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Cited by 3CRISPR and Genetic EngineeringDNA Repair MechanismsGenomic variations and chromosomal abnormalities

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A Recql5 mutant facilitates complex CRISPR/Cas9-mediated chromosomal engineering in mouse zygotes

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Iwata, Satoru, Nagahara, Miki, Ido, Risako, Iwamoto, Takashi·2024·GENETICS
DOI 10.1093/genetics/iyae054PMC PMC11151919

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Abstract

Complex chromosomal rearrangements (CCRs) are often observed in clinical samples from patients with cancer and congenital diseases but are difficult to induce experimentally. Here, we report the first success in establishing animal models for CCRs. Mutation in Recql5, a crucial member of the DNA helicase RecQ family involved in DNA replication, transcription, and repair, enabled CRISPR/Cas9-mediated CCRs, establishing a mouse model containing triple fusion genes and megabase-sized inversions. Some of these structural features of individual chromosomal rearrangements use template switching and microhomology-mediated break-induced replication mechanisms and are reminiscent of the newly described phenomenon “chromoanasynthesis.” These data show that Recql5 mutant mice could be a powerful tool to analyze the pathogenesis of CCRs (particularly chromoanasynthesis) whose underlying mechanisms are poorly understood. The Recql5 mutants generated in this study are to be deposited at key animal research facilities, thereby making them accessible for future research on CCRs.

