Generating herbicide resistant and dwarf rice germplasms through precise sequence insertion or replacement

Precise sequence insertion or replacement in plants is technically challenging but is of great importance in crop breeding because many agronomic traits are affected by DNA fragment variations. Although prime editing (PE) has been continuously optimized to improve its activity in plants (Jiang et al., 2022; Li et al., 2022a,b; Zong et al., 2022), it is still inefficient for targeted insertion or replacement of longer sequences. Similar strategies, twinPE (Anzalone et al., 2022) and GRAND editing (Wang et al., 2022), in which a pair of PE guide RNAs (pegRNAs) are partially complementary to each other in their reverse transcriptase template (RTT) but are not homologous to the genomic sequences, were recently developed to facilitate longer sequence insertion (Figure 1a). HPPD-inhibitor herbicides such as β-triketones are effective in controlling resistant weeds that have emerged. The HIS1 gene in rice confers broad-spectrum resistance to triketone herbicides, whereas a dysfunctional his1 allele with a 28-bp fragment deletion was found in triketone-sensitive Indica varieties (Maeda et al., 2019). A genetic survey for 631 Indica varieties commonly used in rice breeding revealed that the 28-bp deletion is widely distributed, including 50.7% 3-line restorers, 40.7% 2-line restorers and 18.1% conventional varieties (Lv et al., 2021), which causes a huge risk for applying HPPD-inhibitor herbicides in Indica rice cultivating area. S1035 is an elite conventional Indica cultivar that sensitive to triketone due to the 28-bp deletion at HIS1. PE and GRAND editing strategies were tested to targeted insert the 28-bp fragment. Different from the design of PE (Figure S1), GRAND editing uses a pair of pegRNAs to delete the 18-bp genomic sequence between the two nicks and to insert a 46-bp designed sequence, including the to-be-inserted 28-bp and the to-be-replaced 18-bp sequences in which synonymous mutations were introduced to reduce the homology between RTT and genomic sequences (Figure 1b). It was reported that the sequence complementarity within the RTTs significantly affects the insertion efficiency (Wang et al., 2022), 10-, 18- or 26-bp sequence complementarity was designed in our test (Figure 1b). PE yielded 1.46% precise insertion events after protoplast transfection, indicating its low efficiency for DNA fragment insertion. GRAND editing with 10-bp complementary RTT sequences (RTT-10) achieved higher efficiency (9.88%) than that of RTT-18 (3.76%) or RTT-26 (0.59%) (Figure 1c). We then further evaluated GRAND editing during stable transformation. The transgenic lines were directly treated by 60 μm mesotrione, a widely used β-triketone herbicide. Nine (11.5%) resistant lines were generated from RTT-10 transformation, while only one was obtained each from RTT-18 and RTT-26 transformations (Figure 1d; Figures S2 and S3). Then RTT-10 construct was used to edit MingHui86, an elite 3-line restorer with the 28-bp deletion at HIS1. 13 (15.5%) of the 84 transgenic lines were recovered mesotrione-resistance (Figure 1d and Figure S4). T-DNA free, homozygous offsprings were identified from S1035 edited lines in the T1 generation (Figure 1e and Figure S5, Table S1). The expression level of the repaired HIS1 gene was comparable with that of wild type (WT; Figure 1f), but the mesotrione-resistance of the edited plants was similar with that of XiuShui134, a functional HIS1 gene-containing Japonica rice variety (Figure 1g). These results indicated that GRAND editing can be used to rescue other defective varieties for quickly solving the risk of applying HPPD-inhibitor herbicides in Indica rice cultivating area. PE and GRAND editing strategies were also tested for introducing the glyphosate-resistant T173IP177S mutations (C518T + C529T) into the OsEPSPS gene (Figure 1h; Figure S6). Protoplast test showed the efficiency of GRAND editing was 1.86 fold of that of PE (9.98% vs. 5.37%; Figure S7). Only 3 (2.0%) heterozygous plants from PE contained desired TIPS mutations, other mutants were either chimeras or that the C518T and C529T substitutions occurred separately (Figures S6 and S8). In contrast, GRAND editing generated 5 (3.4%) homozygous and 18 (12.2%) heterozygous edited lines (Figure 1i; Figure S8). Since the plants carrying homozygous TIPS mutations were seriously affected in their growth or even died after transplanting to soil (Figure S9), the heterozygous TIPS mutants were tested by glyphosate and showed no symptoms of damage but the WT plants withered (Figure S10). Their offsprings in the T1 generation inherited the glyphosate-resistant trait (Figure 1j,k; Table S1). To further evaluate the activity of GRAND editing, we designed to replace a 28-bp fragment (containing three amino acid substitutions) in the TVHYNP domain of the OsSLR1 gene (Figure 1l). We obtained 4 (6.9%) homozygous, 19 (32.8%) heterozygous plants from 58 transgenic lines (Figure 1m). As expected, both homozygous and heterozygous mutants displayed a dwarf phenotype (Figure 1n,o). During preparation and review of our manuscript, Li et al. (2023) reported the use of GRAND editing in knock-in of protein tags. In combination with site-specific recombinases, large DNA fragments of up to 11.1 kb were targeted inserted in the rice genome (Sun et al., 2023). Collectively, ours and these results demonstrated the technical advancement for insertion or replacement of DNA fragments in the plant genome, which is of great importance in genetic research and crop breeding. This work was supported by Bellagen Biotechnology Co. Ltd., Nanfan special project, CAAS (ZDXM04, ZDXM23014), National Key R&D Program of China (2021YFA1300404 to J.-K.Z.), National Natural Science Foundation of China (32188102 to J.-K.Z., 32271524 and 31901046 to M.W.), the Youth Innovation Promotion Association, CAS (2020272) to M.W., the Key R&D Program of Ningxia (2021BEB04075) and Ningxia Natural Science Foundation (2022AAC03007) to X.L. The authors declare no competing interests. J.-K.Z. and M.W. conceived of and designed the research. X.L., Y.W., H.W., Y.H., Y.S., Z.L., M.L., C.W., Y.D. L.X. and J.Z. conducted the experiments and analysed the data. J.-K.Z. and M.W. wrote the manuscript. Figures S1-S10 Supplementary Figures Tables S1-S2 Supplementary Tables Data S1 Materials and methods. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.