1 Introduction

The yield of wheat is mainly related to grain weight, which is generally expressed as "thousand-grain weight" (TGW). The larger the grain weight, the higher the yield, so increasing grain weight has always been a key goal in breeding (Wang et al., 2018).

TaGW2 is a gene that controls grain weight. It produces a protein called "RING finger E3 ubiquitin ligase". This gene is a "negative regulator", which means that it inhibits grain enlargement. If this gene is knocked out or expressed less, the grain will become larger and the yield will increase. However, if this gene is expressed too much, although it can enhance drought resistance, it may reduce yield under ideal planting conditions (Wang et al., 2018; Li et al., 2023). This gene also plays a role in crop response to adverse environments. It not only affects yield, but is also related to stress resistance. Now, CRISPR/Cas9 technology can be used to "edit" this gene very accurately. Researchers have successfully created plants with single, double, and triple mutations, all of which can make the grains larger, and this change can be inherited (Zhao et al., 2024). Recently, research has also improved CRISPR editing methods, such as the ability to modify multiple genes at a time and more efficient gene introduction methods. These new advances make the application of this technology in wheat breeding faster and more accurate.

This study reviews the structure and expression of TaGW2, summarizes the latest progress in using CRISPR/Cas9-mediated TaGW2 gene editing technology to increase wheat grain size, and explores the challenges, opportunities, and future development directions of this technology in wheat breeding. This study emphasizes the potential of CRISPR/Cas9 technology as a transformative tool for sustainable genetic improvement of crops, hoping to better utilize the targeted editing technology of the TaGW2 gene to increase wheat yield.

2 Structure and Expression Characteristics of TaGW2

2.1 Gene structure and conserved sequence analysis

TaGW2 is a gene related to grain size. It encodes a protein called "E3 ubiquitin ligase". This protein has a special "ring finger" structure, which is critical to its function. In hexaploid wheat, this gene has three versions, namely TaGW2-6A, 6B and 6D. Its coding sequence is 1 275 bases, which can produce a protein containing 424 amino acids. Studies have found that the coding part of this gene is not much different in different wheat varieties, that is, it is very conservative. But its promoter region is more different, especially in the 6A and 6B versions. These differences affect grain size and weight and are the reason for the formation of different haplotypes (Su et al., 2010; Qin et al., 2014). The C-terminus of the protein, that is, the LXLX region near the end (amino acids 376 to 424), is particularly important for identifying the target protein. This segment helps TaGW2 bind to other proteins involved in grain development, thereby exerting its effects (Lü et al., 2022).

2.2 Expression patterns in different tissues and developmental stages

TaGW2 is always expressed in wheat. All three versions (6A, 6B, and 6D) are expressed, but with different intensities. Generally speaking, 6A is expressed the most, 6B is in the middle, and 6D is the least, especially at the critical stage of grain development (Qin et al., 2014). This gene is not only expressed in grains, but can also be seen in leaves, stems and other parts, indicating that it plays a wide range of roles in the growth and development of wheat (Figure 1) (Su et al., 2010; Zhang et al., 2022).

Figure 1 Subcellular localization and spatiotemporal expression of TaGW2L and TaGW2 in wheat (Adopted from Zhang et al., 2022) Image caption: (A) Subcellular localization of TaGW2L and TaGW2 in wheat protoplasts. Scale bars, 10 µm. (B) Expression pattern analysis of TaGW2L and TaGW2 in wheat. SR, seedling roots; SS, seedling stems; SL, seedling leaves; ER, roots at elongation stage; ES, stems at elongation stage; EL, leaves at elongation stage; HR, roots at heading stage; HS, stems at heading stage; HL, leaves at heading stage; 1YS-7YS, 1 cm-7 cm young spikes; 5D-25D, grains at 5-25 days after pollination. Values are presented as mean ± SD (Adopted from Zhang et al., 2022)

2.3 Spatiotemporal expression during key grain developmental stages

During grain development, the expression of TaGW2 is particularly active during the rapid cell division period and the late filling period. This suggests that it may play a regulatory role in endosperm cell growth and nutrient accumulation (Bednarek et al., 2012; Qin et al., 2014). Studies have found that the expression of this gene is related to the width of the grain. The lower the expression, the larger the grain and the higher the thousand-grain weight (TGW) (Su et al., 2010). The variation in its promoter region will further affect the expression level of this gene at different stages, thereby affecting the final grain size.

3 CRISPR/Cas9 System Design and Editing Strategy

3.1 Target site selection and gRNA construction

To edit TaGW2, the first step is to find a suitable target. Generally, a more conservative region of this gene is selected, which makes it easier to interrupt its function. gRNA (guide RNA) is specially designed to simultaneously recognize three versions of TaGW2 in wheat (i.e. 6A, 6B, and 6D). We will use some computational tools to help with the design, which can reduce accidental damage to other genes (Wang et al., 2018). In order to knock out multiple versions of genes at the same time, multiple gRNAs can be strung together and connected with tRNA. In this way, a transcript can carry multiple gRNAs, which will be accurately cut in the cell, improving the overall editing efficiency (Xie et al., 2015).

