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<p>Cancer functional genomics using CRISPR base editors (BEs) holds great promise for molecular characterization and new target discovery. However, traditional BEs, using intact DNA deaminases as mutators, are often constrained by limited control and nonspecific toxicities. Here we developed a small-molecule-controllable system using split-engineered BEs (seBEs). By placing deaminase activity under small-molecule control, seBEs significantly reduced cellular toxicity and enabled robust and inducible in vivo functional genomics screens. High-density seBE genetic screens using ~11,000 single guide RNAs in vitro and ~3,700 single guide RNAs in vivo reveal known and previously unknown loss-of-function and dominant-negative mutations in cancer therapeutic targets. A deeper tiling seBE screen against <i>Adar1</i>, a key mediator in cancer immunotherapy, reveals critical residues within functional domains that show no phenotype in vitro but distinctively elicit non-cell-autonomous cancer dependencies in vivo. Overall, our seBE system offers a generalizable, controllable and highly efficient method to systematically identify key residues in cancer functional genomics.</p>

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Inducible, split base editors for in vivo cancer functional genomics

  • 期刊论文

  • 出版模式：

    开放获取

  • 发表日期：

    2026年4月15日

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  • Diqiu Ren,
  • Shangshang Wang,
  • Keisuke Yamada,
  • Yuqiao Liu,
  • Robert Hapke,
  • Aktan Alpsoy,
  • Yugong Ho,
  • Canjing Zhang,
  • Yemin Lan,
  • Shuo Zhang,
  • Joseph P. Milazzo,
  • Ruchi Lohia,
  • Kiara N. Berríos,
  • Yongjun Li,
  • Evan W. Weber,
  • Qin Li,
  • Christopher R. Vakoc,
  • Andy J. Minn,
  • Rahul M. Kohli,
  • Junwei Shi

摘要

Cancer functional genomics using CRISPR base editors (BEs) holds great promise for molecular characterization and new target discovery. However, traditional BEs, using intact DNA deaminases as mutators, are often constrained by limited control and nonspecific toxicities. Here we developed a small-molecule-controllable system using split-engineered BEs (seBEs). By placing deaminase activity under small-molecule control, seBEs significantly reduced cellular toxicity and enabled robust and inducible in vivo functional genomics screens. High-density seBE genetic screens using ~11,000 single guide RNAs in vitro and ~3,700 single guide RNAs in vivo reveal known and previously unknown loss-of-function and dominant-negative mutations in cancer therapeutic targets. A deeper tiling seBE screen against Adar1, a key mediator in cancer immunotherapy, reveals critical residues within functional domains that show no phenotype in vitro but distinctively elicit non-cell-autonomous cancer dependencies in vivo. Overall, our seBE system offers a generalizable, controllable and highly efficient method to systematically identify key residues in cancer functional genomics.