Base editing represents a major breakthrough in the field of genome editing in recent years. By fusing deaminases with the CRISPR/Cas system, it enables precise single-base modifications of DNA. This review systematically summarizes the development of base editing technologies, including cytosine base editors (CBEs), adenine base editors (ABEs), and glycosylase base editors (GBEs), with a particular focus on their applications in crop improvement as well as future trends and prospects. We highlight advances in the creation of novel germplasm with enhanced stress resistance and desirable agronomic traits through base editing in rice, wheat, maize, potato, and other crops, particularly for improving herbicide resistance, disease resistance, and grain quality. Furthermore, we analyze factors that influence base editing efficiency, noting that challenges remain, such as PAM sequence constraints, limited base conversion types, off-target effects, narrow editing windows, and efficiency variation. Future efforts should aim to optimize deaminase activity, expand PAM compatibility, and develop versatile tools to facilitate the broad application of base editing in molecular breeding. This review provides a timely reference for researchers and breeders, offering theoretical guidance and practical insights into harnessing base editing for crop genetic improvement.
Histone acetylation is the earliest and most well-characterized of post-translation modifications. It is mediated by histone acetyltransferases (HAT) and histone deacetylases (HDAC). Histone acetylation could change the chromatin structure and status and further regulate gene transcription. In this study, nicotinamide, a histone deacetylase inhibitor (HDACi), was used to enhance the efficiency of gene editing in wheat. Transgenic immature and mature wheat embryos harboring a non-mutated GUS gene, the Cas9 and a GUS-targeting sgRNA were treated with nicotinamide in two concentrations (2.5 and 5 mM) for 2, 7, and 14 days in comparison with a no-treatment control. The nicotinamide treatment resulted in GUS mutations in up to 36% of regenerated plants, whereas no mutants were obtained from the non-treated embryos. The highest efficiency was achieved when treated with 2.5 mM nicotinamide for 14 days. To further validate the impact of nicotinamide treatment on the effectiveness of genome editing, the endogenous TaWaxy gene, which is responsible for amylose synthesis, was tested. Utilizing the aforementioned nicotinamide concentration to treat embryos containing the molecular components for editing the TaWaxy gene, the editing efficiency could be increased to 30.3% and 13.3%, respectively, for immature and mature embryos in comparison to the 0% efficiency observed in the control group. In addition, nicotinamide treatment during transformation progress could also improve the efficiency of genome editing approximately threefold in a base editing experiment. Nicotinamide, as a novel approach, may be employed to improve the editing efficacy of low-efficiency genome editing tools such as base editing and prime editing (PE) systems in wheat.
Wheat is a globally important food crop, and its yield is crucial for ensuring food security. Lunxuan 13 is an elite wheat variety developed by the Institute of Crop Sciences, Chinese Academy of Agricultural Sciences. It has high yield potential and outstanding agronomic traits, such as excellent seed setting rate, plump kernels, and good lodging resistance. However, this variety is highly susceptible to pre-harvest sprouting (PHS) when exposed to rain during the maturation period, leading to premature grain germination on the spike, which causes yield losses and quality deterioration, severely restricting its popularization. This study focused on addressing the PHS susceptibility of Lunxuan 13 by employing CRISPR/Cas9 technology for the targeted knockout of the three homoeologous copies (A, B, and D subgenomes) of TaQsd1, a key gene regulating seed dormancy. A total of 41 transgenic plants were obtained, achieving a transformation efficiency of 52.6%, among which 27 plants exhibited edits at the target sites, resulting in an editing efficiency of 65.9%. Phenotypic analysis of homozygous T2 edited lines revealed significant functional redundancy among the three TaQsd1 homoeologs: a significant extension of the seed dormancy period and a substantial increase in PHS resistance were achieved only when all three A, B, and D copies underwent loss-of-function mutation (aabbdd genotype). After-ripened seeds from these mutants showed normal germination ability, indicating enhanced dormancy rather than loss of germination capacity. Importantly, all of the edited lines exhibited no significant differences compared to the wild type in key agronomic traits such as plant height, spike length, and grains per spike, thus retaining the excellent characteristics of Lunxuan 13. This study successfully optimized Lunxuan 13 for significantly enhanced PHS resistance while retaining its superior agronomic traits. This work provides an effective approach for improving PHS resistance in white-grained wheat and removes a key barrier to the potential commercialization of this variety.
Wheat is one of the globally important food crops, but its genetically modified application lags behind other staple crops due to its complex genome and low transformation efficiency. Normally, traditional transgenic and gene‐edited wheat requires multiple selfing generations for obtaining homozygous lines, which delayed the development of genetically modified wheat varieties. In this study, we crossed a haploid inducer line with purple embryos (HIPE) as a male parent with a wheat cultivar Fielder, and then chose the haploid immature embryos without purple colour to be transformed with the Agrobacterium cells carrying the expression vectors with GUS , RUBY , and Cas9 and sgRNAs for targeting TaWaxy for generating both transgenic and gene editing wheat plants. Consequently, 139 haploid transgenic wheat plants were obtained with transformation efficiencies ranging from 66.7% to 78.4% according to molecular analysis, histochemical staining assay and colour observation. After chromosome doubling treatment to the haploid transgenic plantlets, homozygous transgenic and gene‐edited wheat lines were produced in T 0 generation. Genetic analysis of T 1 generation plants confirmed the stable inheritance of the transgenes and edited alleles without segregation. Finally, we established, for the first time, an efficient genetic transformation system based on the use of wheat haploid immature embryos, enabling direct acquisition of homozygous transgenic and gene‐edited wheat in T 0 generation. This approach can significantly shorten the wheat transgenic and gene‐edited breeding cycle and will provide a novel strategy for the genetic improvement of other polyploid crops.