A large-scale genome and transcriptome sequencing analysis reveals the mutation landscapes induced by high-activity adenine base editors in plants

Abstract BackgroundThe high-activity adenine base editors (ABEs), engineered with the recently-developed tRNA adenosine deaminases (TadA8e and TadA9), show robust base editing activity but raise concerns about off-target effects. ResultsIn this study, we performed a comprehensive evaluation of ABE8e- and ABE9-induced DNA and RNA mutations in Oryza sativa. Whole-genome sequencing analysis of plants transformed with four ABEs, including SpCas9n-TadA8e, SpCas9n-TadA9, SpCas9n-NG-TadA8e, and SpCas9n-NG-TadA9, revealed that ABEs harboring TadA9 lead to a higher number of off-target A-to-G (A>G) single-nucleotide variants (SNVs), and that those harboring the CRISPR/SpCas9n-NG lead to a higher total number of off-target SNVs in the rice genome. An analysis of the T-DNAs carrying the ABEs indicated that the on-target mutations could be introduced before and/or after T-DNA integration into plant genomes, with more off-target A>G SNVs forming after the ABEs had integrated into the plant genome. Furthermore, we detected off-target A>G RNA mutations in plants with high expression of ABEs but not in plants with low expression of ABEs. The off-target A>G RNA mutations tended to cluster, while off-target A>G DNA mutations rarely clustered. ConclusionOur findings that Cas proteins, TadA variants, temporal expression of ABEs, and expression levels of ABEs contribute to ABE specificity in rice provide insight into the specificity of ABEs and suggest alternative ways to increase ABE specificity besides engineering TadA variants.