Rational selection of SpCas9 and variant-derived adenine base editors for human genome editing

Adenine base editors (ABEs), composed of Cas9 (i.e., SpCas9) nickase, adenosine deaminase, and sgRNA, can convert A•T base pairs to G•C base pairs in the genome without causing DNA double-strand breaks, thereby offering broad application prospects. The strict protospacer adjacent motif requirement and off-target effects of wild-type SpCas9 notably restricted its application until the identification of two SpCas9 variants, SpCas9-NG and SpRY, which have overcome these limitations. However, how to select appropriate ABEs from various SpCas9 variants remains unclear. In this study, we evaluated NGG-ABE8e, NG-ABE8e, and SpRY-ABE8e in human cells and explored the impact of sgRNA design on their performance. The results indicated that NGG-ABE8e showed higher editing activity at most tested NGG-PAM sites, whereas SpRY-ABE8e showed stronger mismatch discrimination and lower detectable A-to-G editing at the selected predicted off-target sites with NGG-PAM sites. Regarding sgRNA design, we found that spacer length and 5' guanine (G) addition affected editing outcomes in a site- and editor-dependent manner. Notably, adding an extra 5' G was not uniformly beneficial and could reduce editing efficiency, especially when the native spacer already began with G. In conclusion, this study provides insights into selecting optimal ABEs and designing effective sgRNAs for the treatment of human genetic diseases.