PhieABEs: a PAM‐less/free high‐efficiency adenine base editor toolbox with wide target scope in plants

Adenine base editors (ABEs), which are generally engineered adenosine deaminases and Cas variants, introduce site‐specific A‐to‐G mutations for agronomic trait improvement. However, notably varying editing efficiencies, restrictive requirements for protospacer‐adjacent motifs (PAMs) and a narrow editing window greatly limit their application. Here, we developed a robust high‐efficiency ABE (PhieABE) toolbox for plants by fusing an evolved, highly active form of the adenosine deaminase TadA8e and a single‐stranded DNA‐binding domain (DBD), based on PAM‐less/free Streptococcus pyogenes Cas9 (SpCas9) nickase variants that recognize the PAM NGN (for SpCas9n‐NG and SpGn) or NNN (for SpRYn). By targeting 29 representative targets in rice and assessing the results, we demonstrate that PhieABEs have significantly improved base‐editing activity, expanded target range and broader editing windows compared to the ABE7.10 and general ABE8e systems. Among these PhieABEs, hyper ABE8e‐DBD‐SpRYn (hyABE8e‐SpRY) showed nearly 100% editing efficiency at some tested sites, with a high proportion of homozygous base substitutions in the editing windows and no single guide RNA (sgRNA)‐dependent off‐target changes. The original sgRNA was more compatible with PhieABEs than the evolved sgRNA. In conclusion, the DBD fusion effectively promotes base‐editing efficiency, and this novel PhieABE toolbox should have wide applications in plant functional genomics and crop improvement.