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<p>CRISPR-associated (Cas) transposases (CAST) are RNA-guided systems capable of programmable integration of large segments of DNA without creating double-strand breaks. Engineered Cascade CAST function in human cells but are challenging to deploy due to the complexity of the targeting components. Unlike Cascade, which require three Cas proteins, type V-K CAST require a single Cas12k effector for targeting. Here, we show that compact type V-K CAST from uncultivated microbes are repurposable for programmable DNA integration into the genome of human cells. Engineering for nuclear localization and function enables integration of a therapeutically relevant transgene at a safe-harbor site in multiple human cell types. Notably, off-targets are rare events reproducibly found in specific genomic regions. These CAST advancements are expected to accelerate applications of genome editing to therapeutic development, biotechnology, and synthetic biology.</p>

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Integration of therapeutic cargo into the human genome with programmable type V-K CAST

  • 期刊论文

  • 出版模式：

    开放获取

  • 发表日期：

    2025年3月13日

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  • Jason Liu,
  • Daniela S. Aliaga Goltsman,
  • Lisa M. Alexander,
  • Khak Khak Khayi,
  • Jennifer H. Hong,
  • Drew T. Dunham,
  • Christine A. Romano,
  • Morayma M. Temoche-Diaz,
  • Shailaja Chadha,
  • Rodrigo Fregoso Ocampo,
  • Jennifer Oki-O’Connell,
  • Owen P. Janson,
  • Keirstinne Turcios,
  • Liliana Gonzalez-Osorio,
  • Jared Muysson,
  • Jenat Rahman,
  • Sarah M. Laperriere,
  • Audra E. Devoto,
  • Cindy J. Castelle,
  • Cristina N. Butterfield,
  • Gregory J. Cost,
  • Christopher T. Brown,
  • Brian C. Thomas

摘要

CRISPR-associated (Cas) transposases (CAST) are RNA-guided systems capable of programmable integration of large segments of DNA without creating double-strand breaks. Engineered Cascade CAST function in human cells but are challenging to deploy due to the complexity of the targeting components. Unlike Cascade, which require three Cas proteins, type V-K CAST require a single Cas12k effector for targeting. Here, we show that compact type V-K CAST from uncultivated microbes are repurposable for programmable DNA integration into the genome of human cells. Engineering for nuclear localization and function enables integration of a therapeutically relevant transgene at a safe-harbor site in multiple human cell types. Notably, off-targets are rare events reproducibly found in specific genomic regions. These CAST advancements are expected to accelerate applications of genome editing to therapeutic development, biotechnology, and synthetic biology.