Chen Zhang, Jingying Li, Yucai Li, Lei Yan, Christina S.Y. Yong, Shaoya Li, Yubing He, Lanqin Xia

Journal of Integrative Plant Biology; 2026; IF: 12.5

DOI:10.1111/jipb.70154

Abstract

CRISPR/Cas12i3 belongs to type V-I Cas system, characterized by its smaller protein size and less-restricted canonical ‘TTN’ protospacer adjacent motif (PAM). Developments of Cas12i3-mediated base editing systems for either C-to-T or A-to-G transitions will expand the editing scope and enrich the plant base editing toolkits for crop improvement. However, while the Cas12i3-based cytosine base editor (CBE) only exhibits very low editing efficiency in plants, its adenine base editor (ABE) has not been documented yet. Here, we engineered a series of Cas12i3(5M)-based CBEs (V0-V5) and ABEs (V0-V5), by fusing a deactivated dCas12i3(5M) with a transactivation module VP64, a single-stranded DNA binding domain Rad51, or a double-stranded DNA binding domain HMG-D, or in combinations, respectively, and systemically evaluated their performances in rice protoplasts. Our results demonstrated that synergistic combinations of both VP64 and HMG-D outperformed other architectures and significantly boosted the efficiencies of Cas12i3(5M)-based CBE and ABE for either C-to-T or A-to-G base editing and expand the editing window. In stable lines, in comparison to the non-fusion control, the optimized Cas12i3(5M)-based CBE-V5 and ABE-V5 enabled up to 4.78- and 3.35-fold higher editing efficiencies, with maximum C-to-T and A-to-G efficiencies reaching 32.35% and 38.24%, and a higher proportion of homozygous mutants in T0 generation, respectively. Furthermore, we generated herbicide-resistant rice germplasm by using CBE-V5 and ABE-V5, respectively, demonstrating their potential for precision breeding in crops. Together, we here report the novel Cas12i3(5M)-based CBE and ABE that substantially enrich base editing toolkits for improvement of rice and potentially other crops.