Hess Gaelen T, Frésard Laure, Han Kyuho, Lee Cameron H, Li Amy, Cimprich Karlene A, Montgomery Stephen B, Bassik Michael C
Department of Genetics, Stanford University, Stanford, California, USA.
Department of Pathology, Stanford University, Stanford, California, USA.
Nat Methods. 2016 Dec;13(12):1036-1042. doi: 10.1038/nmeth.4038. Epub 2016 Oct 31.
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.
通过定向进化对蛋白质功能进行工程改造和研究一直受到使用全局诱变或引入DNA文库的技术要求的限制。在此,我们开发了CRISPR-X,这是一种将体细胞超突变机制重新用于原位蛋白质工程的策略。使用催化失活的dCas9与MS2修饰的sgRNA招募胞苷脱氨酶(AID)变体,我们可以特异性地诱变内源性靶点,同时减少脱靶损伤。这产生了多样化的局部点突变文库,并可同时靶向多个基因组位置。我们诱变绿色荧光蛋白(GFP)并筛选光谱偏移变体,包括增强型绿色荧光蛋白(EGFP)。此外,我们对癌症治疗药物硼替佐米的靶点PSMB5进行诱变,并鉴定出赋予硼替佐米抗性的已知和新突变。最后,使用一种高活性AID变体,我们对转录起始位点上游和下游的基因座进行诱变。这些实验说明了一种在天然环境中创建复杂遗传变异文库的强大方法,该方法广泛适用于研究和改善蛋白质功能。