High-throughput functional evaluation of human cancer-associated mutations using base editors

Comprehensive phenotypic characterization of the many mutations found in cancer tissues is one of the biggest challenges in cancer genomics. In this study, we evaluated the functional effects of 29,060 cancer-related transition mutations that result in protein variants on the survival and proliferat...

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Bibliographic Details
Published in:	Nature biotechnology Vol. 40; no. 6; pp. 874 - 884
Main Authors:	Kim, Younggwang, Lee, Seungho, Cho, Soohyuk, Park, Jinman, Chae, Dongwoo, Park, Taeyoung, Minna, John D., Kim, Hyongbum Henry
Format:	Journal Article
Language:	English
Published:	New York Nature Publishing Group US 01-06-2022 Nature Publishing Group
Subjects:	631/1647/1513/1967/3196 631/208/68 Adenine Agriculture Bioinformatics Biomedical and Life Sciences Biomedical Engineering/Biotechnology Biomedicine Biotechnology Cancer Cell proliferation CRISPR-Cas Systems Cytosine Editing Evaluation Gene Editing Humans Life Sciences Mutation Mutation - genetics Neoplasms - genetics Phenotypes Proteins RNA, Guide, CRISPR-Cas Systems Tumors
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Summary:	Comprehensive phenotypic characterization of the many mutations found in cancer tissues is one of the biggest challenges in cancer genomics. In this study, we evaluated the functional effects of 29,060 cancer-related transition mutations that result in protein variants on the survival and proliferation of non-tumorigenic lung cells using cytosine and adenine base editors and single guide RNA (sgRNA) libraries. By monitoring base editing efficiencies and outcomes using surrogate target sequences paired with sgRNA-encoding sequences on the lentiviral delivery construct, we identified sgRNAs that induced a single primary protein variant per sgRNA, enabling linking those mutations to the cellular phenotypes caused by base editing. The functions of the vast majority of the protein variants (28,458 variants, 98%) were classified as neutral or likely neutral; only 18 (0.06%) and 157 (0.5%) variants caused outgrowing and likely outgrowing phenotypes, respectively. We expect that our approach can be extended to more variants of unknown significance and other tumor types. Cancer-associated variants of unknown significance are identified using base editing.
Bibliography:	ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23 Y.K., S.L. and H.H.K. conceived and designed the study. Y.K. and S.L. performed most of the experiments. J.P. critically contributed to computational analysis. S.C. critically assisted in the wet experiments. Y.K. and S.L. analyzed the data based on comments of H.H.K. J.D.M. generated and provided HBEC30KT-shTP53 cells (P cells). D.C. and T.P. contributed to the mathematical analysis (Supplementary Note 2). Y.K. and H.H.K. wrote the manuscript with input from all authors. Author contributions
ISSN:	1087-0156 1546-1696
DOI:	10.1038/s41587-022-01276-4