Nature Biotechnology
Abstract
Targeted saturation mutagenesis of crop genes could be applied to produce genetic variants with improved agronomic performance. However, tools for directed evolution of plant genes, such as error-prone PCR or DNA shuffling, are limited1. We engineered five saturated targeted endogenous mutagenesis editors (STEMEs) that can generate de novo mutations and facilitate directed evolution of plant genes. In rice protoplasts, STEME-1 edited cytosine and adenine at the same target site with C?>?T efficiency up to 61.61% and simultaneous C?>?T and A?>?G efficiency up to 15.10%. STEME-NG, which incorporates the nickase Cas9-NG protospacer-adjacent motif variant, was used with 20 individual single guide RNAs in rice protoplasts to produce near-saturated mutagenesis (73.21%) for a 56-amino-acid portion of the rice acetyl-coenzyme A carboxylase (OsACC). We also applied STEME-1 and STEME-NG for directed evolution of the OsACC gene in rice and obtained herbicide resistance mutations. This set of two STEMEs will accelerate trait development and should work in any plants amenable to CRISPR-based editing.
论文编号: | DOI:10.1038/s41587-019-0393-7 |
论文题目: | Targeted, Random Mutagenesis of Pant Genes with Dual Cytosine and Adenine Base Editors |
英文论文题目: | Targeted, Random Mutagenesis of Pant Genes with Dual Cytosine and Adenine Base Editors |
第一作者: | Chao Li, Rui Zhang, Xiangbing Meng, Sha Chen, Yuan Zong, Chunju Lu, Jin-Long Qiu, Yu-Hang Chen, Jiayang Li & Caixia Gao |
英文第一作者: | Chao Li, Rui Zhang, Xiangbing Meng, Sha Chen, Yuan Zong, Chunju Lu, Jin-Long Qiu, Yu-Hang Chen, Jiayang Li & Caixia Gao |
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发表年度: | 2020-01-14 |
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摘要: | Targeted saturation mutagenesis of crop genes could be applied to produce genetic variants with improved agronomic performance. However, tools for directed evolution of plant genes, such as error-prone PCR or DNA shuffling, are limited1. We engineered five saturated targeted endogenous mutagenesis editors (STEMEs) that can generate de novo mutations and facilitate directed evolution of plant genes. In rice protoplasts, STEME-1 edited cytosine and adenine at the same target site with C?>?T efficiency up to 61.61% and simultaneous C?>?T and A?>?G efficiency up to 15.10%. STEME-NG, which incorporates the nickase Cas9-NG protospacer-adjacent motif variant, was used with 20 individual single guide RNAs in rice protoplasts to produce near-saturated mutagenesis (73.21%) for a 56-amino-acid portion of the rice acetyl-coenzyme A carboxylase (OsACC). We also applied STEME-1 and STEME-NG for directed evolution of the OsACC gene in rice and obtained herbicide resistance mutations. This set of two STEMEs will accelerate trait development and should work in any plants amenable to CRISPR-based editing. |
英文摘要: | Targeted saturation mutagenesis of crop genes could be applied to produce genetic variants with improved agronomic performance. However, tools for directed evolution of plant genes, such as error-prone PCR or DNA shuffling, are limited1. We engineered five saturated targeted endogenous mutagenesis editors (STEMEs) that can generate de novo mutations and facilitate directed evolution of plant genes. In rice protoplasts, STEME-1 edited cytosine and adenine at the same target site with C?>?T efficiency up to 61.61% and simultaneous C?>?T and A?>?G efficiency up to 15.10%. STEME-NG, which incorporates the nickase Cas9-NG protospacer-adjacent motif variant, was used with 20 individual single guide RNAs in rice protoplasts to produce near-saturated mutagenesis (73.21%) for a 56-amino-acid portion of the rice acetyl-coenzyme A carboxylase (OsACC). We also applied STEME-1 and STEME-NG for directed evolution of the OsACC gene in rice and obtained herbicide resistance mutations. This set of two STEMEs will accelerate trait development and should work in any plants amenable to CRISPR-based editing. |
刊物名称: | Nature Biotechnology |
英文刊物名称: | Nature Biotechnology |
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其它备注: | Chao Li, Rui Zhang, Xiangbing Meng, Sha Chen, Yuan Zong, Chunju Lu, Jin-Long Qiu, Yu-Hang Chen, Jiayang Li & Caixia Gao. Targeted, Random Mutagenesis of Pant Genes with Dual Cytosine and Adenine Base Editors. Nature Biotechnology. DOI:10.1038/s41587-019-0393-7 |
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