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Plant Commun
Plant Commun
Plant Communications
2590-3462
Elsevier

S2590-3462(24)00284-0
10.1016/j.xplc.2024.100976
100976
Correspondence
An improved plant prime editor for efficient generation of multiple-nucleotide variations and structural variations in rice
Zhong Zhaohui 134
Fan Tingting 14
He Yao 1
Liu Shishi 1
Zheng Xuelian 12
Xu Yang 1
Ren Jingqi 1
Yuan Hua 3
Xu Zhengyan 3
Zhang Yong zhangyong916@swu.edu.cn
2∗
1 Department of Biotechnology, School of Life Sciences and Technology, Center for Informational Biology, University of Electronic Science and Technology of China, Chengdu 610054, China
2 Chongqing Key Laboratory of Plant Resource Conservation and Germplasm Innovation, Integrative Science Center of Germplasm Creation in Western China (Chongqing) Science City, School of Life Sciences, Southwest University, Chongqing 400715, China
3 State Key Laboratory of Crop Gene Exploration and Utilization in Southwest China, Rice Research Institute, Sichuan Agricultural University, Chengdu 611130, Sichuan, China
∗ Corresponding author zhangyong916@swu.edu.cn
4 These authors contributed equally to this article.

14 5 2024
09 9 2024
14 5 2024
5 9 10097629 1 2024
29 4 2024
11 5 2024
© 2024 The Author(s)
2024
https://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Published: May 14, 2024
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pmcDear Editor,

The ability to manipulate single-nucleotide variations (SNVs), multiple-nucleotide variations (MNVs), and structural variations (SVs) in rice genomes is crucial for studying gene function and improving agricultural traits. Traditionally, these manipulations were achieved mainly through homology-directed repair, which has limited efficiency in plants. With the advent of CRISPR–Cas systems, particularly SpCas9, researchers found a simpler way to create mutants via DNA double-strand breaks and non-homologous end joining (Jinek et al., 2012). However, this often led to undesirable outcomes such as frameshift mutations or unwanted modifications. Recent advances in base-editing technologies, such as C-to-T, A-to-G, C-to-G, and A-to-Y base editors (Fan et al., 2024), have allowed for targeted nucleotide changes. Yet, challenges remain due to off-target effects and limited editing windows, which hinder the achievement of desired SNVs, MNVs, and SVs.

In response to these challenges, prime editing (PE) technology was introduced in 2019, providing precise single-nucleotide-level editing, encompassing conversions of all four bases (A, C, G, and T), as well as nucleotide deletions and insertions (Anzalone et al., 2019) by SpCas9 nickase (H840A) fused to Moloney Murine Leukemia Virus Reverse Transcriptase (M-MLV RT). Since then, research has focused on improving PE efficiency, resulting in various advances that have made it a more practical tool for manipulation of nucleotide variations and SVs in mammalian cells (Chen and Liu, 2023). However, its application in plants is still in its early stages because of its relatively low efficiency. The limited efficiency of PE in plants poses a particular challenge when attempting to achieve intricately designed SNVs, MNVs, and SVs (Chen and Liu, 2023).

To develop an efficient PE system for rice, we constructed prime editors using several approaches: classical PE2 (Anzalone et al., 2019), Cas9-PE2, N-PE2 (a simplified version of N-PE3 [Xu et al., 2021]), and engineered Plant Prime Editor (ePPE) (Zong et al., 2022) (Figure 1A; Supplemental Figure 1). We also tested engineered pegRNA (epegRNA) with an extra 3′ motif, EvopreQ1 (Nelson et al., 2022) (Figure 1B). In rice protoplast cells, our newly designed N-PE2 with epegRNA showed the highest editing efficiency at all tested sites, ranging from 9.0% to 18.4% (Figures 1C and 1D; Supplemental Figure 2), with an insertion or deletion frequency of less than 0.2% (Supplemental Figure 3). We named this innovative PE tool NEPE (N terminus M-MLV RT fused to Cas9 nickase plus epegRNA prime editor). When compared with the recently developed plant PEmax system, known for its enhanced PE in plants (Jiang et al., 2022; Qiao et al., 2023), NEPE demonstrated surprising superiority at the tested locus (Supplemental Figure 2B). In addition, when the capacity to achieve complex SNVs and MNVs was tested, NEPE consistently outperformed classical PE2 at all tested sites, with lower insertion or deletion frequency (Supplemental Figures 4–6).Figure 1 NEPE efficiently induced SNVs, MNVs, and SVs in rice.

