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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Vydáno v:	Nature methods Ročník 13; číslo 12; s. 1036 - 1042
Hlavní autoři:	Hess, Gaelen T, Frésard, Laure, Han, Kyuho, Lee, Cameron H, Li, Amy, Cimprich, Karlene A, Montgomery, Stephen B, Bassik, Michael C
Médium:	Journal Article
Jazyk:	angličtina
Vydáno:	New York Nature Publishing Group US 01.12.2016 Nature Publishing Group
Témata:	631/208 631/61 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
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Shrnutí:	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.
Bibliografie:	ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 14 content type line 23
ISSN:	1548-7091 1548-7105 1548-7105
DOI:	10.1038/nmeth.4038