3.2 Construction of editing vectors and system validation

Usually, we will put the CRISPR/Cas9 system into a binary vector. This vector contains the Cas9 protein, which is driven by a strong promoter (such as the Ubi promoter in maize), and a gRNA module controlled by the wheat U6 promoter (Zhang et al., 2019). We use Agrobacterium to transfer this system into wheat cells, which is more stable than the gene gun and can reduce the number of transformations and gene silencing (Zhang et al., 2019). In transgenic wheat, we will check whether Cas9 and gRNA are expressed normally and confirm whether the target site is successfully edited.

3.3 Mutant identification and mutation type analysis

We use PCR to amplify the target region of TaGW2 and then use sequencing to confirm whether mutations have occurred. In wheat, the most common mutation is "deletion", which sometimes exceeds 10 base pairs. Homozygous mutants or biallelic mutants can sometimes be obtained in the first generation (T0), and sometimes need to wait until the next few generations to appear (Zhang et al., 2019). The CRISPR/Cas9 system can continue to work in the next generation, sometimes generating new mutations in the offspring, which can bring more genetic changes (Wang et al., 2018). In order to ensure that this system does not cut randomly, we also perform off-target checks. Many studies have found that as long as the gRNA is well designed, off-target problems will hardly occur or cannot be detected at all.

4 Phenotypic Changes and Grain Weight in TaGW2 Mutants

4.1 Analysis of grain length, width, and thousand-kernel weight

Studies have found that TaGW2 gene mutations can increase grain size and thousand-grain weight (TGW). Compared with the wild type, single-gene mutations can increase grain length by about 2%, width by 2%-3%, and TGW by 5%-7% (Simmonds et al., 2016; Wang et al., 2018; Bi et al., 2024). If two genes mutate together, the effect is more obvious, and TGW can be increased by 10%-12%. When all three genes mutate, the increase in TGW is the largest, reaching 16%-21% (Zhang et al., 2018). This increase is quite stable in different wheat varieties and environments. Moreover, the more mutations there are, the more obvious the increase in grain weight is. This shows that these genes have an additive effect. In particular, the two alleles TaGW2-6B and TaGW2-6A have a greater effect on grain width and weight (Yang et al., 2012).

4.2 Effects of gene editing on plant architecture and growth period

Although we mainly want to improve grain traits by editing TaGW2, it sometimes affects other agronomic traits. Studies have found that some TaGW2 mutations are related to early heading and early maturity of wheat, and may also change plant height and ear structure (Jaiswal et al., 2015). However, most edited varieties have no major changes in the number of spikelets and grains. In other words, these mutations mainly make the grains themselves larger and do not affect the appearance of the whole plant much (Simmonds et al., 2016). Sometimes there is a small "trade-off": the grain weight increases, but the number of grains per ear may decrease slightly. But overall, the yield can still be maintained, or even higher (Zhai et al., 2017; Vicentin et al., 2024).

4.3 Correlation analysis between phenotype and genotype

The number of copies of TaGW2 is negatively correlated with grain size and TGW. In other words, the more copies are mutated, the larger and heavier the grains are (Wang et al., 2018; Zhang et al., 2018). Genetic variation in the promoter region can also affect expression levels, which may be one of the reasons for the phenotypic differences between different varieties (Qin et al., 2014; Jaiswal et al., 2015). Now, we can use SNPs or haplotypes related to TaGW2 for molecular marker-assisted selection, so that we can more quickly select lines with larger grain weight and higher yield (Yang et al., 2012). Different combinations of homologous genes and the special reactions between varieties show that the relationship between genotype and phenotype is very critical. Understanding these relationships will help to better optimize breeding strategies (Bi et al., 2024).

5 Comparison of Editing Effects in Different Genetic Backgrounds

5.1 Research findings from the Crop Science Institute, Chinese Academy of Agricultural Sciences

Researchers from the Chinese Academy of Agricultural Sciences found that after editing TaGW2 homologous genes in different wheat varieties, the grains became larger and the thousand-grain weight (TGW) was significantly improved. However, the increase was different for different varieties. For example, when single mutations were made in the A, B and D genomes, the mutation effect of the B and D genomes was the best. The two varieties Paragon and Bobwhite performed best in this regard. The study also found that if two or even three homologous genes were mutated, the improvement of TGW would be more obvious, reaching 16% to 21%. This difference may be related to the gene expression level of the variety itself. This shows that genetic background plays a big role in the results of gene editing (Simmonds et al., 2016; Wang et al., 2018; Zhang et al., 2018).

