Fig 1.
In vivo electroporation-based chromosomal engineering in the murine uterus.
(A) The experimental workflow. Anaesthetised female mice (>8 weeks old) received intrauterine injection of CRISPR–Cas9 RNP with or without ssODN donor (2 µL), followed by electroporation across the uterine horns. Genomic DNA collected ≥7 days post-electroporation for PCR and Sanger sequencing. (B) Distribution of the electroporated material. Dextran (red) was co-delivered with Cas9–RNP. Non-EP, non-electroporated region (cervical side); EP, electroporated region (ovarian side). Nuclei were counterstained with DAPI (blue). The arrow indicates the cervical end.
Fig 2.
CCRs in the uterine epithelium induced via in vivo electroporation.
(A) Schematic of CCR design involving Hmga2, Wif1, and Rassf3 on chromosome 10, showing Cas9 cut sites (scissors) and ssODN donors for precise junction repair. (B) Sequence alignment of PCR products corresponding to the genomic breakpoint junctions, Hmga2–Wif1, Hmga2–Rassf3, and Wif1–Rassf3. Samples are grouped as “with ssODN” and “without ssODN”; “+” and “–” after sample numbers indicate the respective condition. Arrows indicate Cas9 cleavage sites; boxed bases show microhomology; lowercase letters, insertions or mismatches; underlines, PAM sequences. PAM, protospacer adjacent motif; dsDNA, double-stranded DNA; Pred., predicted sequence. (C) CCR induction efficiency in the uterine epithelium with or without ssODN. Precise junctions denote ssODN-consistent joins; rearrangements, all detected junctions.
Fig 3.
Translocations at the Firre locus in the uterine epithelium induced via in vivo electroporation.
(A) Schematic showing the Firre locus on the X chromosome. Translocation design between Eef1a1N (chr. 9) and Atf4N (chr. 15) is shown, with Cas9 cut sites (scissors) and ssODN donors for junction repair. (B) Sequence alignment of the PCR products corresponding to the genomic breakpoint junctions of Eef1a1N and Atf4N. The arrows indicate Cas9 cleavage sites; lowercase letters, insertions or mismatches; underlines, PAM sequences. PAM, protospacer adjacent motif; dsDNA, double-stranded DNA; Pred., predicted sequence. (C) Schematic representation of the translocation between the Ypel4N (chr. 2) and Atf4N (chr. 15). (D) Sequence alignment of the PCR products corresponding to the genomic breakpoint junctions of Ypel4N and Atf4N. Boxed regions indicate the microhomology sequences. (E) Translocation efficiency of Eef1a1N–Atf4N and Ypel4N–Atf4N in the uterine epithelium. The precise junctions denote ssODN-consistent joins; rearrangements, all detected junctions connecting the intended loci.
Fig 4.
Repair of a large-scale chromosomal inversion in the uterine epithelium via in vivo electroporation.
(A) Schematic of the CRISPR/Cas9-based strategy to repair the 57.8-Mb inversion In(6)1J on chromosome 6, showing Cas9 cut sites (scissors) and ssODN donors for junction repair. (B) Sequence alignment of the PCR products corresponding to the left and right breakpoint junctions after repair. Samples are grouped as “with ssODN” and “without ssODN”; “+” and “–” after sample numbers indicate the respective condition. Arrows mark Cas9 cleavage sites; boxed or shaded bases, microhomology or repaired regions; lowercase letters, insertions or mismatches; underlines, PAM sequences. PAM, protospacer adjacent motif; dsDNA, double-stranded DNA; Pred., predicted sequence. (C) Repair efficiency of In(6)1J in the uterine epithelium with or without ssODN. Precise junctions denote ssODN-consistent joins; rearrangements, all detectable junctions connecting the intended loci.
Fig 5.
Oncogenic translocations in the uterine epithelium induced via in vivo electroporation.
(A) Schematic of the translocation between Ncoa2 (chr. 1) and Greb1 (chr. 12) showing Cas9 cut sites (scissors) and ssODN donors for junction repair. (B) Sequence alignment of the PCR products corresponding to the genomic breakpoint junctions of Ncoa2 and Greb1. The arrows indicate Cas9 cleavage sites; boxed bases, microhomology; lowercase letters, insertions or mismatches; underlines, PAM sequences. PAM, protospacer adjacent motif; dsDNA, double-stranded DNA; Pred., predicted sequence. (C) Schematic of the translocation between Ywhae (chr. 11) and Nutm2 (chr. 13). (D) Sequence alignment of the PCR products corresponding to the genomic breakpoint junctions of Ywhae and Nutm2. (E) Translocation efficiency of Ncoa2–Greb1 and Ywhae–Nutm2 in the uterine epithelium. Precise junctions denote ssODN-consistent joins; rearrangements, all detected junctions connecting the intended loci.
Fig 6.
IGV visualization of chromosomal translocations detected by whole-genome sequencing of edited uterine tissue.
(A–C) Integrative Genomics Viewer (IGV) screenshots showing paired-end read alignments spanning the predicted translocation breakpoints. (A) Translocation between chromosomes 2 (purple) and 15 (orange) at the Ypel4N–Atf4N locus. (B) Translocation between chromosomes 1 (purple) and 12 (blue) at the Ncoa2–Greb1 locus. (C) Translocation between chromosomes 11 (orange) and 13 (brown) at the Ywhae–Nutm2 locus. Discordant paired reads bridging the two chromosomes are color-coded by mate-pair orientation, and normal reads are shown in gray.