植物におけるCas12i3変異体ベースのシトシンおよびアデニン編集を増強するトランス活性化モジュールと二本鎖DNA結合ドメインのカップリング

CRISPR/Cas12i3 belongs to the type V-I Cas system, characterized by its smaller protein size and less restricted canonical "TTN" protospacer adjacent motif. 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 shows very low editing efficiency in plants, its adenine base editor (ABE) has not been documented as 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, and systemically evaluated their performance 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 C-to-T and A-to-G base editing and expanded 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 the maximum C-to-T and A-to-G efficiencies reaching 32.35% and 38.24%, respectively, and a higher proportion of homozygous mutants in the T₀ generation. Furthermore, we generated herbicide-resistant rice germplasm by using CBE-V5 and ABE-V5, demonstrating their potential for precision breeding in crops. Together, here, we report novel Cas12i3 (5M)-based CBE and ABE that substantially enrich base editing toolkits for improvement of rice and potentially other crops.