Improving precision base editing of the zebrafish genome by Rad51DBD-incorporated single-base editors

Single-base editors, including cytosine base editors (CBEs) and adenine base editors (ABEs), facilitate accurate C⋅G to T⋅A and A⋅T to G⋅C, respectively, holding promise for the precise modeling and treatment of human hereditary disorders. Efficient base editing and expanded base conversion range have been achieved in human cells through base editors fusing with Rad51 DNA binding domain (Rad51DBD), such as hyA3A-BE4max. Here, we show that hyA3A-BE4max catalyzes C-to-T substitution in the zebrafish genome and extends editing positions (C₁₂-C₁₆) proximal to the protospacer adjacent motif. We develop a codon-optimized counterpart zhyA3A-CBE5, which exhibits substantially high C-to-T conversion with 1.59- to 3.50-fold improvement compared with the original hyA3A-BE4max. With these tools, disease-relevant hereditary mutations can be more efficaciously generated in zebrafish. We introduce human genetic mutation rpl11^Q42∗ and abcc6a^R1463C by zhyA3A-CBE5 in zebrafish, mirroring Diamond-Blackfan anemia and Pseudoxanthoma Elasticum, respectively. Our study expands the base editing platform targeting the zebrafish genomic landscape and the application of single-base editors for disease modeling and gene function study.