Inducible, split base editors for in vivo cancer functional genomics
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.
Authors
- Joseph P. Milazzo (ORCID: https://orcid.org/0009-0000-8320-4794)
- Yemin Lan (ORCID: https://orcid.org/0000-0003-3455-3618)
- Rahul M. Kohli (ORCID: https://orcid.org/0000-0002-7689-5678)
- Yuqiao Liu (ORCID: https://orcid.org/0000-0001-8868-9022)
- Christopher R. Vakoc (ORCID: https://orcid.org/0000-0002-1158-7180)
- Ruchi Lohia
- Shangshang Wang (ORCID: https://orcid.org/0000-0001-7806-0415)
- Evan W. Weber (ORCID: https://orcid.org/0000-0001-5039-2899)
- Yugong Ho (ORCID: https://orcid.org/0000-0002-7642-7700)
- S Zhang
- Diqiu Ren
- Kiara N. Berríos (ORCID: https://orcid.org/0000-0002-0684-5413)
- Robert Hapke (ORCID: https://orcid.org/0000-0002-3827-7772)
- Aktan Alpsoy (ORCID: https://orcid.org/0000-0002-8940-8277)
- Keisuke Yamada (ORCID: https://orcid.org/0000-0003-1256-9299)
- Andy J. Minn (ORCID: https://orcid.org/0000-0003-1418-0906)
- Junwei Shi
- Qin Li
- Yongjun Li (ORCID: https://orcid.org/0000-0002-3618-6041)
- Canjing Zhang
Institutions
- University City Science Center (US)
- Memorial Sloan Kettering Cancer Center (US)
- Children's Hospital of Philadelphia (US)
- Cancer Research Institute (US)
- Cold Spring Harbor Laboratory (US)
- Foundation Center (US)
- UPMC Hillman Cancer Center (US)
- University of Pennsylvania (US)
Publication Details
- Journal
- Nature Biotechnology
- Published
- 2026-04-15
- DOI
- https://doi.org/10.1038/s41587-026-03077-5
- Primary Topic
- CRISPR and Genetic Engineering
- Type
- article
- Field-Weighted Citation Impact
- 0.00
Funders
- St. Jude Children's Research Hospital
- Mark Foundation For Cancer Research
- Toyota Physical and Chemical Research Institute
- National Institutes of Health
- National Cancer Institute
- National Institute of General Medical Sciences