Orthogonal Cas9-Cas9 chimeras provide a versatile platform for genome editing.

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.