Targeted A‐to‐T and A‐to‐C base replacement in maize using an optimized adenine base editor

Base editors, including cytosine and adenine base editors (CBE and ABE), are promising tools for precise genome modification. They enable the generation of single nucleotide variants in plants for research and crop improvement (Li et al., 2020; Manghwar et al., 2019; Ren et al., 2021; Xu et al., 2021; Zeng et al., 2022). However, existing base editors are still limited in the types of base conversions they can induce. Recently, a new base editor was constructed by fusing an engineered N-methylpurine DNA glycosylase (MPG) with ABE to create AYBE. This has achieved efficient A-to-T and A-to-C (A-to-Y, AYBE) transversions in mammalian cells and also timely assessed in rice to induce A-to-T (AKBE) (Li et al., 2023; Tong et al., 2023; Wu et al., 2023). However, the editing activity of AYBE remains unexplored in maize, and its editing efficiency leaves room for further optimization. Here, by fusing the adenine base editor with a codon-optimized N-methylpurine DNA glycosylase (MPG) and co-expressing the maize translesion synthesis DNA polymerase η (Polη), we developed an optimized AYBE base editor (ZmAYBEv3) for both A-to-T and A-to-C base conversions with high efficiency in maize and other monocots plants. First, the human-derived MPG (hMPG) was engineered (G163R, N169S, S198A, K202A, G203A, S206A and K210A) and codon-optimized for maize (MzMPG), then fused to the C-terminus of the maize ABE editor ZmABE8e to construct the initial AYBE editor ZmAYBEv1 (Figure 1a). Two sgRNAs (sgRNA1 and sgRNA2) targeting maize genes ZmGA20ox3 and ZmCT2 were designed. Hundreds of young embryos from the inbred maize variety KN5585 were transformed with Agrobacterium for evaluation. Approximately 50 regenerated shoots from each transformation were pooled and genotyped using the next-generation sequencing (NGS). As expected, only A-to-G substitutions were detected in samples edited with the conventional ZmABE8e, while A-to-Y conversions were found in ZmAYBEv1 edited samples (Figure 1b). For example, at the A8 site of sgRNA1, the A-to-T and A-to-C conversion frequencies were 3.86% and 0.53%, respectively, demonstrating the A-to-Y editing activity of ZmAYBEv1. We then tested it in maize plants. A total of 45 T0 plants were obtained and genotyped by NGS (Liu et al., 2019). The results showed seven T0 plants contained A-to-Y substitutions, further demonstrating ZmAYBEv1's editing capability in plantlet (Figure 1c; Table S1). However, the chimerism state of A-to-Y substitutions (calculated from the proportion of NGS reads, Li et al., 2023) was too low in most mutants. Usually, T0 plants with a chimerism>10% are required to ensure heritability. Thus, only one mutant could be identified as a valid A-to-T edited line, and no A-to-C editing lines were found. This revealed the need for further improvement of ZmAYBEv1. Polη is involved in the replication of damaged DNA and may improve base editing efficiency (Tong et al., 2023). Accordingly, human and maize Polη (hPolη and ZmPolη) were incorporated into ZmAYBEv1 to construct ZmAYBEv2 and ZmAYBEv3, respectively (Figure 1a). Quick tests in maize embryos showed a significant increase in A-to-T and A-to-C editing efficiencies when using ZmAYBEv3 (Figure 1b), indicating positive regulation of ZmPolη on AYBE. We think that different base conversion types between pAYBEv2 and pAYBEv3 might be caused by the different enzymatic activity of hPolη and ZmPolη (Figure S1). To assess them in transgenic plants, 39 and 52 T0 plants were generated using ZmAYBEv2 and ZmAYBEv3, respectively, targeting the same two genes above (Figure 1c). As expected, substantially more A-to-Y edited plants (7 out of 23 with chimerism>10%, the same hereinafter) were identified in ZmAYBEv3 edited lines for ZmGA20ox3. At sgRNA2 of ZmCT2, an uneditable site for ZmAYBEv1 or ZmAYBEv2, an A-to-T editing plant was successfully obtained using ZmAYBEv3. Moreover, three A-to-C edited lines were also identified. To confirm the editing results, we then resequenced the ZmAYBEv3-derived lines by Sanger sequencing and further confirmed these results (Figure 1d; Figure S2). Notably, we also found that homozygous lines could be generated in T0 plants. The homozygous A-to-T editing at the sgRNA1 (A8) of ZmGA20ox3 produced a premature stop codon (AAG to TAG), resulting in a semi-dwarf phenotype of maize, even in T0 generation (Figure 1e,f). These results indicate ZmAYBEv3 has the highest editing efficiency, capable of both A-to-T and A-to-C editing. To further confirm the versatility of ZmAYBEv3, we targeted three additional maize genes (ZmLW2, ZmABH2 and ZmLBD5) for editing. We regenerated 51 T0 maize plants and performed NGS genotyping. The results showed successful A-to-Y editing by ZmAYBEv3 at all three genes, with an average efficiency of 35.3% (18/51). Notably, the A-to-T editing frequency reached 45.5% at the ZmLBD5 locus. Given the known transferability of base editors across monocot species, we also tested ZmAYBEv3 in rice on three genes (OskTN80b, OsWaxy and OsTB1). NGS and Sanger sequencing showed ZmAYBEv3 could efficiently induce A-to-Y editing at the three rice genes with an average efficiency of 21.1% (12/57) (Figure 1c,d; Figure S3). In some locus, the A8 site within a sgRNA seems the best targeting nucleotide (Figure 1g). Together, these results further validate the editing activity of ZmAYBEv3 in both maize and other monocot species. Collectively, the incorporation of ZmPolη enhanced the A-to-Y editing efficiency of ZmAYBEv3. Across five target sites in 103 T0 maize plants, 50 plants had A-to-Y conversions (chimerism >1%), validating its capabilities. Notably, 26 plants (25.2%) showed potentially heritable edits (chimerism>10%). ZmAYBEv3 also enables the possibility of obtaining homozygous edits within the T0 generation. The high editing efficiencies achieved by ZmAYBEv3 in maize and rice highlight its usefulness as an alternative tool to supplement existing base and prime editors for functional studies and trait improvement in crops. Supported by the National Key R&D Program of China (No. 2021YFD1201300) and the National Natural Science Foundation of China (No. 32070396) to Y.L. We thank WIMI for assistance with maize transformation. D.Z. and Y.L. designed the research; D.Z, H.P., K.L., Y.Z., F.Z., L.Y., S.R., Q.D. and J.X. performed experiments; D.Z. and Y.L. wrote and revised the manuscript. The authors declare no competing interests. The data that supports the findings of this study are available in the supplementary material of this article. Data S1 Supplemental methods. 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