Creation of herbicide‐resistance in allotetraploid peanut using CRISPR/Cas9‐meditated cytosine base‐editing

Genome editing has been employed to introduce accurate and predictable genetic mutations in plants, gaining wide acceptance due to its high efficiency, precision, usability and significant potential for crop improvement (Manghwar et al., 2019). The recent discovery of CRISPR-associated base editors, such as the cytidine base editor (CBE) that converts C∙G to T∙A (Komor et al., 2016) and the adenine base editor (ABE) that converts A∙T to G∙C (Gaudelli et al., 2017), has considerably broadened the scope of genome editing by enabling the creation of irreversible point mutations. This base editor approach has facilitated the genetic enhancement of numerous plant species. However, it remains unclear whether the base editing system would function seamlessly in the allotetraploid peanut (Arachis hypogaea), one of the most vital annual oilseed crops. Acetolactate synthase (ALS), which catalyses the initial step in the biosynthetic pathway to valine, isoleucine and leucine, is targeted by over 50 commercial herbicides that are extensively utilized for weed control. A single base mutation at a specific target site in the ALS nucleotide sequence, such as a natural point mutation converting Pro197 to Ser197 or Leu197 in ALS (numbered based on the corresponding Arabidopsis thaliana ALS sequence), leads to resistance to ALS-inhibiting herbicides (Jin et al., 2022). In this study, we generated herbicide-resistant peanut plants by introducing a targeted single-base substitution into the A. hypogaea ALS (AhALS) gene using the CBE system. Based on the released genome data in PeanutBase (https://www.peanutbase.org/gbrowse_peanut1.0), A. hypogaea contains four AhALS copies: AhALS1-A, AhALS1-B, AhALS2-A and AhALS2-B. Phylogenetic analysis revealed that AhALS2-A and AhALS2-B are more closely related to the Arabidopsis homologue than to their paralogs, AhALS1-A and AhALS1-B (Figure S1). Consequently, AhALS2-A and AhALS2-B were selected as the target genes for engineering herbicide resistance using base-editing in peanut in this study. To generate herbicide-resistant peanut plants, we selected a region encompassing Pro197 as the target site and employed the CBE system to replace the C with a T at position Pro197 in both AhALS2-A and AhALS2-B (Figure 1a). A CRISPR construct (Figure 1b) was created by integrating a designed sgRNA, driven by the AtU6 promoter, into the pCSGAP01 vector (Wimi Biotechnology, Changzhou, Jiangsu, China). We used this construct to transform A. hypogaea cultivar Yuhua 9326 via microprojectile bombardment. To evaluate the efficacy of the base-editing procedure, we divided the bombarded calli into two sets. For one set, including about 14 thousand embryogenic calli, 141 hygromycin B (Hyg)-resistant T0 callus lines were selected over three consecutive 3-week periods using MS medium containing Hyg but without bispyribac-sodium (BS). The second set, including about 29 thousand embryogenic calli, was screened for 3 cycles, each lasting 3 weeks on Hyg-BS-containing MS medium, yielding 13 independent lines of Hyg-BS-resistant T0 calli. Next, DNA extracted from the calli that were transformed independently underwent PCR amplification using gene-specific primers targeting the regions of AhALS2-A and AhALS2-B. The resulting amplicons were then genotyped via Sanger sequencing. Six out of the 141 callus lines (4.25%) in the group without BS selection and all 13 lines in the group with BS selection (100%) were precisely base-edited, displaying double peaks on the sequencing chromatogram within the base editing window (Figure 1c). All 19 examined lines exhibited the desired C-T substitution in the protospacer at positions 6–8 (designated PAM as positions 21–23). Seven lines incurred a single base-editing mutation in either AhALS2-A or AhALS2-B, and 12 lines underwent mutations in both genes simultaneously. At these mutation sites, three sites conversed into a single heterozygous C-T substitution at the targeted C base, resulting in P197S; 26 sites converted into two or three heterozygous or homozygous C-T substitutions in the target sequence, resulting in P197F; and two sites converted into a single heterozygous T-G, a single homozygous C-T and two heterozygous C-A substitutions at the targeted C base, leading to the P197N amino acid substitution. Overall, the efficiency of base-editing was approximately 3.5% (10/282) for both AhALS2-A and AhALS2-B. In contrast to the T0 plants that only carried the heterozygous C-T substitution at the corresponding site, the T1 and T2 plants, which derived from the same T0 editing event, harboured both homozygous and heterozygous mutations in addition to the wild type (WT) (Figure 1d). It is, therefore, plausible to assume that T0 plants can pass on the mutations introduced via base-editing to their progeny. All the T1 progenies were sprayed with a 5 mg active ingredient (ai)/L (field-recommended dose) concentration of tribenuron-methyl (Ryan Pingan, Zhengzhou, Henan, China) at the 4 ~ 6-leaf stage. Two weeks after treatment, the WT plants and null segregants were dead. In contrast, the herbicide treatment had no lethal impact on plants with base-editing mutations in either AhALS2-A or AhALS2-B or both (Figure 1e). T2 plants could survive the herbicide treatment at high dosages of 15 mg ai/L and 30 mg ai/L (Figure 1f,g). The gained herbicide resistance appears to be gene dosage-dependent, whereby the T2 plants with simultaneous base conversions in both AhALS2-A and AhALS2-B genes grew and thrived upon herbicide application, while those with mutations in only one ALS exhibited no newly grown leaves (Figure 1f). In light of these findings, it is conceivable that peanut mutants harbouring a combination of multiple herbicide-resistant alleles were more resistant to herbicide exposure than those with a single mutation. Potential off-target sites were predicted using CRISPR-P 2.0 (http://crispr.hzau.edu.cn/CRISPR2/) (Liu et al., 2017). Five of the most likely candidate off-target sites, including AhALS1-A and AhALS1-B, with scores above 0.1 in T1 double mutant lines were chosen and analysed by PCR and Sanger sequencing. The analysis showed no discernible difference between WT and base-edited plants, ruling out the possibility of off-target mutations. In conclusion, we have demonstrated in allotetraploid peanut that the CBE base-editing system can precisely introduce an intended single nucleotide substitution into the targeted ALS gene site, resulting in desired herbicide resistance. This novel germplasm is useful in its own right for integration into peanut breeding programs. Further, the deployment of the CBE system in the genetic modification of various other traits is expected to significantly advance yield and quality improvement as well as functional genomic research in this economically significant oilseed crop. This study was supported by grants from the National Key Research and Development Program of China (2022YFD1200400), the Joint Funds of Natural Science Foundation of Henan Province (222301420026), the Key Project of Science and Technology of Henan Province (201300111000, 221100110300), the Programs for Science and Technology Development of Henan Province (212102110256), China Agriculture Research System of MOF and MARA (CARS-13) and the Henan Province Agriculture Research System (S2012-5). The authors declare no conflict of interest. L.S. and X.Z conceived the study; L.S., X.L., L.X., J.Z., B.H., Z.Z., X.D., S.H. and W.D. performed the experiments. L.S. wrote the manuscript and X.Z. revised the manuscript. All authors read and approved the final manuscript. Figure S1. Phylogenetic tree for ALS homologues in peanut and Arabidopsis. Table S1. sequences of primers used in this study. 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