Generation of RIPK1 autophosphorylation site mutant mice. (A) The residues S161 and S166 in exon 5 of the Ripk1 gene were altered from serine to alanine (S161A), from serine to asparagine (S161N), and from serine to glutamic acid (S161E) using CRISPR/Cas9 technology. Oocytes fertilized with sperm of Ripk1^S166A/S166A males or WT mice were electroporated with knock-in oligos and Cas9 protein for the respective mouse lines. The sequences for all knock-in oligos can be found in the Materials and methods section. (B) DNA sequence alignment (5′-3′) of the region surrounding the mutation site in WT and Ripk1^S161A/S161A mice. Amino acids by triplet code are shown. The mutation is marked in red. (C) DNA sequence alignment (5′-3′) of the region surrounding the mutation site in WT and Ripk1^S161N/S161N mice. Amino acids by triplet code are shown. The mutation is marked in red. (D) DNA sequence alignment (5′-3′) of the region surrounding the mutation site in WT and Ripk1^{S161N_S166A/S161N_S166A} mice. Amino acids by triplet code are shown. The mutations are marked in red. (E) DNA sequence alignment (5′-3′) of the region surrounding the mutation site in WT and Ripk1^{S161A_S166A/S161A_S166A} mice. Amino acids by triplet code are shown. The mutations are marked in red. (F) DNA sequence alignment (5′-3′) of the region surrounding the mutation site in WT and Ripk1^{S161E_S166A/S161E_S166A} mice. Amino acids by triplet code are shown. The mutations are marked in red.