A single-residue mutation variant of ABE8e eliminates its prevalent off-target effects in the genome

Genome-wide off-target effect poses a safety risk for clinical use of adenine deaminase base editors (ABEs), among which ABE8e is one of the most efficient. Two-cell embryo injection (GOTI) analysis showed that the rate of genome-wide single nucleotide variants (SNVs) in ABE8e-edited cells was ~30-fold higher than that of spontaneous SNVs in control cells, indicating prevalent off-target effects of ABE8e, but no off-target effect for ABE7.10, from which ABE8e was derived. We performed saturation mutagenesis of eight amino acid sites of the deaminase (TadA8e) within ABE8e and obtained ABE8e^Y149V that exhibited high editing efficiency without detectable off-target effect. Furthermore, TadA8e^Y149V could be fused with other Cas homologs (PAM-relaxed SpRY, hypercompact SaKKH, or IscB) to expand its target range. Finally, ABE8e^Y149V editing of hydroxyphenylpyruvate dioxygenase (Hpd) gene prevented lethality in hereditary tyrosinemia type I mice. The high efficiency and fidelity of ABE8e^Y149V suggest its potential application in ABE-based gene therapies.