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Figures

Fig. 1. Chromosomal engineering of inversions using the Recql5 mutant. a) Schematic illustration of the chromosomal rearrangements created by an inversion between Adamts20 and the K18N-locus in chromosome 15. b) Alignment of sequences corresponding to the Adamts20 and the K18N-locus genomic breakpoint junctions, obtained via the i-GONAD technique. c) The generated inversion [In(15)#6] on chromosome 15 shows a recessive, white-spotted phenotype. d) Junction sequence obtained through in vitro electroporation, demonstrating complex rearrangements with insertions associated with the FoSTeS/MMBIR mechanism. e) Schematic illustration of the chromosomal rearrangements created by an inversion between the Hmga2 and Wif1 genes in chromosome 10. f) Alignment of sequences corresponding to Hmga2 and Wif1 genomic breakpoint junctions, obtained via the i-GONAD technique. g) PCR amplification of the predicted fusion transcript. NTC, negative control. h) Sanger sequences corresponding to Hmga2–Wif1 cDNA. i) The generated inversion [In(10)#3] on chromosome 10 showed a recessive pygmy phenotype. j) Summary of the experimental efficiency of chromosomal inversions using the i-GONAD technique. k) Summary of chromosomal inversion efficiency and embryonic development stages post in vitro electroporation.
Fig. 1. Chromosomal engineering of inversions using the Recql5 mutant. a) Schematic illustration of the chromosomal rearrangements created by an inversion between Adamts20 and the K18N-locus in chromosome 15. b) Alignment of sequences corresponding to the Adamts20 and the K18N-locus genomic breakpoint junctions, obtained via the i-GONAD technique. c) The generated inversion [In(15)#6] on chromosome 15 shows a recessive, white-spotted phenotype. d) Junction sequence obtained through in vitro electroporation, demonstrating complex rearrangements with insertions associated with the FoSTeS/MMBIR mechanism. e) Schematic illustration of the chromosomal rearrangements created by an inversion between the Hmga2 and Wif1 genes in chromosome 10. f) Alignment of sequences corresponding to Hmga2 and Wif1 genomic breakpoint junctions, obtained via the i-GONAD technique. g) PCR amplification of the predicted fusion transcript. NTC, negative control. h) Sanger sequences corresponding to Hmga2–Wif1 cDNA. i) The generated inversion [In(10)#3] on chromosome 10 showed a recessive pygmy phenotype. j) Summary of the experimental efficiency of chromosomal inversions using the i-GONAD technique. k) Summary of chromosomal inversion efficiency and embryonic development stages post in vitro electroporation.CC BY · PubMed Central
Fig. 2. Induction of Hmga2–Wif1, Hmga2–Rassf3, and Wif1–Rassf3 complex rearrangements in mouse zygotes using the Recql5 mutant. a) Schematic representation of the CCRs created between Hmga2, Wif1, and Rassf3 in chromosome 10. b) PCR amplification of the predicted breakpoint junction and fusion transcript. Sanger sequences corresponding to Hmga2–Wif1, Hmga2–Rassf3, and Wif1–Rassf3 cDNA. NTC, negative control. c) Summary of the experimental efficiency of CCRs using the i-GONAD technique. d) Summary of CCR efficiency and embryonic development stages post in vitro electroporation. e) Appearance of heterozygous and homozygous CCRs(10)#4a male mice at 6 weeks of age. f) Growth curves of WT (+/+) (n = 11/10), heterozygous CCRs(10)#4a (#4a/+) (n = 7/8), and homozygous CCRs(10)#4a (#4a/#4a) (n = 9/12) mice (female/male). Error bars, mean ± SD. *P < 0.05 (2-tailed Student's t-test). g) Food intake (g/day) of WT (+/+) and CCRs(10)#4a/#4a male mice (20 weeks of age). Error bars, mean ± SD. *P < 0.05 (2-tailed Student's t-test; n = 3∼5). h) Comparison of litter sizes. Error bars, mean ± SD. Different letters above the bars indicate significant differences at P < 0.05 by Tukey's honest significance test (HSD; n = 6). i) Mendelian ratios of newborn mice from CCRs(10)#4a heterozygous crossings.
Fig. 2. Induction of Hmga2–Wif1, Hmga2–Rassf3, and Wif1–Rassf3 complex rearrangements in mouse zygotes using the Recql5 mutant. a) Schematic representation of the CCRs created between Hmga2, Wif1, and Rassf3 in chromosome 10. b) PCR amplification of the predicted breakpoint junction and fusion transcript. Sanger sequences corresponding to Hmga2–Wif1, Hmga2–Rassf3, and Wif1–Rassf3 cDNA. NTC, negative control. c) Summary of the experimental efficiency of CCRs using the i-GONAD technique. d) Summary of CCR efficiency and embryonic development stages post in vitro electroporation. e) Appearance of heterozygous and homozygous CCRs(10)#4a male mice at 6 weeks of age. f) Growth curves of WT (+/+) (n = 11/10), heterozygous CCRs(10)#4a (#4a/+) (n = 7/8), and homozygous CCRs(10)#4a (#4a/#4a) (n = 9/12) mice (female/male). Error bars, mean ± SD. *P < 0.05 (2-tailed Student's t-test). g) Food intake (g/day) of WT (+/+) and CCRs(10)#4a/#4a male mice (20 weeks of age). Error bars, mean ± SD. *P < 0.05 (2-tailed Student's t-test; n = 3∼5). h) Comparison of litter sizes. Error bars, mean ± SD. Different letters above the bars indicate significant differences at P < 0.05 by Tukey's honest significance test (HSD; n = 6). i) Mendelian ratios of newborn mice from CCRs(10)#4a heterozygous crossings.CC BY · PubMed Central
Fig. 3. Validation of genome-wide target specificities in the Recql5 mutant. a) WGS results. IGV browser of CCR data aligned to the mouse genome (mm10). The gRNA cut sites are shown using arrowheads. b) Schematic representation of paired-end read interpretation in IGV for complex rearrangements. Each arrow indicates the type and orientation of read pairs as aligned to the reference genome. c) Alignment of sequences from PCR products corresponding to the Hmga2–Wif1, Hmga2–Rassf3, and Wif1–Rassf3 genomic breakpoint junctions. PAM, protospacer adjacent motif; μH, microhomology. d) Manta calls supporting chromosomal breakpoints.
Fig. 3. Validation of genome-wide target specificities in the Recql5 mutant. a) WGS results. IGV browser of CCR data aligned to the mouse genome (mm10). The gRNA cut sites are shown using arrowheads. b) Schematic representation of paired-end read interpretation in IGV for complex rearrangements. Each arrow indicates the type and orientation of read pairs as aligned to the reference genome. c) Alignment of sequences from PCR products corresponding to the Hmga2–Wif1, Hmga2–Rassf3, and Wif1–Rassf3 genomic breakpoint junctions. PAM, protospacer adjacent motif; μH, microhomology. d) Manta calls supporting chromosomal breakpoints.CC BY · PubMed Central
Fig. 4. Efficiency of chromosomal rearrangement and blastocyst development rates following in vitro electroporation in the Recql5 mutant. a) Chromosomal rearrangement efficiencies at 3 distinct breakpoints following in vitro electroporation in WT and Recql5em1/em1 strains. The dashed lines indicate inversions, and the solid lines represent CCRs. b) Chromosomal rearrangement efficiency across all targeted gene loci. Results are presented as the mean ± SD, including data represented by scatter blots. *P < 0.05, Mann–Whitney U test (n = 13). c) Blastocyst development rate across all targeted gene loci. *P < 0.05, Mann–Whitney U test (n = 5). The dashed lines indicate inversions, and the solid lines represent CCRs.
Fig. 4. Efficiency of chromosomal rearrangement and blastocyst development rates following in vitro electroporation in the Recql5 mutant. a) Chromosomal rearrangement efficiencies at 3 distinct breakpoints following in vitro electroporation in WT and Recql5em1/em1 strains. The dashed lines indicate inversions, and the solid lines represent CCRs. b) Chromosomal rearrangement efficiency across all targeted gene loci. Results are presented as the mean ± SD, including data represented by scatter blots. *P < 0.05, Mann–Whitney U test (n = 13). c) Blastocyst development rate across all targeted gene loci. *P < 0.05, Mann–Whitney U test (n = 5). The dashed lines indicate inversions, and the solid lines represent CCRs.CC BY · PubMed Central

