High-efficiency and multiplex adenine base editing in plants using new TadA variants.

Recently reported adenine base editors (ABEs) exhibit powerful potential for targeted gene correction as well as developing gain-of-function mutants and novel germplasms for both gene function studies and crop breeding. However, editing efficiency varies significantly among different target sites. Here, we investigated the activities of three evolved E. coli adenosine deaminase TadA variants (TadA8e, TadA8.17, and TadA8.20) side-by-side in transgenic rice. We found that TadA8e outperforms TadA8.17 and TadA8.20, and induces efficient A-to-G conversion at all tested sites in the rice genome, including those that were uneditable by ABE7.10 in our previous experiments. Furthermore, V82S/Q154R mutations were incorporated into TadA8e, resulting in a new variant that we named TadA9. Our data show that TadA9 is broadly compatible with CRISPR/SpCas9, CRISPR/SpCas9-NG, and CRISPR/SpRY, as well as CRISPR/ScCas9 nickase systems, achieving comparable or enhanced editing in a larger editing window at diverse PAM sites as compared with TadA8e. Finally, TadA9 was used to simultaneously install novel SNPs in four endogenous herbicide target genes in the commercial rice cultivar Nangeng 46 for potential field application in weed control. Collectively, we successfully generated a series of novel ABEs that can efficiently edit adenosines in the rice genome. Our findings suggest that TadA9 and TadA8e have great potentials in the development of plant base editors and crop molecular breeding.

SpRY greatly expands the genome editing scope in rice with highly flexible PAM recognition.

BACKGROUND: Plant genome engineering mediated by various CRISPR-based tools requires specific protospacer adjacent motifs (PAMs), such as the well-performed NGG, NG, and NNG, to initiate target recognition, which notably restricts the editable range of the plant genome. RESULTS: In this study, we thoroughly investigate the nuclease activity and the PAM preference of two structurally engineered SpCas9 variants, SpG and SpRY, in transgenic rice. Our study shows that SpG nuclease favors NGD PAMs, albeit less efficiently than the previously described SpCas9-NG, and that SpRY nuclease achieves efficient editing across a wide range of genomic loci, exhibiting a preference of NGD as well as NAN PAMs. Furthermore, SpRY-fused cytidine deaminase hAID*Δ and adenosine deaminase TadA8e are generated, respectively. These constructs efficiently induce C-to-T and A-to-G conversions in the target genes toward various non-canonical PAMs, including non-G PAMs. Remarkably, high-frequency self-editing events (indels and DNA fragments deletion) in the integrated T-DNA fragments as a result of the nuclease activity of SpRY are observed, whereas the self-editing of SpRY nickase-mediated base editor is quite low in transgenic rice lines. CONCLUSIONS: The broad PAM compatibility of SpRY greatly expands the targeting scope of CRISPR-based tools in plant genome engineering.