The DOI matches its Crossref record; 17 of the 17 references it cites were found in a registry or an index.
Directed evolution using dCas9-targeted somatic hypermutation in mammalian cells
Open accessAbstract
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.
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Cites
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Self-citation
Cited by
Genome editing with CRISPR–Cas nucleases, base editors, transposases and prime editors
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Metadata
- Authors
- Hess, G. T., Frésard, L., Han, K., Lee, C. H., Li, A., Cimprich, K. A., Montgomery, S. B., & Bassik, M. C.
- Venue
- Nature Methods
- Published
- 2016-10-31
- Volume
- 13
- Issue
- 12
- Pages
- 1036-1042
- ISSN
- 1548-7091 (ISSN-L)
- 1548-7105
- OpenAlex
- W2542621333
- Cited by
- 476
- References
- 17
- FWCI
- 26.03
- Type
- journal article
- Language
- en
- Open access
- green
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