Development of Multiplexed Orthogonal Base Editor (MOBE) systems

Base editors (BEs) enable efficient, programmable installation of point mutations while avoiding the use of double-strand breaks. Simultaneous application of two or more different BEs, such as an ABE and a CBE, is not feasible because gRNA crosstalk results in non-orthogonal editing, with all BEs modifying all target loci. Here, we engineer CBEs and ABEs that can be orthogonally multiplexed by using RNA aptamer/coat protein systems to recruit the DNA modifying enzymes directly to the gRNAs. We generate four multiplexed orthogonal base editor (MOBE) systems that enable rates of co-occurring edits of up to 7.1% without enrichment or selection strategies. Addition of a fluorescent enrichment strategy increases co-occurring editing rates up to 24.8% in HEK293T cells. These systems are compatible with expanded-PAM and high-fidelity Cas9 variants, function well in multiple cell types, have equivalent or reduced off-target propensities compared to their parental systems, and can model disease-relevant point mutation combinations.