Directed evolution using dCas9-targeted somatic hypermutation in mammalian cells

Nat Methods. 2016 Dec;13(12):1036-1042. doi: 10.1038/nmeth.4038. Epub 2016 Oct 31.

Abstract

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.

MeSH terms

  • Bortezomib / pharmacology
  • CRISPR-Associated Proteins / genetics*
  • Clustered Regularly Interspaced Short Palindromic Repeats / genetics*
  • Cytidine Deaminase / genetics
  • Directed Molecular Evolution / methods*
  • Drug Resistance / genetics
  • Green Fluorescent Proteins / genetics
  • Humans
  • K562 Cells
  • Levivirus / genetics
  • Point Mutation*
  • Proteasome Endopeptidase Complex / genetics
  • Protein Engineering / methods*
  • RNA, Guide / genetics*

Substances

  • CRISPR-Associated Proteins
  • RNA, Guide
  • enhanced green fluorescent protein
  • Green Fluorescent Proteins
  • Bortezomib
  • PSMB5 protein, human
  • Proteasome Endopeptidase Complex
  • Cytidine Deaminase