A parts list of promoters and gRNA scaffolds for mammalian genome engineering and molecular recording
A standardized 'parts list' of sequences for genetic engineering of microbes has been indispensable to progress in synthetic biology, but few analogous parts exist for mammalian systems. Here we design libraries of extant, ancestral, mutagenized or miniaturized variants of polymerase III promoters and guide RNA (gRNA) scaffolds and quantify their abilities to mediate precise edits to the mammalian genome through multiplex prime editing. We identify thousands of parts for reproducible editing in human and mouse cell lines, including hundreds with greater activity than commonly used sequences. Saturation mutagenesis screens identify tolerated sequence variants that further enhance sequence diversity. In an application to molecular recording, we design a 'ten key' array that, in mammalian cells, achieves balanced activity of pegRNAs as predicted by the activity of the component parts. The data reported here will aid the design of synthetic loci encoding arrays of gRNAs exhibiting predictable, differentiated levels of activity for applications in multiplexed perturbation, biological recorders and complex genetic circuits.
Authors
- Troy A. McDiarmid (ORCID: https://orcid.org/0000-0002-4893-9733)
- Alessandro Luis Venega Coradini (ORCID: https://orcid.org/0000-0001-7923-9994)
- Beth Martin (ORCID: https://orcid.org/0000-0002-9661-014X)
- Jean‐Benoît Lalanne (ORCID: https://orcid.org/0000-0001-8753-0669)
- Florence M. Chardon (ORCID: https://orcid.org/0000-0001-8858-989X)
- Tony Li (ORCID: https://orcid.org/0000-0002-1234-1334)
- Hanna Liao (ORCID: https://orcid.org/0000-0003-4890-7990)
- Megan L. Taylor (ORCID: https://orcid.org/0009-0002-2511-1498)
- Sudarshan Pinglay (ORCID: https://orcid.org/0000-0002-8781-1476)
- Jenny F. Nathans (ORCID: https://orcid.org/0000-0002-0276-734X)
- Junhong Choi (ORCID: https://orcid.org/0000-0001-9291-5977)
- Jay Shendure (ORCID: https://orcid.org/0000-0002-1516-1865)
- Wei Chen (ORCID: https://orcid.org/0000-0002-5255-4166)
- Xiaoyi Li (ORCID: https://orcid.org/0000-0001-6433-5262)
- Haedong Kim (ORCID: https://orcid.org/0000-0003-3589-0042)
- Jesse M. Gray (ORCID: https://orcid.org/0000-0003-4560-8813)
- Jordan Knuth (ORCID: https://orcid.org/0009-0007-0176-7093)
Institutions
- Memorial Sloan Kettering Cancer Center (US)
- Howard Hughes Medical Institute (US)
- University of Washington (US)
- Lineage Cell Therapeutics (United States) (US)
- Brotman Baty Institute (US)
- Allen Institute (US)
- Seattle University (US)
Publication Details
- Journal
- Nature Biotechnology
- Published
- 2025-11-11
- DOI
- https://doi.org/10.1038/s41587-025-02896-2
- Citations
- 2
- Primary Topic
- CRISPR and Genetic Engineering
- Type
- article
- Field-Weighted Citation Impact
- 1.61
Funders
- Damon Runyon Cancer Research Foundation
- Washington Research Foundation
- University of Washington
- National Institutes of Health
- Natural Sciences and Engineering Research Council of Canada