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Metadata

Authors
Iwata, S., Nagahara, M., Ido, R., & Iwamoto, T.
Affiliations
Chubu University (JP)
Corresponding author
Iwata, Satoru
ORCID
Venue
GENETICS
Publisher
Oxford University Press (OUP)
Published
2024-04-05
Volume
227
Issue
2
ISSN
  • 0016-6731 (ISSN-L)
  • 1943-2631
DOI
10.1093/genetics/iyae054
PMC
PMC11151919
OpenAlex
W4393991893
Cited by
3
References
38
Citations by year
2026: 2 · 2025: 1
FWCI
0.41
Type
journal article
Language
en
Open access
Licence
Locations
  • Genetics (publishedVersion, cc-by)
  • bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
Funders
  • Chubu University
  • Promotion and Mutual Aid Corporation for Private Schools of Japan
  • Japan Society for the Promotion of Science (23K21287)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › CRISPR and Genetic Engineering
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › DNA Repair Mechanisms
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Genomic variations and chromosomal abnormalities
Keywords
RECQL5, complex chromosomal rearrangements, mouse zygotes, RecQ helicase, template switching, microhomology-mediated break-induced replication, DNA replication and repair
MeSH terms
Animals, Chromosome Aberrations, Chromosomes, Mutation, Zygote, Mice, RecQ Helicases, CRISPR-Cas Systems

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CoChat. (2026). Citation check: A Recql5 mutant facilitates complex CRISPR/Cas9-mediated chromosomal engineering in mouse zygotes. https://app.cochat.ai/papers/10.1093/genetics/iyae054
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Iwata, S., Nagahara, M., Ido, R., & Iwamoto, T. (2024). A Recql5 mutant facilitates complex CRISPR/Cas9-mediated chromosomal engineering in mouse zygotes. GENETICS. https://doi.org/10.1093/genetics/iyae054

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