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Title: Directed evolution using dCas9-targeted somatic hypermutation in mammalian cells.

Authors: Hess, Gaelen T; Frésard, Laure; Han, Kyuho; Lee, Cameron H; Li, Amy; Cimprich, Karlene A; Montgomery, Stephen B; Bassik, Michael C

Published In Nat Methods, (2016 Dec)

Abstract: Engineering and study of protein function by directed evolution has been limited by the technical requirement to use global mutagenesis or introduce DNA libraries. Here, we develop CRISPR-X, a strategy to repurpose the somatic hypermutation machinery for protein engineering in situ. Using catalytically inactive dCas9 to recruit variants of cytidine deaminase (AID) with MS2-modified sgRNAs, we can specifically mutagenize endogenous targets with limited off-target damage. This generates diverse libraries of localized point mutations and can target multiple genomic locations simultaneously. We mutagenize GFP and select for spectrum-shifted variants, including EGFP. Additionally, we mutate the target of the cancer therapeutic bortezomib, PSMB5, and identify known and novel mutations that confer bortezomib resistance. Finally, using a hyperactive AID variant, we mutagenize loci both upstream and downstream of transcriptional start sites. These experiments illustrate a powerful approach to create complex libraries of genetic variants in native context, which is broadly applicable to investigate and improve protein function.

PubMed ID: 27798611 Exiting the NIEHS site

MeSH Terms: Bortezomib/pharmacology; CRISPR-Associated Proteins/genetics*; Clustered Regularly Interspaced Short Palindromic Repeats/genetics*; Cytidine Deaminase/genetics; Directed Molecular Evolution/methods*; Drug Resistance/genetics; Green Fluorescent Proteins/genetics; Humans; K562 Cells; Levivirus/genetics; Point Mutation*; Proteasome Endopeptidase Complex/genetics; Protein Engineering/methods*; RNA, Guide, CRISPR-Cas Systems/genetics*

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