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<p>CRISPR nuclease-mediated gene knock-out is limited by suboptimal sgRNAs, inaccessible target sites, and undesired repair outcomes. Here, we present a Cas12a-based system in <i>Drosophila</i> that targets each gene with four sgRNAs to overcome these limitations. Multiplexed sgRNAs act through redundancy and synergism, frequently creating deletions between target sites and increasing the fraction of loss-of-function mutations. We show that multiplexed gene targeting is well tolerated and does not cause widespread proximity effects. To visualize CRISPR-nuclease activity in living animals, we developed a screening assay and used it to assess Cas12a activity across 33% of the <i>Drosophila</i> genome in combination with over 2000 sgRNAs. This revealed remarkably high on-target (&gt;99%) and very low (&lt;1%) off-target activity of multiplexed Cas12a sgRNA arrays. Quantitative side-by-side comparisons with current Cas9-based systems targeting over 100 genes in parallel demonstrate that multiplexed Cas12a gene targeting achieves superior performance and reveals phenotypes missed by established methods. The system described here provides a framework for reliable gene knock-out in multicellular systems.</p>

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Improved in vivo gene knockout with high specificity using multiplexed Cas12a sgRNAs

  • 期刊论文

  • 出版模式：

    开放获取

  • 发表日期：

    2026年1月15日

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  • Fillip Port,
  • Martha A. Buhmann,
  • Jun Zhou,
  • Mona Stricker,
  • Alexander Vaughan-Brown,
  • Ann-Christin Michalsen,
  • Eva Roßmanith,
  • Amélie Pöltl,
  • Lena Großkurth,
  • Julia Huber,
  • Laura B. Menendez Kury,
  • Bea Weberbauer,
  • Maria Hübl,
  • Elli Puscher,
  • Florian Heigwer,
  • Michael Boutros

摘要

CRISPR nuclease-mediated gene knock-out is limited by suboptimal sgRNAs, inaccessible target sites, and undesired repair outcomes. Here, we present a Cas12a-based system in Drosophila that targets each gene with four sgRNAs to overcome these limitations. Multiplexed sgRNAs act through redundancy and synergism, frequently creating deletions between target sites and increasing the fraction of loss-of-function mutations. We show that multiplexed gene targeting is well tolerated and does not cause widespread proximity effects. To visualize CRISPR-nuclease activity in living animals, we developed a screening assay and used it to assess Cas12a activity across 33% of the Drosophila genome in combination with over 2000 sgRNAs. This revealed remarkably high on-target (>99%) and very low (<1%) off-target activity of multiplexed Cas12a sgRNA arrays. Quantitative side-by-side comparisons with current Cas9-based systems targeting over 100 genes in parallel demonstrate that multiplexed Cas12a gene targeting achieves superior performance and reveals phenotypes missed by established methods. The system described here provides a framework for reliable gene knock-out in multicellular systems.