PE6c greatly enhances prime editing in transgenic rice plants

Abstract

Prime editing is a versatile CRISPR/Cas-based precise genome-editing technique for crop breeding. Four new types of prime editors (PEs) named PE6a–d were recently generated using evolved and engineered reverse transcriptase (RT) variants from three different sources. In this study, we tested the editing efficiencies of four PE6 variants and two additional PE6 constructs with double-RT modules in transgenic rice (Oryza sativa) plants. PE6c, with an evolved and engineered RT variant from the yeast Tf1 retrotransposon, yielded the highest prime-editing efficiency. The average fold change in the editing efficiency of PE6c compared with PEmax exceeded 3.5 across 18 agronomically important target sites from 15 genes. We also demonstrated the feasibility of using two RT modules to improve prime-editing efficiency. Our results suggest that PE6c or its derivatives would be an excellent choice for prime editing in monocot plants. In addition, our findings have laid a foundation for prime-editing-based breeding of rice varieties with enhanced agronomically important traits.

Keywords

genome editing / PE6 / PE6c / prime editing / prime editor / rice

References

[1] Anzalone, A.V.,Randolph, P.B.,Davis, J.R.,Sousa, A.A.,Koblan, L.W.,Levy, J.M.,Chen, P.J.,Wilson, C.,Newby, G.A.,Raguram, A., et al. (2019). Search-and-replace genome editing without double-strand breaks or donor DNA. Nature 576:149–157.

[2] Bai, M.,Lin, W.,Peng, C.,Song, P.,Kuang, H.,Lin, J.,Zhang, J.,Wang, J.,Chen, B.,Li, H., et al. (2024). Expressing a human RNA demethylase as an assister improves gene-editing efficiency in plants. Mol. Plant 17:363–366.

[3] Chen, P.J.,Hussmann, J.A.,Yan, J.,Knipping, F.,Ravisankar, P.,Chen, P.F.,Chen, C.,Nelson, J.W.,Newby, G.A.,Sahin, M., et al. (2021). Enhanced prime editing systems by manipulating cellular determinants of editing outcomes. Cell 184:5635–5652.e29.

[4] Doman, J.L.,Pandey, S.,Neugebauer, M.E.,An, M.,Davis, J.R.,Randolph, P.B.,McElroy, A.,Gao, X.D.,Raguram, A.,Richter, M.F., et al. (2023). Phage-assisted evolution and protein engineering yield compact, efficient prime editors. Cell 186:3983–4002.e26.

[5] Jiang, Y.,Chai, Y.,Qiao, D.,Wang, J.,Xin, C.,Sun, W.,Cao, Z.,Zhang, Y.,Zhou, Y.,Wang, X.C., et al. (2022). Optimized prime editing efficiently generates glyphosate-resistant rice plants carrying homozygous TAP-IVS mutation in EPSPS. Mol. Plant 15:1646–1649.

[6] Li, H.,Zhu, Z.,Li, S.,Li, J.,Yan, L.,Zhang, C.,Ma, Y., and Xia, L. (2022a). Multiplex precision gene editing by a surrogate prime editor in rice. Mol. Plant 15:1077–1080.

[7] Li, J.,Chen, L.,Liang, J.,Xu, R.,Jiang, Y.,Li, Y.,Ding, J.,Li, M.,Qin, R., and Wei, P. (2022b). Development of a highly efficient prime editor 2 system in plants. Genome Biol. 23:161.

[8] Li, J.,Ding, J.,Zhu, J.,Xu, R.,Gu, D.,Liu, X.,Liang, J.,Qiu, C.,Wang, H.,Li, M., et al. (2023). Prime editing-mediated precise knockin of protein tag sequences in the rice genome. Plant Commun. 4:100572.

[9] Li, X.,Chen, W.,Martin, B.K.,Calderon, D.,Lee, C.,Choi, J.,Chardon, F.M.,McDiarmid, T.A.,Daza, R.M.,Kim, H., et al. (2024). Chromatin context-dependent regulation and epigenetic manipulation of prime editing. Cell 187:2411–2427.e25.

[10] Liang, R.,He, Z.,Zhao, K.T.,Zhu, H.,Hu, J.,Liu, G.,Gao, Q.,Liu, M.,Zhang, R.,Qiu, J.L., et al. (2024). Prime editing using CRISPR-Cas12a and circular RNAs in human cells. Nat. Biotechnol.

