Directed evolution using dCas9-targeted somatic hypermutation in mammalian cells

• Published in: Nature methods Vol. 13; no. 12; pp. 1036 - 1042
• Main Authors: Hess, Gaelen T, Frésard, Laure, Han, Kyuho, Lee, Cameron H, Li, Amy, Cimprich, Karlene A, Montgomery, Stephen B, Bassik, Michael C
• Format: Journal Article
• Language: English
• Published: New York Nature Publishing Group US 01.12.2016; Nature Publishing Group
• Subjects: 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
• DOI: 10.1038/nmeth.4038

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.