A Molecular Glue Approach to Control the Half-Life of CRISPR-Based Technologies
Cas9 is a programmable nuclease that has furnished transformative technologies, including base editors and transcription modulators (e.g., CRISPRi/a), but several applications of these technologies, including therapeutics, mandatorily require precision control of their half-life. For example, such control can help avert any potential immunological and adverse events in clinical trials. Current genome editing technologies to control the half-life of Cas9 are slow, have lower activity, involve fusion of large response elements (>230 amino acids), utilize expensive controllers with poor pharmacological attributes, and cannot be implemented <i>in vivo</i> on several CRISPR-based technologies. We report a general platform for half-life control using the molecular glue, pomalidomide, that binds to a ubiquitin ligase complex and a response-element bearing CRISPR-based technology, thereby causing the latter's rapid ubiquitination and degradation. Using pomalidomide, we were able to control the half-life of large CRISPR-based technologies (e.g., base editors and CRISPRi) and small anti-CRISPRs that inhibit such technologies, allowing us to build the first examples of on-switch for base editors. The ability to switch on, fine-tune, and switch-off CRISPR-based technologies with pomalidomide allowed complete control over their activity, specificity, and genome editing outcome. Importantly, the miniature size of the response element and favorable pharmacological attributes of the drug pomalidomide allowed control of activity of base editor <i>in vivo</i> using AAV as the delivery vehicle. These studies provide methods and reagents to precisely control the dosage and half-life of CRISPR-based technologies, propelling their therapeutic development.
Authors
- Veronica Kovalcik
- Amit Choudhary (ORCID: https://orcid.org/0000-0002-8437-0150)
- Richard I. Sherwood (ORCID: https://orcid.org/0000-0001-7427-7713)
- Benjamin K. Law
- James D. Fife (ORCID: https://orcid.org/0000-0001-7561-3651)
- Ruilin Tian (ORCID: https://orcid.org/0000-0001-7680-6682)
- Kevin T. Zhao (ORCID: https://orcid.org/0000-0003-3440-2094)
- Vedagopuram Sreekanth (ORCID: https://orcid.org/0000-0002-7065-2675)
- Jessie R. Davis (ORCID: https://orcid.org/0000-0002-6911-0908)
- Donghyun Lim (ORCID: https://orcid.org/0000-0002-6070-198X)
- Max Jan (ORCID: https://orcid.org/0000-0002-7071-5517)
- Sam Barkal
- Michael Vinyard (ORCID: https://orcid.org/0000-0001-8167-1807)
- Basheer Becerra (ORCID: https://orcid.org/0000-0003-4403-7959)
- David R. Liu (ORCID: https://orcid.org/0000-0002-9943-7557)
- Benjamin L. Ebert (ORCID: https://orcid.org/0000-0003-0197-5451)
- Martin Kampmann (ORCID: https://orcid.org/0000-0002-3819-7019)
- Luca Pinello (ORCID: https://orcid.org/0000-0003-1195-9607)
- Marie McConkey (ORCID: https://orcid.org/0000-0002-9500-018X)
- Surached Siriwongsup
Institutions
- Broad Institute (US)
- Brigham and Women's Hospital (US)
- Howard Hughes Medical Institute (US)
- Harvard University (US)
- University of California, San Francisco (US)
- German Center for Neurodegenerative Diseases (DE)
- Harvard University Press (US)
- Massachusetts General Hospital (US)
- Dana-Farber Cancer Institute (US)
- Pediatrics and Genetics (US)
- Center for Cancer Research (US)
Publication Details
- Journal
- Journal of the American Chemical Society
- Published
- 2025-06-30
- DOI
- https://doi.org/10.1021/jacs.5c06230
- Citations
- 5
- Primary Topic
- CRISPR and Genetic Engineering
- Type
- article
- Field-Weighted Citation Impact
- 4.03
Funders
- Howard Hughes Medical Institute
- Defense Advanced Research Projects Agency
- National Human Genome Research Institute
- National Institute of General Medical Sciences
- National Institute of Allergy and Infectious Diseases
- Division of Diabetes, Endocrinology, and Metabolic Diseases