Fig 1: Southern blot analysis of Nup93 locus targetingDNA samples were extracted from mouse-tail biopsies of F0 offspring and digested with BamHI. The following groups of samples were analyzed: Samples 1, 2, 112–129: microinjected with denatured DNA template and crRNA1–crRNA7 pair. Samples 15–34: microinjected with denatured DNA template, crRNA1–crRNA7, and RAD52. Sample 39: microinjected with denatured DNA template and crRNA2–crRNA8 pair. Samples 50–78: microinjected with denatured DNA template, crRNA2–crRNA8, and RAD52. Samples 79–111: microinjected with denatured DNA template and crRNA1–crRNA8. Southern blot analysis was performed using a specific probe (Figure 1) following BamHI digestion. This detected the wild-type (wt) allele (21.7 kb) and two fragments (2.8 kb and 19.0 kb) corresponding to the correctly targeted allele. Notably, under the given conditions, the 21.7 kb and 19.0 kb fragments were not resolved on agarose gels (see wt control animals). The presence of a single 2.8 kb band in DNA samples 21, 106, and 121 indicates successful targeting of the Nup93 allele (Nup93+/−). Size marker positions (in bp) are shown on the left. All other animals, except 19, 20, and 24, which contained an unmodified Nup93 locus, exhibited either head-to-tail or aberrant integrations of the DNA template.