(A) Diagram of the PE2, Cas9-PE2, N-PE2, and ePPE systems.

(B) Diagram of pegRNA and epegRNA. The 3′ motif (EvopreQ1) was used to prevent degradation by RNase.

(C) Editing frequency summary of PE2, Cas9-PE2, N-PE2, and ePPE at three target sites.

(D) Editing efficiency of prime editors with pegRNA or epegRNA.

(E) Diagram of RTT design for fragment deletion by NEPE. The homology sequence labeled in cyan is upstream of the deletion region, and that labeled in purple is downstream of the deletion region.

(F) Diagram of deletion regions in the OsWx promoter area.

(G) Gel electrophoresis results from four types of genomic deletion by PE2, Cas9-PE2, and NEPE. The lengths of the wild-type fragment and the deletion fragment are labeled with red arrows. The deletion ratio was measured with ImageJ software.

(H) Overall deletion frequency achieved by PE2, Cas9-PE2, and NEPE.

(I) Frequency of NEPE-induced fragment deletions in rice T0 lines.

(J) Diagram of RTT design for domain modification by NEPE. The domain modification includes domain deletion, insertion, and replacement. The HAs for deletion are shown in cyan and purple, and the insertion fragment is shown in orange.

(K) Genotype of a homozygous FLAG-tag insertion line (#594-26) created by NEPE at the OsMSP target site. The insertion sequence is highlighted in cyan.

(L) Western blot of the wild type and FLAG-tag insertion lines.

(M) Genotype of a homozygous 6×His tag insertion line (#597-17) created by NEPE at the OsMSP target site. The insertion sequence is highlighted in cyan.

(N) Western blot of the wild type and 6×His tag insertion lines.

(O) Frequency of NEPE-induced precise domain modifications in rice T0 lines.

(P) Genotype of a rice T0 line with precise domain deletion (#590-1). The replaced region is labeled in cyan, and the amino acid change is labeled above. The new amino acid numbers are labeled in salmon.

Data are presented as mean values ± SD. Each dot represents a biological replicate. Data were analyzed using two-tailed unpaired t-tests. ns, p > 0.05; ∗∗∗p < 0.001; and ∗∗∗∗p < 0.0001. Solid line, median; dashed lines, quartiles.

In stable transgenic T0 rice lines, NEPE demonstrated higher editing efficiency (22.9%–30.0%) than PE2 (3.7%–22.2%), with biallelic editing frequencies between 2.1% and 10.0% (Supplemental Figure 7A). The editing events were confirmed through phenotype selection and Sanger sequencing (Supplemental Figures 7B–7E, 8, and 9). Notably, NEPE induced a 7-bp nucleotide conversion at the OsGRF4 locus, removing the microRNA binding site and enhancing agricultural traits (Gupta et al., 2024) (Supplemental Figures 7F–7O and 10).

To address the low efficiency and accuracy of SV creation with previous tools, we investigated the potential of NEPE for induction of targeted SVs in rice (Figure 1E). For targeted fragment deletions, NEPE was designed to generate double homology arms (HAs) downstream of each strand, specifically targeting the seven key regions (KRs) of the OsWx promoter (Zhou et al., 2023) (Figure 1F). We strategically designed deletions of various lengths, including a 98-bp deletion to remove the valley between KR-B and KR-C, a 121-bp deletion for KR-D, a 156-bp deletion for the two high peaks in KR-A, and a 523-bp deletion to remove part of KR-B, all of KR-C, and KR-D (Figure 1F). Efficiency assessments in rice protoplasts showed that NEPE was the most effective in inducing these deletions, with deep sequencing confirming its accuracy and efficiency (Figures 1G and 1H; Supplemental Figures 11 and 12). Further validation in rice T0 lines revealed that NEPE could induce efficient and precise fragment deletions in the OsWx promoter region with editing efficiencies ranging from 18.8% to 30.7% and biallelic mutation rates between 11.5% and 13.3% (Figure 1I; Supplemental Figures 13A–13C and 14A–14C).

