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Background <p>After Cas12a cleaves its DNA target, it generates a DNA double strand break (DSB) with two compatible 5′-staggered ends. The Cas12a-gRNA complex remains at the protospacer adjacent motif (PAM)-proximal end (PPE) while releasing the PAM-distal end (PDE). The effects of this asymmetric retention on DSB repair are currently unknown.</p> Results <p>Post-cleavage retention of <i>Lb</i>Cas12a at PPEs suppresses the recruitment of classical non-homologous end joining (c-NHEJ) core factors, leading to longer deletions at PPEs compared to PDEs. This asymmetry in c-NHEJ engagement results in approximately tenfold more accurate ligation between two compatible PDEs induced by paired <i>Lb</i>Cas12a than ligation involving a compatible PPE. Moreover, ligation to a given end of <i>Sp</i>Cas9-induced DSBs demonstrates more efficient ligation with a PDE from Cas12a-induced DSBs than with a PPE. In <i>Lb</i>Cas12a-induced NHEJ-mediated targeted integration, only two compatible PDEs from <i>Lb</i>Cas12a-induced DSBs—one from donor templates and the other from target sites—promote accurate and directional ligation. Based on these findings, we developed a strategy called Cas12a-induced PDE ligation (CIPDEL) for NHEJ-mediated efficient and precise gene correction and insertion.</p> Conclusions <p>The asymmetric retention of CRISPR-<i>Lb</i>Cas12a at DSB ends suppresses c-NHEJ at PPEs, not at PDEs. This unique repair mechanism can be utilized in the CIPDEL strategy, offering a potentially better alternative for homology-directed targeted integration.</p>

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Post-cleavage target residence determines asymmetry in non-homologous end joining of Cas12a-induced DNA double strand breaks

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2025年4月14日

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  • Ruo-Dan Chen,
  • Yi Yang,
  • Kun-Ming Liu,
  • Jing-Zhen Hu,
  • Yi-Li Feng,
  • Chun-Yi Yang,
  • Rui-Rui Jiang,
  • Si-Cheng Liu,
  • Yue Wang,
  • Ping-An Han,
  • Ru-Gang Tian,
  • Yu-Long Wang,
  • Shi-Ming Xu,
  • An-Yong Xie

摘要

Background

After Cas12a cleaves its DNA target, it generates a DNA double strand break (DSB) with two compatible 5′-staggered ends. The Cas12a-gRNA complex remains at the protospacer adjacent motif (PAM)-proximal end (PPE) while releasing the PAM-distal end (PDE). The effects of this asymmetric retention on DSB repair are currently unknown.

Results

Post-cleavage retention of LbCas12a at PPEs suppresses the recruitment of classical non-homologous end joining (c-NHEJ) core factors, leading to longer deletions at PPEs compared to PDEs. This asymmetry in c-NHEJ engagement results in approximately tenfold more accurate ligation between two compatible PDEs induced by paired LbCas12a than ligation involving a compatible PPE. Moreover, ligation to a given end of SpCas9-induced DSBs demonstrates more efficient ligation with a PDE from Cas12a-induced DSBs than with a PPE. In LbCas12a-induced NHEJ-mediated targeted integration, only two compatible PDEs from LbCas12a-induced DSBs—one from donor templates and the other from target sites—promote accurate and directional ligation. Based on these findings, we developed a strategy called Cas12a-induced PDE ligation (CIPDEL) for NHEJ-mediated efficient and precise gene correction and insertion.

Conclusions

The asymmetric retention of CRISPR-LbCas12a at DSB ends suppresses c-NHEJ at PPEs, not at PDEs. This unique repair mechanism can be utilized in the CIPDEL strategy, offering a potentially better alternative for homology-directed targeted integration.