAGAACAAGATCACCGCT-3′) targeting the first exon of rat FcεRIγ was designed using the http://crispr.mit.edu/ portal and then subcloned into PX458 vector (Addgene plasmid #48138) for simultaneous expression of gRNA, WT Cas9, and a GFP reporter. For gRNA subcloning, two partially complementary oligonucleotides (Integrated DNA Technologies) were assembled by PCR. Gel-purified PCR products were cloned into BbsI-digested PX458 using Gibson assembly (NEB) following the manufacturer’s specifications. GFP-positive cells were selected on an iCyt cell sorter 24 h after transfection using the Amaxa system and then dispensed at single-cell density into 96-well plates. Subclones were screened for lack of fluorescent IgE binding by flow cytometry, followed by Western blotting to confirm selected clones completely lack FcεRIγ expression.

Cas9-mediated DNA cleavage was used to knock out the endogenous gene coding for the Syk protein in RBL-2H3 cells via the insertion of a premature stop codon in the first exon of the gene. A highly specific single guide RNA (gRNA) (5′-GGCCAGAGCCGCAATTACCT-3′) targeting the first exon of rat Syk was designed using the http://crispr.mit.edu portal and then subcloned into PX458 vector (Addgene plasmid #48138) for simultaneous expression of the gRNA, WT Cas9, and a green fluorescent protein (GFP) reporter. For the gRNA subcloning, two partially complementary oligonucleotides (Integrated DNA Technologies) were assembled by PCR. Gel-purified PCR products were cloned into BbsI-digested PX458 using Gibson Assembly (NEB) following the manufacturer’s specifications. After cloning and sequencing, the final plasmid was used to transiently transfect RBL-2H3 cells using the Amaxa system (Lonza) following the manufacturer’s recommendations. Positive, GFP-expressing cells were selected by flow cytometry using an iCyt cell sorter and immediately plated at suboptimal concentration in 96-well plates. Subclones were screened using Western blotting to identify clones with no Syk expression. The absence of residual GFP expression in Syk KO clones was assessed using a Nikon TE2000 epifluorescence microscope.