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<p>Since their discovery, CRISPR systems have been repurposed for programmable targeted genomic editing, leading to applications for gene disruption, single base editing, insertion, deletion, and manipulation of short genomic sequences. Pairing Cas9 nickase with reverse transcriptase allows applications for insertion, substitution, and deletion of short genomic sequences from an RNA template without generating double stranded breaks&#xa0;however this technology typically shows reduced efficacy in post mitotic cells, limiting its translatability in vivo. Here we present a novel, ligase-based method that addresses these limiations.&#xa0;We introduce edits through delivery and ligation of a synthetic DNA donor to genomic nicks created with Cas9 nickase and report editing activity in cell lines, primary cell cultures, and adult mice via nonviral delivery. With favorable on target outcomes compared to transcription-based editing in key cell types, good tolerability, and deliverability, ligation-mediated gene editing has the potential to further advance genomic medicine.</p>

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Ligase-mediated programmable genomic integration (L-PGI)

  • 期刊论文

  • 出版模式：

    开放获取

  • 发表日期：

    2025年12月10日

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  • Angela X. Nan,
  • Michael Chickering,
  • Christopher L. Bartolome,
  • Neeta Shadija,
  • Dan Li,
  • Brett J. G. Estes,
  • Jessica Von Stetina,
  • Wei Li,
  • Jason Andresen,
  • Kaivalya Molugu,
  • Ravindra Amunugama,
  • Minggang Fang,
  • Chen Bai,
  • Jie Wang,
  • Davood Norouzi,
  • Jesse C. Cochrane,
  • Jason T. Gatlin,
  • Maike Thamsen Dunyak,
  • Sandeep Kumar,
  • Leonard Chavez,
  • Anmol Seth,
  • Shakked Halperin,
  • Jonathan D. Finn,
  • Jenny Xie

摘要

Since their discovery, CRISPR systems have been repurposed for programmable targeted genomic editing, leading to applications for gene disruption, single base editing, insertion, deletion, and manipulation of short genomic sequences. Pairing Cas9 nickase with reverse transcriptase allows applications for insertion, substitution, and deletion of short genomic sequences from an RNA template without generating double stranded breaks however this technology typically shows reduced efficacy in post mitotic cells, limiting its translatability in vivo. Here we present a novel, ligase-based method that addresses these limiations. We introduce edits through delivery and ligation of a synthetic DNA donor to genomic nicks created with Cas9 nickase and report editing activity in cell lines, primary cell cultures, and adult mice via nonviral delivery. With favorable on target outcomes compared to transcription-based editing in key cell types, good tolerability, and deliverability, ligation-mediated gene editing has the potential to further advance genomic medicine.