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Fig. 4 Impact of inter-loxP distances on Cre-mediated recombination and mosaicism in DIO mouse strains: from genotyping to single-cell analysis. a Study design for generating conditional alleles at a fixed locus (R26) using the Cas9+Bxb1 toolbox. The Cas9+Bxb1 toolbox allows for accurate and efficient integration of large DNA constructs into a specific location, achieving higher efficiency compared to Cas9-mediated homology-directed repair (HDR). Bxb1 uses attP and attB attachment sites for DNA transgenesis. In our study, we strategically placed an attP attachment site within the R26 safe harbor locus of the B6/J mouse strain using CRISPR/Cas9-mediated HDR. This approach enabled us to seamlessly integrate DNA constructs with DIO alleles ranging from approximately 1 to 8 kb in size. Consequently, we successfully generated conditional knockout mice with single-copy transgenic modifications. b, c, and d Homozygous floxed male mice were mated with either female Ella-cre (b), CMV-cre (c), or Sox2-cre (d) mice, and the F1 offspring were genotyped using PCR from tail DNA to screen for floxed and recombined alleles. Offspring with complete recombination were categorized as recombined, those with no recombination as still floxed, and those with both recombined and floxed alleles as mosaic. The x-axis denotes the inter-loxP distance, and the y-axis denotes the percentage of recombination or mosaicism. The number of offspring genotyped to assess the percent of three possible outcomes: complete recombination, mosaicism, or no recombination are summarized in Additional file 1: Fig. S3b. e Mosaicism at the single-cell level: Confocal image of an 8-kb Myo15a-Halo DIO floxed allele x CMV-cre F1 cochlear sensory epithelium at postnatal day 2. Phalloidin stains filamentous actin (F-Actin), labeling the sensory hair cell cytoskeleton. TMR-conjugated Halo Tag ligand labels Halo-Myo15a, the product of recombination. Halo-Myo15a enriches at the tips of hair cell cytoskeletal projections. White asterisks indicate recombined cells expressing Halo-Myo15a. Yellow arrowheads indicate cells that escaped recombination, where the allele is still floxed. f Quantification of percentages from four different pups as points and the averages by bar plot.