A prime editor mouse to model a broad spectrum of somatic mutations in vivo

Genetically engineered mouse models only capture a small fraction of the genetic lesions that drive human cancer. Current CRISPR–Cas9 models can expand this fraction but are limited by their reliance on error-prone DNA repair. Here we develop a system for in vivo prime editing by encoding a Cre-indu...

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Published in	Nature biotechnology Vol. 42; no. 3; pp. 424 - 436
Main Authors	Ely, Zackery A., Mathey-Andrews, Nicolas, Naranjo, Santiago, Gould, Samuel I., Mercer, Kim L., Newby, Gregory A., Cabana, Christina M., Rideout, William M., Jaramillo, Grissel Cervantes, Khirallah, Jennifer M., Holland, Katie, Randolph, Peyton B., Freed-Pastor, William A., Davis, Jessie R., Kulstad, Zachary, Westcott, Peter M. K., Lin, Lin, Anzalone, Andrew V., Horton, Brendan L., Pattada, Nimisha B., Shanahan, Sean-Luc, Ye, Zhongfeng, Spranger, Stefani, Xu, Qiaobing, Sánchez-Rivera, Francisco J., Liu, David R., Jacks, Tyler
Format	Journal Article
Language	English
Published	New York Nature Publishing Group US 01.03.2024 Nature Publishing Group
Subjects	631/1647/1511 631/67/68 631/67/70 Agriculture Animal models Animals Bioinformatics Biomedical and Life Sciences Biomedical Engineering/Biotechnology Biomedicine Biotechnology Cancer Cell Line CRISPR CRISPR-Cas Systems - genetics DNA repair Drug resistance Editing Gene Editing Genetic engineering Humans In vivo methods and tests Life Sciences Lipids Lung cancer Mice Mice, Transgenic Mutation Mutation - genetics Mutation hot spots Nanoparticles Organoids Pancreatic cancer Pancreatic Neoplasms - genetics RNA, Guide, CRISPR-Cas Systems Transplantation
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ISSN	1087-0156 1546-1696 1546-1696
DOI	10.1038/s41587-023-01783-y

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Summary:	Genetically engineered mouse models only capture a small fraction of the genetic lesions that drive human cancer. Current CRISPR–Cas9 models can expand this fraction but are limited by their reliance on error-prone DNA repair. Here we develop a system for in vivo prime editing by encoding a Cre-inducible prime editor in the mouse germline. This model allows rapid, precise engineering of a wide range of mutations in cell lines and organoids derived from primary tissues, including a clinically relevant Kras mutation associated with drug resistance and Trp53 hotspot mutations commonly observed in pancreatic cancer. With this system, we demonstrate somatic prime editing in vivo using lipid nanoparticles, and we model lung and pancreatic cancer through viral delivery of prime editing guide RNAs or orthotopic transplantation of prime-edited organoids. We believe that this approach will accelerate functional studies of cancer-associated mutations and complex genetic combinations that are challenging to construct with traditional models. Prime-editing mouse models enable the study of specific cancer mutations in vivo.
Bibliography:	ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 14 content type line 23 These authors contributed equally. Z.A.E. and T.J. conceived of the study. Z.A.E., N.M.A., and T.J. designed the experiments. S.N. and Z.A.E. designed and constructed all DNA vectors described, including the transgene cassette. N.M.A. conducted all autochthonous lung modeling. Z.A.E. designed pegRNAs, with contributions from N.M.A., P.B.R., J.R.D., K.H., and G.A.N. S.I.G. and F.J.S.R. designed the computational pipeline to analyze patient mutation data, and S.I.G. conducted related bioinformatics analyses. W.A.F.-P., G.C.J., and Z.K. conducted pancreatic orthotopic transplant experiments. L.L and G.C.J. conducted autochthonous pancreatic modeling. J.M.K., B.L.H., G.A.N., and N.M.A. conducted LNP experiments. C.M.C. and S.N. conducted experiments involving lung organoids. N.M.A., Z.A.E., N.B.P., and W.A.F.-P. conducted in vitro experiments using pancreatic organoids. W.M.R. conducted mESC targeting and chimera generation. K.L.M. provided animal husbandry expertise and conceptual advice. S.N., G.A.N., P.B.R., A.V.A., W.A.F.-P., P.M.K.W., K.L.M., S.L.S., F.J.S.R., and D.R.L. provided conceptual advice. Z.A.E., N.M.A., S.I.G., F.J.S.R., and T.J. wrote the manuscript with input from all authors. Author contributions
ISSN:	1087-0156 1546-1696 1546-1696
DOI:	10.1038/s41587-023-01783-y