Abstract: Background: The 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 of-target efects. Results: In this study, we perform 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, reveal that ABEs harboring TadA9 lead to a higher number of of-target A-to-G (A>G) single-nucleotide variants (SNVs), and that those harboring CRISPR/SpCas9n-NG lead to a higher total number of of-target SNVs in the rice genome. An analysis of the T-DNAs carrying the ABEs indicates that the ontarget mutations could be introduced before and/or after T-DNA integration into plant genomes, with more of-target A>G SNVs forming after the ABEs had integrated into the genome. Furthermore, we detect of-target A>G RNA mutations in plants with high expression of ABEs but not in plants with low expression of ABEs. The of-target A>G RNA mutations tend to cluster, while of-target A>G DNA mutations rarely clustered. Conclusion: Our fndings that Cas proteins, TadA variants, temporal expression of ABEs, and expression levels of ABEs contribute to ABE specifcity in rice provide insight into the specifcity of ABEs and suggest alternative ways to increase ABE specifcity besides engineering TadA variants.

Genome Biology,  IF: 13.58

DOI：doi.org/10.1186/s13059-022-02618-w