CABE-RY: A PAM-flexible dual-mutation base editor for reliable modeling of multi-nucleotide variants

Multi-nucleotide variants (MNVs) represent an important type of genetic variation and have biological and clinical significance. To simulate MNVs, we designed four dual-mutation base editors combining hA3A(Y130F), TadA8e(V106W), and protospacer adjacent motif (PAM)-flexible SpRY and selected cytosine and adenine base editor-SpRY (CABE-RY), which had the best editing performance, for further study. Characterization and comparison showed that CABE-RY had a smaller DNA editing window and lower RNA off-target edits than the corresponding single base editors. Thus, we have established a versatile tool to efficiently simulate MNVs over the genome, which could be very useful for functional studies on MNVs in humans. [Display omitted] Tao et al. established a new dual-mutation base editor (CABE-RY) to efficiently simulate multi-nucleotide variants (MNVs). Further characterization and comparison showed that CABE-RY had a smaller DNA editing window and lower RNA off-target edits than the corresponding single base editors, providing a new tool for functional studies on MNVs in humans.