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High-throughput evaluation of genetic variants with prime editing sensor libraries.
Gould, Samuel I; Wuest, Alexandra N; Dong, Kexin; Johnson, Grace A; Hsu, Alvin; Narendra, Varun K; Atwa, Ondine; Levine, Stuart S; Liu, David R; Sánchez Rivera, Francisco J.
Afiliação
  • Gould SI; Department of Biology, Massachusetts Institute of Technology, Cambridge, MA, USA.
  • Wuest AN; David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA, USA.
  • Dong K; Department of Biology, Massachusetts Institute of Technology, Cambridge, MA, USA.
  • Johnson GA; David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA, USA.
  • Hsu A; David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA, USA.
  • Narendra VK; University of Chinese Academy of Sciences, Beijing, China.
  • Atwa O; Department of Biology, Massachusetts Institute of Technology, Cambridge, MA, USA.
  • Levine SS; David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA, USA.
  • Liu DR; Merkin Institute of Transformative Technologies in Healthcare, Broad Institute of MIT and Harvard, Cambridge, MA, USA.
  • Sánchez Rivera FJ; Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA, USA.
Nat Biotechnol ; 2024 Mar 12.
Article em En | MEDLINE | ID: mdl-38472508
ABSTRACT
Tumor genomes often harbor a complex spectrum of single nucleotide alterations and chromosomal rearrangements that can perturb protein function. Prime editing has been applied to install and evaluate genetic variants, but previous approaches have been limited by the variable efficiency of prime editing guide RNAs. Here we present a high-throughput prime editing sensor strategy that couples prime editing guide RNAs with synthetic versions of their cognate target sites to quantitatively assess the functional impact of endogenous genetic variants. We screen over 1,000 endogenous cancer-associated variants of TP53-the most frequently mutated gene in cancer-to identify alleles that impact p53 function in mechanistically diverse ways. We find that certain endogenous TP53 variants, particularly those in the p53 oligomerization domain, display opposite phenotypes in exogenous overexpression systems. Our results emphasize the physiological importance of gene dosage in shaping native protein stoichiometry and protein-protein interactions, and establish a framework for studying genetic variants in their endogenous sequence context at scale.

Texto completo: 1 Coleções: 01-internacional Base de dados: MEDLINE Idioma: En Revista: Nat Biotechnol Assunto da revista: BIOTECNOLOGIA Ano de publicação: 2024 Tipo de documento: Article País de afiliação: Estados Unidos

Texto completo: 1 Coleções: 01-internacional Base de dados: MEDLINE Idioma: En Revista: Nat Biotechnol Assunto da revista: BIOTECNOLOGIA Ano de publicação: 2024 Tipo de documento: Article País de afiliação: Estados Unidos