PhieABEs demonstrate nearly 100% editing efficiency at some tested sites in rice.
PhieABEs are a new toolbox combining an evolved adenosine deaminase and a DNA-binding domain that enhances editing capabilities.
This toolbox significantly improves base-editing activity and expands the target range compared to existing systems.
The hyper ABE8e-DBD-SpRYn variant showed a high proportion of homozygous base substitutions within the editing windows.
No off-target changes were observed with the hyper ABE8e-DBD-SpRYn variant under the tested conditions.
The original single guide RNA was found to be more compatible with PhieABEs than the evolved version.
Simplified
(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 () 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.
Key numbers
100%
Editing Efficiency
Editing efficiency at select target sites with .
78.5%
Average Editing Efficiency
Average editing efficiency of hyABE8e-SpRY across tested sites.
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