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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Data availability
All data supporting the findings of this study are available in the article and its supplementary figures and tables or can be obtained from the corresponding authors upon reasonable request. For sequence data, rice locus identifiers (http://rice.plantbiology.msu.edu/) are as follows:
LOC_Os01g55540 (OsAAT), LOC_Os05g22940 (OsACC), LOC_Os03g54790 (OsALS), LOC_Os03g05730 (OsCDC48), LOC_Os09g26999 (OsDEP1), LOC_Os06g04280 (OsEPSPS), LOC_Os08g39890 (OsIPA1); LOC_Os10g40600 (OsNRT1.1B), LOC_Os08g03290 (OsGAPDH), LOC_Os03g08570 (OsPDS) and LOC_Os06g35970 (OsROC5). The deep sequencing data have been deposited in a National Center for Biotechnology Information BioProject database (accession code PRJNA702010). Plasmids pSpG-PPE, pOsU3, pTaU3 and pH-nCas9-PPE-V2 will be available through Addgene.
Code availability
The PlantPegDesigner web application code is available at GitHub (https://github.com/JinShuai001/PlantPegDesigner). The web portal server is accessible at http://www.plantgenomeediting.net for non-profit use.
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Acknowledgements
This work was supported by grants from the National Transgenic Science and Technology Program of China (2019ZX08010-001 and 2019ZX08010-003), the Strategic Priority Research Program of the Chinese Academy of Sciences (Precision Seed Design and Breeding, XDA24020310 and XDA24030504) and the National Natural Science Foundation of China (31788103 and 32001060).
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Q.L., S.J., Y.Z., J.Q., J.L. and C.G. designed the project. Q.L., Y.Z., S.J., Z.Z., G.L. and Y.W. performed the experiments. H.Y., S.J. and L.K. developed the web application. Q.L., S.J., Y.Z., J.Q., J.L. and C.G. wrote the manuscript. J.L. and C.G. supervised the project.
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Lin, Q., Jin, S., Zong, Y. et al. High-efficiency prime editing with optimized, paired pegRNAs in plants. Nat Biotechnol 39, 923–927 (2021). https://doi.org/10.1038/s41587-021-00868-w
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DOI: https://doi.org/10.1038/s41587-021-00868-w
