Directed evolution using dCas9-targeted somatic hypermutation in mammalian cells

Recruiting a hyperactive cytidine deaminase via the guide RNA to dCas9 allows for the introduction of diverse point mutations at the CRISPR target locus to create complex libraries of variants for protein engineering or dissection of protein function. Engineering and study of protein function by dir...

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Veröffentlicht in:Nature methods Jg. 13; H. 12; S. 1036 - 1042
Hauptverfasser: Hess, Gaelen T, Frésard, Laure, Han, Kyuho, Lee, Cameron H, Li, Amy, Cimprich, Karlene A, Montgomery, Stephen B, Bassik, Michael C
Format: Journal Article
Sprache:Englisch
Veröffentlicht: New York Nature Publishing Group US 01.12.2016
Nature Publishing Group
Schlagworte:
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Analysis
Bioinformatics
Biological Microscopy
Biological Techniques
Biomedical Engineering/Biotechnology
Bortezomib - pharmacology
Cells
Clustered Regularly Interspaced Short Palindromic Repeats - genetics
CRISPR-Associated Proteins - genetics
Cytidine Deaminase - genetics
Deoxyribonucleic acid
Directed Molecular Evolution - methods
DNA
Drug Resistance - genetics
Evolutionary biology
Gene mutation
Genetic variance
Green Fluorescent Proteins - genetics
Health aspects
Humans
K562 Cells
Levivirus - genetics
Life Sciences
Mammals
Mutation
Point Mutation
Proteasome Endopeptidase Complex - genetics
Protein Engineering - methods
Proteins
Proteomics
RNA, Guide, CRISPR-Cas Systems - genetics
Tissue engineering
Transcription (Genetics)
United States
ISSN:1548-7091, 1548-7105, 1548-7105
Online-Zugang:Volltext
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Zusammenfassung:Recruiting a hyperactive cytidine deaminase via the guide RNA to dCas9 allows for the introduction of diverse point mutations at the CRISPR target locus to create complex libraries of variants for protein engineering or dissection of protein function. 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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ISSN:1548-7091
1548-7105
1548-7105
DOI:10.1038/nmeth.4038