Engineering and study of protein function by directed evolution has been limited by the requirement to introduce DNA libraries of defined size or to use global mutagenesis. Here, we develop a strategy to repurpose the somatic hypermutation machinery used in antibody affinity maturation to efficiently perform protein engineering in situ. Using catalytically inactive Cas9 (dCas9) to recruit variants of the deaminase AID (CRISPR-X), we can specifically mutagenize endogenous targets with limited off-target damage. This generates diverse libraries of localized point mutations, in contrast to insertions and deletions created by active Cas9, and can be used to mutagenize multiple genomic locations simultaneously. With this technology, we mutagenize GFP and select for spectrum-shifted variants, including EGFP. In addition, we mutate the target of the cancer therapeutic bortezomib, PSMB5, and identify known and novel mutations that confer resistance to treatment. Finally, we utilize a hyperactive AID variant with dramatically increased activity to mutagenize endogenous loci both upstream and downstream of transcriptional start sites. These experiments illustrate a powerful new approach to create highly complex libraries of genetic variants in native context, which can be broadly applied to investigate and improve protein function.
展开▼