[11] Lin, Q.,Jin, S.,Zong, Y.,Yu, H.,Zhu, Z.,Liu, G.,Kou, L.,Wang, Y.,Qiu, J.L.,Li, J., et al. (2021). High-efficiency prime editing with optimized, paired pegRNAs in plants. Nat. Biotechnol. 39:923–927.

[12] Liu, Q.,Wang, C.,Jiao, X.,Zhang, H.,Song, L.,Li, Y.,Gao, C., and Wang, K. (2019). Hi-TOM: A platform for high-throughput tracking of mutations induced by CRISPR/Cas systems. Sci. China: Life Sci. 62:1–7.

[13] Liu, X.,Gu, D.,Zhang, Y.,Jiang, Y.,Xiao, Z.,Xu, R.,Qin, R.,Li, J., and Wei, P. (2024). Conditional knockdown of OsMLH1 to improve plant prime editing systems without disturbing fertility in rice. Genome Biol. 25:131.

[14] Nelson, J.W.,Randolph, P.B.,Shen, S.P.,Everette, K.A.,Chen, P.J.,Anzalone, A.V.,An, M.,Newby, G.A.,Chen, J.C.,Hsu, A., et al. (2022). Engineered pegRNAs improve prime editing efficiency. Nat. Biotechnol. 40:402–410.

[15] Ni, P.,Zhao, Y.,Zhou, X.,Liu, Z.,Huang, Z.,Ni, Z.,Sun, Q., and Zong, Y. (2023). Efficient and versatile multiplex prime editing in hexaploid wheat. Genome Biol. 24:156.

[16] Qiao, D.,Wang, J.,Lu, M.H.,Xin, C.,Chai, Y.,Jiang, Y.,Sun, W.,Cao, Z.,Guo, S.,Wang, X.C., et al. (2023). Optimized prime editing efficiently generates heritable mutations in maize. J. Integr. Plant Biol. 65:900–906.

[17] Sun, C.,Lei, Y.,Li, B.,Gao, Q.,Li, Y.,Cao, W.,Yang, C.,Li, H.,Wang, Z.,Li, Y., et al. (2023). Precise integration of large DNA sequences in plant genomes using PrimeRoot editors. Nat. Biotechnol. 42:316–327.

[18] Xu W.,Yang Y.,Yang B.,Krueger C.J.,Xiao Q.,Zhao S.,Zhang L.,Kang G.,Wang F.,Yi H., et al. (2022). A design optimized prime editor with expanded scope and capability in plants. Nat. Plants 8:45–52.

[19] Yan, J.,Oyler-Castrillo, P.,Ravisankar, P.,Ward, C.C.,Levesque, S.,Jing, Y.,Simpson, D.,Zhao, A.,Li, H.,Yan, W., et al. (2024). Improving prime editing with an endogenous small RNA-binding protein. Nature 628:639–647.

[20] Zhang, Q.,Zhang, Y.,Lu, M.H.,Chai, Y.P.,Jiang, Y.Y.,Zhou, Y.,Wang, X.C., and Chen, Q.J. (2019). A novel ternary vector system united with morphogenic genes enhances CRISPR/Cas delivery in maize. Plant Physiol. 181:1441–1448.

[21] Zhong, Z.,Fan, T.,He, Y.,Liu, S.,Zheng, X.,Xu, Y.,Ren, J.,Yuan, H.,Xu, Z., and Zhang, Y. (2024). An improved plant prime editor for efficient generation of multiple-nucleotide variations and structural variations in rice. Plant Commun.

[22] Zong, Y.,Liu, Y.,Xue, C.,Li, B.,Li, X.,Wang, Y.,Li, J.,Liu, G.,Huang, X.,Cao, X., et al. (2022). An engineered prime editor with enhanced editing efficiency in plants. Nat. Biotechnol. 40:1394–1402.

[23] Zou, J.,Meng, X.,Liu, Q.,Shang, M.,Wang, K.,Li, J.,Yu, H., and Wang, C. (2022). Improving the efficiency of prime editing with epegRNAs and high-temperature treatment in rice. Sci. China Life Sci. 65:2328–2331.

RIGHTS & PERMISSIONS

2024 The Author(s). Journal of Integrative Plant Biology published by John Wiley & Sons Australia, Ltd on behalf of Institute of Botany, Chinese Academy of Sciences.