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 Cas12aCBEs wer...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 Cas12aCBEs 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 Cas12aCBEs in base editing efficiency. In T0transgenic rice plants, ttCas12a-hyA3Bctd edited an average of42.01% 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.展开更多
基金funded by the National Natural Science Foundation of China (U19A2022 and 32000284)the Natural Science Foundation of Anhui Province (2208085Y11, 2108085Y07,2008085QC101, and 2008085MC71)+2 种基金the University Synergy Innovation Program of Anhui Province (GXXT-2021-056)Open Research Fund Program of Anhui Province Key Laboratory of Rice Genetics and Breeding (SDKF-2021-01 and SDKF-2022-04)Natural Science Research Project for Anhui Universities(KJ2021A0196)。
文摘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 Cas12aCBEs 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 Cas12aCBEs in base editing efficiency. In T0transgenic rice plants, ttCas12a-hyA3Bctd edited an average of42.01% 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.
