Nuñez James K, Harrington Lucas B, Doudna Jennifer A
Department of Molecular and Cell Biology, University of California, Berkeley , Berkeley, California 94720, United States.
Department of Chemistry, Howard Hughes Medical Institute, Innovative Genomics Institute, Center for RNA Systems Biology, University of California, Berkeley , Berkeley, California 94720, United States.
ACS Chem Biol. 2016 Mar 18;11(3):681-8. doi: 10.1021/acschembio.5b01019. Epub 2016 Feb 9.
The application of the CRISPR-Cas9 system for genome engineering has revolutionized the ability to interrogate genomes of mammalian cells. Programming the Cas9 endonuclease to induce DNA breaks at specified sites is achieved by simply modifying the sequence of its cognate guide RNA. Although Cas9-mediated genome editing has been shown to be highly specific, cleavage events at off-target sites have also been reported. Minimizing, and eventually abolishing, unwanted off-target cleavage remains a major goal of the CRISPR-Cas9 technology before its implementation for therapeutic use. Recent efforts have turned to chemical biology and biophysical approaches to engineer inducible genome editing systems for controlling Cas9 activity at the transcriptional and protein levels. Here, we review recent advancements to modulate Cas9-mediated genome editing by engineering split-Cas9 constructs, inteins, small molecules, protein-based dimerizing domains, and light-inducible systems.
CRISPR-Cas9系统在基因组工程中的应用彻底改变了对哺乳动物细胞基因组进行研究的能力。通过简单修改其同源引导RNA的序列，即可对Cas9核酸内切酶进行编程，使其在特定位点诱导DNA断裂。尽管Cas9介导的基因组编辑已被证明具有高度特异性，但也有报道称存在脱靶位点的切割事件。在将CRISPR-Cas9技术用于治疗之前，尽量减少并最终消除不必要的脱靶切割仍是该技术的主要目标。最近的研究致力于化学生物学和生物物理方法，以构建可诱导的基因组编辑系统，从而在转录和蛋白质水平上控制Cas9的活性。在此，我们综述了通过构建分裂Cas9结构、内含肽、小分子、基于蛋白质的二聚化结构域和光诱导系统来调节Cas9介导的基因组编辑的最新进展。