We also investigated the ability of NEPE to precisely modify protein domains by incorporating additional HA sequences (Figure 1J). Focusing on the OsMSP gene, we designed fragment insertions, including an eight-amino-acid FLAG tag (24 bp) and a six-amino-acid 6×His tag (18 bp), near the N terminus of the OsMSP protein. We performed Agrobacterium-mediated stable transformation to obtain rice lines with the inserted fragments, achieving insertion rates of 20.0% for the FLAG tag and 15.0% for the 6×His tag. Biallelic insertion lines were obtained with frequencies of 4.0% and 5.0%, respectively (Figure 1O). Sanger sequencing confirmed the accuracy of these insertions in homozygous lines (Figures 1K and 1M), and western blotting demonstrated the successful in-frame insertion of tags into the target protein (Figures 1L and 1N). We next tested the ability of NEPE to insert a 75-bp GFP fragment at the OsRBCS2 gene, with successful insertion verified through both fluorescence microscopy and deep sequencing (Supplemental Figure 15). In addition, we examined the capacity of NEPE to simultaneously induce a precise deletion and insertion, enabling protein domain replacement (Figure 1J). At the OsSRFR1 site, NEPE was designed to generate a 3866-bp modification of the coding region, comprising a 3900-bp deletion and a 34-bp insertion, leading to a precise 405-amino-acid deletion in the OsSRFR1 protein (Figures 1O and 1P; Supplemental Figures 13D, 13E, and 14D).

On the basis of these results, we developed a set of design principles to enable multi-type precise editing for plant genome functional manipulation using NEPE (Supplemental Figure 16). Its applications are categorized into four primary types: conversion, deletion, insertion, and replacement. For prime binding-site (PBS) design, we recommend a length of 8–10 bp or a melting temperature equal to or higher than the culture temperature to ensure efficient genome binding. For reverse transcription template (RTT) design, the principles vary based on the application. For conversion, both single and dual epegRNA designs are effective, with a ∼15-bp RTT template including a 6–8-bp HA for single-nucleotide conversion. In cases of multiple nucleotide conversion, a longer HA is recommended. For deletion, dual epegRNA is advised, with an RTT containing a ∼15-bp HA for upstream or downstream regions for each epegRNA. In insertion scenarios, a dual epegRNA strategy is recommended, with an RTT containing a ∼10–15-bp HA, and the inserted regions should overlap. For replacement applications, a ∼15-bp HA is required for deletion, and the insertion region should overlap.

In summary, our newly developed NEPE system has proven highly efficient for PE in rice, excelling at the induction of SNVs, MNVs, and SVs. This advance not only provides a powerful tool for plant genome research but also enables new possibilities for molecular breeding, allowing for precise and versatile genetic modifications.

Construct availability

All the backbone plasmids in this study have been submitted to Addgene. The backbone plasmids include PE2 (pGEL682, Addgene #205591), N-PE2 (pGEL685, Addgene #205592), Cas9-PE2 (pGEL683, Addgene #205593), and ePPE (pGEL686, Addgene #207510).

Funding

This research was supported by the STI 2030 -Major Projects (2023ZD04074 ) to Y.Z. and the 10.13039/501100001809 National Natural Science Foundation of China (award nos. 32301245 and 32072045 ) to Z.Z. and X.Z.

Author contributions

Y.Z. proposed the project and designed the experiments. Z.Z. constructed the vectors. Z.Z. and T.F. performed the rice protoplast transformation. Z.Z. and T.F. analyzed the editing frequencies in protoplasts. Z.Z., T.F., Y.H., S.L., Y.X., and X.Z. performed the rice stable transformation. H.Y. and Z.X. cultured the rice stable lines. J.R. performed the western blotting. Z.Z. and T.F. analyzed genotypes and phenotypes of the rice stable lines. Y.Z., Z.Z., and T.F. analyzed the data and wrote the manuscript with input from other authors. All authors read and approved the final manuscript.

Supplemental information

Document S1. Supplemental Figures 1–16, Supplemental Tables 1 and 2, and Supplemental materials and methods

Document S2. Article plus supplemental information

Acknowledgments

No conflict of interest is declared.

Published by the Plant Communications Shanghai Editorial Office in association with Cell Press, an imprint of Elsevier Inc., on behalf of CSPB and CEMPS, CAS.

Supplemental information is available at Plant Communications Online.
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