期刊
NATURE METHODS
卷 13, 期 12, 页码 1036-+出版社
NATURE PUBLISHING GROUP
DOI: 10.1038/NMETH.4038
关键词
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资金
- NIH S10 Shared Instrument Grant [S10RR025518-01]
- NIH [T32HG000044, ES016486, R01HG008150, 1DP2HD084069-01]
- CEHG Fellowship
- Walter V. and Idun Berry postdoctoral fellowship
- NSF [DGE-114747]
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 sgRNRNAs, 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.
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