Scholarlink

<p>The design and screening of sgRNA in CRISPR-dependent gene knock-in is always laborious. Therefore, a universal and highly efficient knock-in strategy suitable for different sgRNA target sites is necessary. In our mouse embryo study, we find that the knock-in efficiency guided by adjacent sgRNAs varies greatly, although similar indel frequency. MMEJ-biased sgRNAs usually lead to high knock-in efficiency, whereas NHEJ-biased sgRNAs result in low knock-in efficiency. Blocking MMEJ repair by knocking down <i>Polq</i> can enhance knock-in efficiency, but inhibiting NHEJ repair shows variable effects. We identify a compound, AZD7648, that can shift DSBs repair towards MMEJ. Finally, by combining AZD7648 treatment with <i>Polq</i> knockdown, we develop a universal and highly efficient knock-in strategy in mouse embryos. This approach is validated at more than ten genomic loci, achieving up to 90% knock-in efficiency, marking a significant advancement toward predictable and highly efficient CRISPR-mediated gene integration.</p>

错误：搜索内容不能为空，请输入英文关键词
错误：关键词超出字数限制，请精简
高级检索

Refined DNA repair manipulation enables a universal knock-in strategy in mouse embryos

  • 期刊论文

  • 出版模式：

    开放获取

  • 发表日期：

    2025年7月15日

查看全文
  • Hongyu Chen,
  • Qingtong Tan,
  • Li Li,
  • Lanxin Li,
  • Jiqiang Fu,
  • Wencheng Zhu,
  • Jie Li,
  • Yining Wang,
  • Shiyan Li,
  • Huimin Li,
  • Yidi Sun,
  • Qiang Sun,
  • Zongyang Lu,
  • Zhen Liu

摘要

The design and screening of sgRNA in CRISPR-dependent gene knock-in is always laborious. Therefore, a universal and highly efficient knock-in strategy suitable for different sgRNA target sites is necessary. In our mouse embryo study, we find that the knock-in efficiency guided by adjacent sgRNAs varies greatly, although similar indel frequency. MMEJ-biased sgRNAs usually lead to high knock-in efficiency, whereas NHEJ-biased sgRNAs result in low knock-in efficiency. Blocking MMEJ repair by knocking down Polq can enhance knock-in efficiency, but inhibiting NHEJ repair shows variable effects. We identify a compound, AZD7648, that can shift DSBs repair towards MMEJ. Finally, by combining AZD7648 treatment with Polq knockdown, we develop a universal and highly efficient knock-in strategy in mouse embryos. This approach is validated at more than ten genomic loci, achieving up to 90% knock-in efficiency, marking a significant advancement toward predictable and highly efficient CRISPR-mediated gene integration.