Development of PmCDA1-based high-efficiency cytidine base editors (ChyCBEs) incorporating a GmRad51 DNA-binding domain in soybean

Abstract

Although many efficient base editing tools have been developed, there is still a need for more efficient CBEs in many important crop species. For instance, the BE editing efficiency in soybean still falls behind other model crops. In this study, we first generated an improved CBE configuration, using two copy UGI and a single transcript unit, and found that PmCDA1 is more effective than other tested base editors in soybean. We next developed a novel PmCDA1-based hyperdrive CBE (ChyBE03) by introducing a newly designed linker using a soybean GmRad51 ssDBD domain. We next used ChyBE03 to install a predefined stop codon in the Nod factor receptor gene (NFR1a). ChyBE03 showed up to 52.6% efficiency and can generate homozygous nfr1a mutants with desired non-nodulation phenotype in stable transgenic soybean. We also developed a ChyBE-NG with an expanded targeting scope and high editing efficiency. In conclusion, we developed a new PmCDA1-based high efficiency CBE tool, which is highly efficient and can be expanded for relaxed targeting scope with the SpCas9-NG variants.