UC San Diego

Base editors (BEs) enable the programmable installation of point mutations with high efficiency and precision while avoiding the use of double-strand breaks. Two major classes of BEs exist that convert C•G base pairs to T•A (CBEs) and A•T base pairs to G•C (ABEs). Although these current tools are in theory well-suited for multiplexing, in practice researchers are limited to multiplexing with a single type of BE due to the covalent fusion of the DNA modifying enzyme to the Cas9 effector causing “gRNA crosstalk”, or non-orthogonal editing. Here, we engineer and characterize aptamer-derived CBEs and ABEs that can be multiplexed with each other orthogonally. We generated four multiplexed orthogonal base editor (MOBE) systems that enable rates of co-occurring edits of up to 7.1% without enrichment or selection strategies. We also develop a simple fluorescent enrichment strategy, which increased co-occurring edit rates up to 24.8% in HEK293T cells. We show that 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 be used to model disease-relevant point mutation combinations. These MOBE systems are a valuable addition to the genome editing toolbox, and will aid significantly in efforts to study/model or correct combinations of genetic variants, such as those found in haplotypes, polygenic disorders, and driver-passenger mutation pairs.