title: Directed evolution using dCas9-targeted somatic hypermutation in mammalian cells.
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.
authors: Department of Genetics, Stanford University, Stanford, California, USA.
authors: Gaelen T
authors: GT
authors: Hess
authors: Department of Pathology, Stanford University, Stanford, California, USA.
authors: Laure
authors: L
authors: Frésard
authors: Department of Genetics, Stanford University, Stanford, California, USA.
authors: Kyuho
authors: K
authors: Han
authors: Department of Genetics, Stanford University, Stanford, California, USA.
authors: Cameron H
authors: CH
authors: Lee
authors: Department of Genetics, Stanford University, Stanford, California, USA.
authors: Amy
authors: A
authors: Li
authors: Department of Chemical and Systems Biology, Stanford University, Stanford, California, USA.
authors: Karlene A
authors: KA
authors: Cimprich
authors: Department of Genetics, Stanford University, Stanford, California, USA.|Department of Pathology, Stanford University, Stanford, California, USA.
authors: Stephen B
authors: SB
authors: Montgomery
authors: Department of Genetics, Stanford University, Stanford, California, USA.|Stanford University Chemistry, Engineering, and Medicine for Human Health (ChEM-H), Stanford, California, USA.
authors: Michael C
authors: MC
authors: Bassik
mesh_terms: D000069286:Bortezomib / Q000494:pharmacology; D064130:CRISPR-Associated Proteins / Q000235:genetics*; D064112:Clustered Regularly Interspaced Short Palindromic Repeats / Q000235:genetics*; D003564:Cytidine Deaminase / Q000235:genetics; D019020:Directed Molecular Evolution / Q000379:methods*; D004351:Drug Resistance / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000235:genetics; D006801:Humans; D020014:K562 Cells; D017909:Levivirus / Q000235:genetics; D017354:Point Mutation; D046988:Proteasome Endopeptidase Complex / Q000235:genetics; D015202:Protein Engineering / Q000379:methods*; D000094704:RNA, Guide, CRISPR-Cas Systems / Q000235:genetics*
chemical_list: D064130:CRISPR-Associated Proteins; D000094704:RNA, Guide, CRISPR-Cas Systems; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D000069286:Bortezomib; C088874:PSMB5 protein, human; D046988:Proteasome Endopeptidase Complex; D003564:Cytidine Deaminase