Researchers at the Agricultural Genomics Institute at Shenzhen, Chinese Academy of Agricultural Sciences (CAAS), have engineered a high-fidelity adenine base editor variant, ABE8e^Y149V, to overcome the severe genome-wide off-target risks associated with the hyperactive ABE8e. Through comprehensive genome-wide off-target analysis by two-cell embryo injection (GOTI), the team first revealed that ABE8e induces massive sgRNA-independent single-nucleotide variants (SNVs), nearly 30-fold higher than spontaneous background levels. By performing systematic saturated mutagenesis on the critical amino acids of the TadA8e deaminase, they identified the Y149V mutation. From a structural perspective, replacing the tyrosine at the entrance of the catalytic pocket with a hydrophobic valine cleverly weakens the non-specific binding to the non-target single-stranded DNA. Functionally, ABE8e^Y149V maintains exceptionally high on-target editing efficiency while strictly narrowing the editing window to A3–A7, reducing both genome-wide DNA and transcriptome-wide RNA off-target mutations to absolute background levels.
The study further demonstrates the extraordinary platform compatibility and therapeutic potential of ABE8e^Y149V. It integrates seamlessly with various Cas homologs, such as SpRY, SaKKH, and IscB, to significantly expand the targeting scope, successfully correcting pathogenic mutations associated with thalassemia, cardiomyopathy, and Dravet syndrome in human cell lines with remarkable precision. To validate its in vivo therapeutic efficacy, the team delivered ABE8e^Y149V via a dual-AAV system into a mouse model of hereditary tyrosinemia type I (HTI), a lethal metabolic liver disease. The treatment precisely disrupted the start codon of the Hpd gene, fully restoring liver function, normalizing weight gain, and rescuing the lethal phenotype without the need for the life-saving drug nitisinone. This high-fidelity optimization successfully bridges the gap between highly efficient laboratory editing and safe in vivo applications, offering a robust and reliable platform for the clinical translation of gene therapies.
DOI：10.1093/procel/pwag006