Off-target analysis of Cas9/gRNA system-induced mutations in KO founders. (A) T7EI assays of the PCR products of candidate off-target sites using the pooled DNA of all KO founders for each gene as the template (primer sequences listed in Additional file 3: Table S2). The IOT5 could be cleaved (red arrowheads). (B) Sequencing diagram of IOT5 in #1 IL2rg KO new-born rabbits showing a double curve after the mutation around the PAM region. (C) Detailed mutations of IOT5 in the newborn rabbits. The number of founder KO rabbits is shown in the left column. The WT sequence is shown at the top with the target sites in underline. Deletions are indicated by dashes, insertions are indicated in blue and substitutions are indicated in pink, and the sizes of the deletions (-) or insertions (+) are shown in the right column. The fractions indicate the read number of the mutant allele (numerator) out of total read number (denominator).