High-efficiency prime editing with optimized, paired pegRNAs in plants

Qiupeng Lin^#^{1

2}, Shuai Jin^#^{1

2}, Yuan Zong^#¹, Hong Yu^#³, Zixu Zhu^{1

2}, Guanwen Liu^{2

4}, Liquan Kou³, Yanpeng Wang^{1

2}, Jin-Long Qiu^{4

5}, Jiayang Li^{6

7}, Caixia Gao^{8

9}

Affiliations

¹ State Key Laboratory of Plant Cell and Chromosome Engineering, Center for Genome Editing, Institute of Genetics and Developmental Biology, Innovation Academy for Seed Design, Chinese Academy of Sciences, Beijing, China.
² College of Advanced Agricultural Sciences, University of Chinese Academy of Sciences, Beijing, China.
³ State Key Laboratory of Plant Genomics, and National Center for Plant Gene Research, Institute of Genetics and Developmental Biology, Innovation Academy for Seed Design, Chinese Academy of Sciences, Beijing, China.
⁴ State Key Laboratory of Plant Genomics, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China.
⁵ CAS Center for Excellence in Biotic Interactions, University of Chinese Academy of Sciences, Beijing, China.
⁶ College of Advanced Agricultural Sciences, University of Chinese Academy of Sciences, Beijing, China. jyli@genetics.ac.cn.
⁷ State Key Laboratory of Plant Genomics, and National Center for Plant Gene Research, Institute of Genetics and Developmental Biology, Innovation Academy for Seed Design, Chinese Academy of Sciences, Beijing, China. jyli@genetics.ac.cn.
⁸ State Key Laboratory of Plant Cell and Chromosome Engineering, Center for Genome Editing, Institute of Genetics and Developmental Biology, Innovation Academy for Seed Design, Chinese Academy of Sciences, Beijing, China. cxgao@genetics.ac.cn.
⁹ College of Advanced Agricultural Sciences, University of Chinese Academy of Sciences, Beijing, China. cxgao@genetics.ac.cn.

^# Contributed equally.

PMID: 33767395
DOI: 10.1038/s41587-021-00868-w

High-efficiency prime editing with optimized, paired pegRNAs in plants

Qiupeng Lin et al. Nat Biotechnol. 2021 Aug.

. 2021 Aug;39(8):923-927.

doi: 10.1038/s41587-021-00868-w. Epub 2021 Mar 25.

Authors

Qiupeng Lin^#^{1

2}, Shuai Jin^#^{1

2}, Yuan Zong^#¹, Hong Yu^#³, Zixu Zhu^{1

2}, Guanwen Liu^{2

4}, Liquan Kou³, Yanpeng Wang^{1

2}, Jin-Long Qiu^{4

5}, Jiayang Li^{6

7}, Caixia Gao^{8

9}

Affiliations

¹ State Key Laboratory of Plant Cell and Chromosome Engineering, Center for Genome Editing, Institute of Genetics and Developmental Biology, Innovation Academy for Seed Design, Chinese Academy of Sciences, Beijing, China.
² College of Advanced Agricultural Sciences, University of Chinese Academy of Sciences, Beijing, China.
³ State Key Laboratory of Plant Genomics, and National Center for Plant Gene Research, Institute of Genetics and Developmental Biology, Innovation Academy for Seed Design, Chinese Academy of Sciences, Beijing, China.
⁴ State Key Laboratory of Plant Genomics, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China.
⁵ CAS Center for Excellence in Biotic Interactions, University of Chinese Academy of Sciences, Beijing, China.
⁶ College of Advanced Agricultural Sciences, University of Chinese Academy of Sciences, Beijing, China. jyli@genetics.ac.cn.
⁷ State Key Laboratory of Plant Genomics, and National Center for Plant Gene Research, Institute of Genetics and Developmental Biology, Innovation Academy for Seed Design, Chinese Academy of Sciences, Beijing, China. jyli@genetics.ac.cn.
⁸ State Key Laboratory of Plant Cell and Chromosome Engineering, Center for Genome Editing, Institute of Genetics and Developmental Biology, Innovation Academy for Seed Design, Chinese Academy of Sciences, Beijing, China. cxgao@genetics.ac.cn.
⁹ College of Advanced Agricultural Sciences, University of Chinese Academy of Sciences, Beijing, China. cxgao@genetics.ac.cn.

^# Contributed equally.

PMID: 33767395
DOI: 10.1038/s41587-021-00868-w

Abstract

Prime editing (PE) applications are limited by low editing efficiency. Here we show that designing prime binding sites with a melting temperature of 30 °C leads to optimal performance in rice and that using two prime editing guide (peg) RNAs in trans encoding the same edits substantially enhances PE efficiency. Together, these approaches boost PE efficiency from 2.9-fold to 17.4-fold. Optimal pegRNAs or pegRNA pairs can be designed with our web application, PlantPegDesigner.

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Comment in

Prime editing progress.
Tang L. Tang L. Nat Methods. 2021 Jun;18(6):592. doi: 10.1038/s41592-021-01188-2. Nat Methods. 2021. PMID: 34099936 No abstract available.

References

1. Chen, K., Wang, Y., Zhang, R., Zhang, H. & Gao, C. CRISPR/Cas genome editing and precision plant breeding in agriculture. Annu. Rev. Plant Biol. 70, 667–697 (2019). - DOI - PubMed
1. Jiao, R. & Gao, C. Anything impossible with CRISPR/Cas9? Sci. China Life Sci. 60, 445–446 (2017). - DOI - PubMed
1. Anzalone, A. V. et al. Search-and-replace genome editing without double-strand breaks or donor DNA. Nature 576, 149–157 (2019). - DOI - PubMed - PMC
1. Lin, Q. et al. Prime genome editing in rice and wheat. Nat. Biotechnol. 38, 582–585 (2020). - DOI - PubMed
1. Tang, X. et al. Plant prime editors enable precise gene editing in rice cells. Mol. Plant 13, 667–670 (2020). - DOI - PubMed

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High-efficiency prime editing with optimized, paired pegRNAs in plants

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High-efficiency prime editing with optimized, paired pegRNAs in plants

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