Development of plant cytosine base editors with the Cas12a system
Huanhuan Wang,
Jing Liang,
Like Chen,
Bufang Deng,
Dongfang Gu,
Xiaoshuang Liu,
Shan Jin,
Rongfang Xu,
Ruiying Qin,
Yitong Zhu,
Liangxia Zhao,
Dourong Kou,
Yanjun Chen,
Yingli Jiang,
Juan Li,
Pengcheng Wei
Affiliations
Huanhuan Wang
College of Agronomy, Anhui Agricultural University, Hefei 230036, Anhui, China
Jing Liang
College of Agronomy, Anhui Agricultural University, Hefei 230036, Anhui, China
Like Chen
College of Agronomy, Anhui Agricultural University, Hefei 230036, Anhui, China
Bufang Deng
College of Agronomy, Anhui Agricultural University, Hefei 230036, Anhui, China
Dongfang Gu
Key Laboratory of Rice Genetic Breeding of Anhui Province, Rice Research Institute, Anhui Academy of Agricultural Sciences, Hefei 230031, Anhui, China
Xiaoshuang Liu
College of Agronomy, Anhui Agricultural University, Hefei 230036, Anhui, China
Shan Jin
Key Laboratory of Rice Genetic Breeding of Anhui Province, Rice Research Institute, Anhui Academy of Agricultural Sciences, Hefei 230031, Anhui, China
Rongfang Xu
Key Laboratory of Rice Genetic Breeding of Anhui Province, Rice Research Institute, Anhui Academy of Agricultural Sciences, Hefei 230031, Anhui, China
Ruiying Qin
Key Laboratory of Rice Genetic Breeding of Anhui Province, Rice Research Institute, Anhui Academy of Agricultural Sciences, Hefei 230031, Anhui, China
Yitong Zhu
College of Agronomy, Anhui Agricultural University, Hefei 230036, Anhui, China
Liangxia Zhao
College of Agronomy, Anhui Agricultural University, Hefei 230036, Anhui, China
Dourong Kou
Key Laboratory of Rice Genetic Breeding of Anhui Province, Rice Research Institute, Anhui Academy of Agricultural Sciences, Hefei 230031, Anhui, China
Yanjun Chen
College of Agronomy, Anhui Agricultural University, Hefei 230036, Anhui, China
Yingli Jiang
College of Agronomy, Anhui Agricultural University, Hefei 230036, Anhui, China
Juan Li
Key Laboratory of Rice Genetic Breeding of Anhui Province, Rice Research Institute, Anhui Academy of Agricultural Sciences, Hefei 230031, Anhui, China; Corresponding authors.
Pengcheng Wei
College of Agronomy, Anhui Agricultural University, Hefei 230036, Anhui, China; Corresponding authors.
Base editors of the Cas9 system have been widely used for precise nucleotide substitution in crops. In this study, Cas12a was applied to construct plant cytosine base editors (CBEs). The main elements of Cas12a-CBEs were engineered and their efficiency was evaluated in stably transformed rice cells. An optimized ttCas12a-hyA3Bctd editor, consisting of a LbCas12a variant carrying catalytic inactive D832A and temperature-tolerance D156R double mutations, a truncated human APOBEC3B deaminase, a human RAD51 single-stranded DNA-binding domain, and double copies of UGI, outperformed other Cas12a-CBEs in base editing efficiency. In T0 transgenic rice plants, ttCas12a-hyA3Bctd edited an average of 42.01% and a maximum of 68.75% of lines at six genomic targets. A-to-G conversions were generated in rice by an adenine base editor with a similar architecture to the optimized CBE. Our results provide preliminary evidence for the feasibility of robust and efficient plant Cas12a base editing systems, which could be useful for precise crop breeding.
