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.
Subscribe to Journal
Get full journal access for 1 year
only $21.58 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Tax calculation will be finalised during checkout.
Rent or Buy article
Get time limited or full article access on ReadCube.
All prices are NET prices.
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.
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).
Jiao, R. & Gao, C. Anything impossible with CRISPR/Cas9? Sci. China Life Sci. 60, 445–446 (2017).
Anzalone, A. V. et al. Search-and-replace genome editing without double-strand breaks or donor DNA. Nature 576, 149–157 (2019).
Lin, Q. et al. Prime genome editing in rice and wheat. Nat. Biotechnol. 38, 582–585 (2020).
Tang, X. et al. Plant prime editors enable precise gene editing in rice cells. Mol. Plant 13, 667–670 (2020).
Li, H., Li, J., Chen, J., Yan, L. & Xia, L. Precise modifications of both exogenous and endogenous genes in rice by prime editing. Mol. Plant 13, 671–674 (2020).
Xu, W. et al. Versatile nucleotides substitution in plant using an improved prime editing system. Mol. Plant 13, 675–678 (2020).
Xu, R. et al. Development of plant prime-editing systems for precise genome editing. Plant Commun. 1, 100043 (2020).
Hua, K., Jiang, Y., Tao, X. & Zhu, J. K. Precision genome engineering in rice using prime editing system. Plant Biotechnol. J. 18, 2167–2169 (2020).
Butt, H. et al. Engineering herbicide resistance via prime editing in rice. Plant Biotechnol. J. 18, 2370–2372 (2020).
Jiang, Y. et al. Prime editing efficiently generates W542L and S621I double mutations in two ALS genes of maize. Genome Biol. 21, 257 (2020).
Liu, Y. et al. Efficient generation of mouse models with the prime editing system. Cell Discov. 6, 27 (2020).
Surun, D. et al. Efficient generation and correction of mutations in human iPS cells utilizing mRNAs of CRISPR base editors and prime editors. Genes 11, 511 (2020).
Kaback, D. B., Angerer, L. M. & Davidson, N. Improved methods for the formation and stabilization of R-loops. Nucleic Acids Res. 6, 2499–2517 (1979).
Walton, R. T. et al. Unconstrained genome targeting with near-PAMless engineered CRISPR–Cas9 variants. Science 368, 290–296 (2020).
Bhagwat, A. M. et al. multicrispr: gRNA design for prime editing and parallel targeting of thousands of targets. Life Sci. Alliance 3, e202000757 (2020).
Chow, R. D., Chen, J. S., Shen, J. & Chen, S. A web tool for the design of prime-editing guide RNAs. Nat. Biomed. Eng. 5, 190–194 (2020).
Hsu, J. Y. et al. PrimeDesign software for rapid and simplified design of prime editing guide RNAs. Nat. Commun. 12, 1034 (2021).
Morris, J. A., Rahman, J. A., Guo, X. & Sanjana, N. E. Automated design of CRISPR prime editors for thousands of human pathogenic variants. Preprint at https://www.biorxiv.org/content/10.1101/2020.05.07.083444v1 (2020)
Kim, H. K. et al. Predicting the efficiency of prime editing guide RNAs in human cells. Nat. Biotechnol. 39, 198–206 (2020).
Shan, Q. et al. Targeted genome modification of crop plants using a CRISPR–Cas system. Nat. Biotechnol. 31, 686–688 (2013).
Zong, Y. et al. Efficient C-to-T base editing in plants using a fusion of nCas9 and human APOBEC3A. Nat. Biotechnol. 36, 950–953 (2018).
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).
The authors have submitted a patent application based on the results reported in this paper.
Peer review information Nature Biotechnology thanks the anonymous reviewers for their contribution to the peer review of this work.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.