## 3 Results
### Off-Target Analysis  
The selected potential off-target sites were amplified by PCR using genomic DNA from both the wild-type animal and founder rabbit #5 and evaluated by DNA sequencing. Eight candidate off-target sites were analyzed. In all these cases, no difference was observed (see Figure 5), so no off-target event was detected at any of the genomic locations. Figure 4: Detection of mutation is performed with PCR and/or T7 digestion. MW = molecular weight marker. (1–2) T7 assay and PCR of F1 heterozygous offspring of \(\Delta\)8 line, respectively; (3–4) PCR of heterozygous offspring of \(\Delta\)104 and \(\Delta\)128 line, respectively; (5) PCR of founder male #5; (6–7) PCR of wild-type animal and no template control. Figure 3: Comparison of mutations originating from the #5 founder male. The gRNA and the target in the gene followed by the PAM sequence are highlighted in red. The yellow spaces show the sites of deletions. The second exon is underlined with a black line. The \(\Delta\)8 mutation is a small deletion of eight base pairs, indicated in yellow. In \(\Delta\)104, a TAAT insertion is present, highlighted in blue, and a couple of single base pair changes appeared in the sequence of \(\Delta\)104 and \(\Delta\)8, which, in the case of the \(\Delta\)104 mutated allele, was probably the consequence of imperfect NHEJ repair. However, in the \(\Delta\)8 mutated allele, it might be a small nucleotide polymorphism not related to NHEJ. in sepsis-induced lung injury in mice [57]. The broad spectrum of NOX4 function creates the need for better animal models to make the best mammalian system for the purposes of translational medicine. The efficiency of CRISPR/Cas9 to induce site specific mutations in rabbits is a complex issue. Chromatin structure of the targeted genomic region affects the availability of the site to CRISPR/Cas9 complexes. An open chromatin structure predicts greater efficiency. The concentration of gRNA is also a crucial element. The encountered 16-18% efficiency of mutated blastocysts is within the normal range. However, the efficiency of targeting varies greatly between 10-100% according to scientific data [1]. Five founder animals were generated in total, of which three acquired identical 8 bp deletions. This phenomenon seems striking, but robust analysis of CRISPR-mediated genome editing in cell culture systems has shown that DNA editing varies considerably between sites, with some targets shown to have a highly preferred indel while others display a wide range of infrequent indels [58]. Presumably, the pattern is dependent on both DNA sequence and chromatin structure. Interestingly, one of the _NOX4_ KO founder males turned out to be a genetic mosaic animal with germline transmission of three distinct knockout alleles besides the wild-type allele to its offspring. The \(\Delta\)8 subline of founder animal #5 carried a short 8 bp deletion directly upstream of the PAM site, which is a common type of modification induced by the CRISPR/Cas9 method according to our own observations and the literature [59]. The other two modified alleles carried longer deletions, a 128 bp deletion in the case of the \(\Delta\)128 subline, and a more exciting deletion-insertion-rearrangement type of modification in the case of the \(\Delta\)104 rabbit subline. Complex targeting events in genome-edited animals have previously been detected [60]. Both modifications--\(\Delta\)104 and \(\Delta\)128--make the detection of the knockout allele a simple one-step procedure, with allele-specific PCR sparing us the burden of T7 assays in our future experiments. We found a deviation of the male/female ratio from the expected Mendelian pattern of inheritance: 14% fewer males were born in the F1 litters of the #5 founder male. These data must be closely examined in our future studies and while evaluating F2-generation data. Mosaic events during genome editing are generally unwanted events. There are known strategies to reduce the genetic mosaicism produced by CRISPR/Cas9 system [61].