Cruciform DNA Structures Act as Legible Templates for Accelerating Homologous Recombination in Transgenic Animals
Inverted repeat (IR) DNA sequences compose cruciform structures. Some genetic disorders are the result of genome inversion or translocation by cruciform DNA structures. The present study examined whether exogenous DNA integration into the chromosomes of transgenic animals was related to cruciform DN...
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MDPI AG
2022-04-01
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| Series: | International Journal of Molecular Sciences |
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| Online Access: | https://www.mdpi.com/1422-0067/23/7/3973 |
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| author | Huan Ou-Yang Shiao-Hsuan Yang Wei Chen Shang-Hsun Yang Abdulkadir Cidem Li-Ying Sung Chuan-Mu Chen |
| author_facet | Huan Ou-Yang Shiao-Hsuan Yang Wei Chen Shang-Hsun Yang Abdulkadir Cidem Li-Ying Sung Chuan-Mu Chen |
| author_sort | Huan Ou-Yang |
| collection | DOAJ |
| description | Inverted repeat (IR) DNA sequences compose cruciform structures. Some genetic disorders are the result of genome inversion or translocation by cruciform DNA structures. The present study examined whether exogenous DNA integration into the chromosomes of transgenic animals was related to cruciform DNA structures. Large imperfect cruciform structures were frequently predicted around predestinated transgene integration sites in host genomes of microinjection-based transgenic (Tg) animals (<i>αLA-LPH</i> Tg goat, <i>Akr1A1<sup>eGFP/eGFP</sup></i> Tg mouse, and <i>NFκB-Luc</i> Tg mouse) or CRISPR/Cas9 gene-editing (GE) animals (<i>αLA-AP1</i> GE mouse). Transgene cassettes were imperfectly matched with their predestinated sequences. According to the analyzed data, we proposed a putative model in which the flexible cruciform DNA structures acted as a legible template for DNA integration into linear DNAs or double-strand break (DSB) alleles. To demonstrate this model, artificial inverted repeat knock-in (KI) reporter plasmids were created to analyze the KI rate using the CRISPR/Cas9 system in NIH3T3 cells. Notably, the KI rate of the 5′ homologous arm inverted repeat donor plasmid (5′IR) with the ROSA gRNA group (31.5%) was significantly higher than the knock-in reporter donor plasmid (KIR) with the ROSA gRNA group (21.3%, <i>p</i> < 0.05). However, the KI rate of the 3′ inverted terminal repeat/inverted repeat donor plasmid (3′ITRIR) group was not different from the KIR group (23.0% vs. 22.0%). These results demonstrated that the legibility of the sequence with the cruciform DNA existing in the transgene promoted homologous recombination (HR) with a higher KI rate. Our findings suggest that flexible cruciform DNAs folded by IR sequences improve the legibility and accelerate DNA 3′-overhang integration into the host genome via homologous recombination machinery. |
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| issn | 1661-6596 1422-0067 |
| language | English |
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| spelling | doaj.art-cdd4ceb54c6d4c6982d4a724f83e2e3a2023-11-30T23:24:46ZengMDPI AGInternational Journal of Molecular Sciences1661-65961422-00672022-04-01237397310.3390/ijms23073973Cruciform DNA Structures Act as Legible Templates for Accelerating Homologous Recombination in Transgenic AnimalsHuan Ou-Yang0Shiao-Hsuan Yang1Wei Chen2Shang-Hsun Yang3Abdulkadir Cidem4Li-Ying Sung5Chuan-Mu Chen6Program in Translational Medicine, Department of Life Sciences, National Chung Hsing University, Taichung 402, TaiwanProgram in Translational Medicine, Department of Life Sciences, National Chung Hsing University, Taichung 402, TaiwanDivision of Pulmonary and Critical Care Medicine, Chia-Yi Christian Hospital, Chiayi 600, TaiwanDepartment of Physiology, National Cheng Kung University, Tainan 701, TaiwanProgram in Translational Medicine, Department of Life Sciences, National Chung Hsing University, Taichung 402, TaiwanInstitute of Biotechnology, College of Bioresources and Agriculture, National Taiwan University, Taipei 106, TaiwanProgram in Translational Medicine, Department of Life Sciences, National Chung Hsing University, Taichung 402, TaiwanInverted repeat (IR) DNA sequences compose cruciform structures. Some genetic disorders are the result of genome inversion or translocation by cruciform DNA structures. The present study examined whether exogenous DNA integration into the chromosomes of transgenic animals was related to cruciform DNA structures. Large imperfect cruciform structures were frequently predicted around predestinated transgene integration sites in host genomes of microinjection-based transgenic (Tg) animals (<i>αLA-LPH</i> Tg goat, <i>Akr1A1<sup>eGFP/eGFP</sup></i> Tg mouse, and <i>NFκB-Luc</i> Tg mouse) or CRISPR/Cas9 gene-editing (GE) animals (<i>αLA-AP1</i> GE mouse). Transgene cassettes were imperfectly matched with their predestinated sequences. According to the analyzed data, we proposed a putative model in which the flexible cruciform DNA structures acted as a legible template for DNA integration into linear DNAs or double-strand break (DSB) alleles. To demonstrate this model, artificial inverted repeat knock-in (KI) reporter plasmids were created to analyze the KI rate using the CRISPR/Cas9 system in NIH3T3 cells. Notably, the KI rate of the 5′ homologous arm inverted repeat donor plasmid (5′IR) with the ROSA gRNA group (31.5%) was significantly higher than the knock-in reporter donor plasmid (KIR) with the ROSA gRNA group (21.3%, <i>p</i> < 0.05). However, the KI rate of the 3′ inverted terminal repeat/inverted repeat donor plasmid (3′ITRIR) group was not different from the KIR group (23.0% vs. 22.0%). These results demonstrated that the legibility of the sequence with the cruciform DNA existing in the transgene promoted homologous recombination (HR) with a higher KI rate. Our findings suggest that flexible cruciform DNAs folded by IR sequences improve the legibility and accelerate DNA 3′-overhang integration into the host genome via homologous recombination machinery.https://www.mdpi.com/1422-0067/23/7/3973inverted repeat (IR)cruciformtransgenedouble-strand break (DSB)DNA repairhomologous recombination (HR) |
| spellingShingle | Huan Ou-Yang Shiao-Hsuan Yang Wei Chen Shang-Hsun Yang Abdulkadir Cidem Li-Ying Sung Chuan-Mu Chen Cruciform DNA Structures Act as Legible Templates for Accelerating Homologous Recombination in Transgenic Animals International Journal of Molecular Sciences inverted repeat (IR) cruciform transgene double-strand break (DSB) DNA repair homologous recombination (HR) |
| title | Cruciform DNA Structures Act as Legible Templates for Accelerating Homologous Recombination in Transgenic Animals |
| title_full | Cruciform DNA Structures Act as Legible Templates for Accelerating Homologous Recombination in Transgenic Animals |
| title_fullStr | Cruciform DNA Structures Act as Legible Templates for Accelerating Homologous Recombination in Transgenic Animals |
| title_full_unstemmed | Cruciform DNA Structures Act as Legible Templates for Accelerating Homologous Recombination in Transgenic Animals |
| title_short | Cruciform DNA Structures Act as Legible Templates for Accelerating Homologous Recombination in Transgenic Animals |
| title_sort | cruciform dna structures act as legible templates for accelerating homologous recombination in transgenic animals |
| topic | inverted repeat (IR) cruciform transgene double-strand break (DSB) DNA repair homologous recombination (HR) |
| url | https://www.mdpi.com/1422-0067/23/7/3973 |
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