Generation of Mmd2 A117V, R127P, and V100L knock-in mice using platinum TALEN and CRISPR-Cas9 and immunophenotyping of patients with p.A116V mutation. (A) The genomic structure of Mmd2, indicating the two binding sites of the TALENs, is shown. TALEN pairs were designed to bind exon 4 of the Mmd2 gene. The sequence information of Mmd2 mutant alleles, specifically, sequences obtained from mutant mice, which were generated by microinjection of TALEN mRNA. The DNA sequences that were used for designing the TALENs are highlighted in red. Nucleotide mutations and indels are shown. The reference nucleotide C was substituted with variant nucleotide T in the mutant sample. A 7-bp deletion resulted in a frameshift and thus in truncated proteins. (B) crRNA was designed to bind exon 5 of the Mmd2 gene. (C) crRNA was designed to bind exon 4 of the Mmd2 gene. (D) MMD2 protein expression in Mmd2^+/+, Mmd2^−/−, Mmd2^V100L/V100L, Mmd2^A117V/A117V, and Mmd2^R127P/R127P mice neutrophils. (E) FPR1 protein expression in Mmd2^+/+, Mmd2^−/−, Mmd2^V100L/V100L, Mmd2^A117V/+, and Mmd2^R127P/+ mice neutrophils. (F) Patient immunophenotyping. (G) Patient IgE levels. (H) Immunoblots were performed using HEK293T cells without transfection (lane 1, no transfection) and HEK293T cells transfected with the empty vector (lane 2), the empty vector and Myc-NRAS vector (Lane 3), the Myc-NRAS vector (lane 4), and Flag-MMD2-WT vector (Lane 5). Source data are available for this figure: SourceData FS3.