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Nat Methods. 2016 Dec;13(12):1036-1042. doi: 10.1038/nmeth.4038. Epub 2016 Oct 31.

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

Author information

1
Department of Genetics, Stanford University, Stanford, California, USA.
2
Department of Pathology, Stanford University, Stanford, California, USA.
3
Department of Chemical and Systems Biology, Stanford University, Stanford, California, USA.
4
Stanford University Chemistry, Engineering, and Medicine for Human Health (ChEM-H), Stanford, California, USA.

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.

PMID:
27798611
PMCID:
PMC5557288
DOI:
10.1038/nmeth.4038
[Indexed for MEDLINE]
Free PMC Article

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