Fig. 5

Mosaic genome-edited F0 mouse production. A Overview of F0 mouse production: Day 4 blastocysts obtained by incubating sperm in the c-TYH medium with 2 mM MBCD and 20 ng/ µl pgRNA-Cas9 were used for embryo transfer in a 0.5 dpc pregnant mouse. Genome extraction was subsequently performed from a dark-coated F0 mouse to investigate the presence of plasmids and evaluate the target sequence. B Detection of gRNA-Cas9 plasmid in the F0 mouse genome: PCR and gel electrophoresis were performed to detect the desired fragment (a 250 bp fragment between the U6 promoter and the guide region), which was not detected in the samples. L1-L4: Kidney, liver, brain, and muscle samples, respectively. L5: Positive control (the pgRNA-Cas9 was used as a template); and L: 50 bp DNA ladder. C Gel electrophoresis image of isolated fragments of the gene of interest (Gdf8, fragment size: 445 bp) from genomic DNA of the kidney (L1), liver (L2), brain (L3), and muscle (L4) of the F0 mouse. D-F Direct indel detection using the sequence trace decomposition method through the inference of CRISPR edits (ICE) software. For this detection, Sanger sequencing results of the 445 bp isolated fragments of the mentioned tissues of the F0 mouse were used. D The relative contribution of the wild-type and edited sequence (E) Sanger sequencing view showing edited sequences in kidney, brain, and muscle tissues, non-edited sequence in liver tissue, and wild-type sequence as the control in the vicinity of the guide region. The guide sequence is represented by the black region that is underlined horizontally. F The discordance plot of brain, kidney, liver, and muscle samples of the F0 mouse provides details about the degree of similarity per base between the wild-type (control) and the samples within the specific region surrounding the cut site (inference window)