Department of Molecular, Cell and Cancer Biology, University of Massachusetts Medical School, Worcester, MA, USA.
Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, MA, USA.
Nat Commun. 2018 Nov 19;9(1):4856. doi: 10.1038/s41467-018-07310-x.
The development of robust, versatile and accurate toolsets is critical to facilitate therapeutic genome editing applications. Here we establish RNA-programmable Cas9-Cas9 chimeras, in single- and dual-nuclease formats, as versatile genome engineering systems. In both of these formats, Cas9-Cas9 fusions display an expanded targeting repertoire and achieve highly specific genome editing. Dual-nuclease Cas9-Cas9 chimeras have distinct advantages over monomeric Cas9s including higher target site activity and the generation of predictable precise deletion products between their target sites. At a therapeutically relevant site within the BCL11A erythroid enhancer, Cas9-Cas9 nucleases produced precise deletions that comprised up to 97% of all sequence alterations. Thus Cas9-Cas9 chimeras represent an important tool that could be particularly valuable for therapeutic genome editing applications where a precise cleavage position and defined sequence end products are desirable.
开发强大、多功能且精确的工具集对于促进治疗性基因组编辑应用至关重要。在这里,我们建立了 RNA 可编程的 Cas9-Cas9 嵌合体,包括单酶和双酶两种形式,作为多功能基因组工程系统。在这两种形式中,Cas9-Cas9 融合体显示出扩展的靶向谱,并实现了高度特异性的基因组编辑。与单体 Cas9 相比,双酶 Cas9-Cas9 嵌合体具有明显的优势,包括更高的靶位点活性和在靶位点之间产生可预测的精确缺失产物。在 BCL11A 红细胞增强子内的一个治疗相关的位点上,Cas9-Cas9 核酸酶产生了精确的缺失,其中多达 97%的序列改变。因此,Cas9-Cas9 嵌合体代表了一种重要的工具,对于治疗性基因组编辑应用特别有价值,在这些应用中需要精确的切割位置和定义的序列末端产物。