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<p>Chromosomal inversion is a key structural variation impacting cellular fitness and genomic integrity. Here we developed prime-editing-based inversion with enhanced performance (PIE) to efficiently induce large-scale inversions in mammalian cells. PIEv1 uses a prime-editing guide RNA (pegRNA) pair but yields one imprecise junction. PIEv2 and PIEv3 add a second pegRNA pair for precise inversion, with PIEv3b further enhancing coupling precise inversion through improved plasmid design. PIEv3b achieves inversion efficiencies up to 61.7% for 1 Mb and 14.2% for 50 Mb segments and shows 4–20-fold higher efficiency compared to twin prime editing with integrase, across ranges of 100 kb to 30 Mb. Additionally, PIEv3b outperforms nuclease-based approaches in both inversion efficiency and precision. Using PIE, we convert human chromosomes from metacentric to telocentric configurations by inverting 30-Mb and 100-Mb chromosomal segments. Our work represents a powerful tool for engineering chromosomal structural variations, with broad implications for medicine and biotechnology.</p><p></p>

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Efficient and precise inversion of genomic DNA from large to chromosomal scale

  • 期刊论文

  • 发表日期：

    2025年10月8日

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  • Ao Zhang,
  • Xiangkun Sun,
  • Ying Wu,
  • Pan Gao,
  • Ruiwen Zhang,
  • Man Zhang,
  • Sisi Xie,
  • Wei Fan,
  • Ying Zhang,
  • Hao Yin

摘要

Chromosomal inversion is a key structural variation impacting cellular fitness and genomic integrity. Here we developed prime-editing-based inversion with enhanced performance (PIE) to efficiently induce large-scale inversions in mammalian cells. PIEv1 uses a prime-editing guide RNA (pegRNA) pair but yields one imprecise junction. PIEv2 and PIEv3 add a second pegRNA pair for precise inversion, with PIEv3b further enhancing coupling precise inversion through improved plasmid design. PIEv3b achieves inversion efficiencies up to 61.7% for 1 Mb and 14.2% for 50 Mb segments and shows 4–20-fold higher efficiency compared to twin prime editing with integrase, across ranges of 100 kb to 30 Mb. Additionally, PIEv3b outperforms nuclease-based approaches in both inversion efficiency and precision. Using PIE, we convert human chromosomes from metacentric to telocentric configurations by inverting 30-Mb and 100-Mb chromosomal segments. Our work represents a powerful tool for engineering chromosomal structural variations, with broad implications for medicine and biotechnology.