(A) High-resolution melting analysis identified a putative mutagenesis event at a predicted off-target site (chr4:143,096,216-143,096,238; GRCm38/mm10) for the guide RNA targeting sequences upstream of the Gm26878 transcriptional start site. (B) Sequence of the off-target site on chromosome 4. Bases that match the on target sgRNA target site on chromosome 8 are shown in red, mismatches are shown in green, and the PAM sequence is underlined. Flanking sequences are in black. Breakpoints (chr4; GRCm38/mm10) of a 13 base deletion allele segregating in wild-type and heterozygous and homozygous Gm26878 G2 progeny are indicated with horizontal lines. (C) Chromatogram showing DNA sequences of the nonhomologous end joining (NHEJ) repair junction at the chromosome 4 off-target mutagenesis site.