## 3 Results  
### Testing of Guides and Production of Founder Knockout Animals  
The mutation efficiency of guide RNAs was tested in cultured blastocysts. Both guides were effective in inducing mutations in the rabbit _NOX4_ gene. However, the guide RNA targeting the second exon of _NOX4_ had slightly higher mutation efficiency. Table 3 shows the ratio of total injected zygotes that developed to blastocyst stage to mutant blastocysts for the two guides designed for the second and fourth exons (18% and 16%, respectively).  
\begin{table}
\begin{tabular}{c c c c c c} \hline \hline
**Off-Targets** & \begin{tabular}{c} **Off-Target Sequence Compared** \\ **to Target DNA** \\ \end{tabular} & **Chromosome** & **Location** & \
\begin{tabular}{c} **Number of** \\ **Mismatches** \\ \end{tabular} & **Primers** \\ \hline _NOX4_ & 2 nd & & & & \\ exon target site & & & & & \\ Off-target-1 & TGGTGAGAGTAATGGTACTNGGG & chr1 & 127339745 & 0 & \\  & & & & & \\  & & & & & \\ Off-target-2 & TGGTGAGAGTAATGGTGTGG & chr1 & 135497670 & 3 &
\begin{tabular}{c} forward AAGAGAGAGAGAGAGAG \\ reverse ATTAGAAGAAGACATGTAGGGG \\ forward AAAAGIn addition, it is beneficial to introduce an early stop codon in the second exon in order to avoid the presence of a functional truncated peptide. Hence, we chose the RNA guide targeting the second exon of the _NOX4_ gene to produce live animals.  
Based on the results of embryo testing, a total of five knockout founder animals were produced, two males and three females, as summarized in Table 4. All mutations were heterozygous 7 bp and 8 bp deletions directly upstream of the PAM site with a couple of base differences: Animals #2, #3 and #4 acquired identical mutations (Figure 2), while animal #5 carried three different mutated alleles. We established heterozygous and homozygous genome-edited rabbit sublines by consecutive brother-sister mating from founder animal #5.  
Animal #5 passed on four different alleles to the F1 generation (Figures 3 and 4). The most frequent allele was an 8 bp deletion (\(\Delta\)8). The other allele contained a 128 bp deletion (\(\Delta\)128), including an intronic region. The third allele type was the most irregular, with deletions and insertions adding up to a 104 bp deletion in total (\(\Delta\)104) upstream of the PAM sequence. A wild-type allele also appeared in the F1 generation, demonstrating the presence of a fourth allele. We analyzed the expression level of NOX4 in different tissues of a selected homozygous individual from line \(\Delta\)8 by RT-qPCR. Expression pattern matched to an expected -/- phenotype (data not shown).  
\begin{table}
\begin{tabular}{c c c c} \hline \hline
**Code of Founder Animal** & **Sex** & **Mutation** & **Consequence** \\ \hline \#1 & \(\mathfrak{c}^{\mathfrak{d}}\) & 7 bp deletion (GGTACTC) & early stop codon \\ \#2 & \(\mathfrak{g}\) & 8 bp deletion (TGGTACTC) & early stop codon \\ \#3 & \(\mathfrak{g}\) & 8 bp deletion (TGGTACTC) & early stop codon \\ \#4 & \(\mathfrak{g}\) & 8 bp deletion (TGGTACTC) & early stop codon \\ \#5 & \(\mathfrak{c}^{\mathfrak{d}}\) & allele A: 8 bp deletion (ACTCTGGC) & early stop codon \\  & & allele B: 128 bp deletion & early stop codon \\  & & allele C: 104 bp deletion in total & early stop codon \\ \hline \hline \end{tabular}
\end{table}
Table 4: Summary of mutations in _NOX4_ KO founder rabbits. One male (#1) and three female (#2–4) rabbits had 7–8 bp deletions. Male #5 carried three distinct alleles with alternative mutations.  
Figure 2: Comparison of mutations in _NOX4_ knockout rabbit founders. The gRNA and the target in the gene followed by the PAM sequence are highlighted in red. The yellow spaces indicate the sites of mutation. The second exon is underlined with a black line. Animal #1 had a 7 bp deletion; #2–4 had identical 8 bp deletions. All four deletions were directly followed by the PAM sequence.