5.2 Multi-gene editing experiments conducted by CSIRO, Australia

CSIRO, Australia, and its partners conducted multi-gene editing experiments. They used CRISPR/Cas9 to simultaneously edit TaGW2 and other agronomic trait-related genes in hexaploid wheat. The experimental results showed that after knocking out all three homologous TaGW2 genes, the grains became larger, TGW was also greatly improved, and these traits could be stably inherited (Figure 2). More importantly, this editing system can continue to work for several generations. They also hybridized these edited lines with other wheat varieties to further transfer these excellent traits. This shows that this method can be used in different genetic backgrounds and is a very efficient breeding method (Wang et al., 2018).

Figure 2 The effects of single-, double-, and triple- KO mutations in the TaGW2 gene homoeologues on the grain morphometric and TGW traits in Bobwhite. The image of twenty seeds from wild-type, single-, double- and triple- mutant plants (scale bar 1 cm). b-e Box and whisker plots show the distribution of TGW (b), grain area (c), grain width (d), and grain length (e) for wild-type and mutant wheat lines. The datasets from Bobwhite and the T0 progeny plants carrying wild-type TaGW2 alleles were combined because they did not show statistical differences. The mean value for each genotype is shown as a red circle. The genotypes of the TaGW2 homoeologues are shown in all panels with lower and uppercase letters corresponding to the mutant and wild-type alleles, respectively, for the A, B, and D genome homoeologues (color figure online) (Adopted from Wang et al., 2018)

5.3 Field evaluation of mutants by Nagoya University, Japan

The team at Nagoya University in Japan conducted field trials. They introduced TaGW2 mutations into tetraploid and hexaploid wheat, such as splice acceptor site mutations in TaGW2-A1. These mutations significantly increased wheat grain weight, grain width and grain length under various environments. Moreover, they did not affect the number of spikelets or grains. This shows that editing TaGW2 can not only increase yield, but also has no negative effects (Simmonds et al., 2016).

Editing TaGW2 does have a significant effect. However, the performance will vary between different varieties and different homologous genes. From global research, editing multiple genes together will have a better effect (Simmonds et al., 2016; Wang et al., 2018; Zhang et al., 2018).

6 Integration of CRISPR/Cas9 with Breeding Pipelines for Grain Weight Improvement

6.1 Combination with genomic selection (GS) for selection accuracy

CRISPR/Cas9 can accurately edit key genes such as TaGW2. But if it is used with genomic selection (GS), the breeding effect will be better. GS uses genome-wide markers to predict which plants have potential. In this way, breeders can select plants that not only have the target edited gene but also have a good genetic background. This combination can find materials with larger grains and higher yields more quickly. This method has been proven to be effective in other crops (Awan et al., 2022; Ahmar et al., 2023).

6.2 Integration with high-throughput phenotyping (HTP) platforms

High-throughput phenotyping (HTP) can quickly measure the grain size, weight and other agronomic traits of many wheat plants using machines and sensors. If HTP is combined with CRISPR/Cas9 editing, the best performing mutants can be quickly screened. This will allow for faster evaluation of the effects of these edits in different environments and genetic backgrounds. This approach will make the breeding process simpler and more efficient, and ensure that only the most promising lines advance to the next stage of promotion (Awan et al., 2022; Ahmar et al., 2023).

6.3 Introgression of edited traits into commercial cultivars

Once we have found CRISPR-edited lines with heavy grains and good traits, we can use conventional hybridization methods to introduce these good genes into existing commercial varieties. In this process, marker-assisted selection can also improve efficiency. Rapid breeding and accelerated generation advancement technology can also make this process faster. This approach has been successful in other cereals. This shows that this strategy is also promising for wheat, and can help us breed high-quality non-GMO wheat varieties suitable for large-scale planting (Liang et al., 2017; Awan et al., 2022).

7 Concluding Remarks

Site-directed editing of the TaGW2 gene homolog in wheat using CRISPR/Cas9 technology has been shown to significantly affect grain size and thousand-grain weight (TGW). The study found that the more sites that were mutated, the more obvious the improvement in TGW. TGW increased in single, double, and triple mutations, with the triple mutant increasing TGW by 16% to 21%. However, this phenotypic effect also varies depending on the wheat variety and the location of the edit in the genome. This suggests that the expression level of TaGW2 may be different in different varieties. Overall, these results prove that TaGW2 is a gene that suppresses grain size, and "knocking it out" can help increase yield.

CRISPR/Cas9 technology can quickly and stably change key yield genes like TaGW2. It can also reduce the possibility of off-target effects, allowing the modified traits to continue in future generations. This technology can also edit multiple genes at once, which is very helpful for improving complex traits. These advances also show that gene editing technology can be gradually applied to conventional wheat breeding, helping us breed varieties with strong adaptability and high yield.

In order to maximize the advantages of CRISPR/Cas9, future breeding work will also need to combine it with genomic selection, high-throughput phenotyping, and efficient gene introduction methods. This combined strategy will accelerate the discovery and utilization of superior alleles, thereby rapidly increasing wheat yields. It is also important to continue studying the different functions of TaGW2 and its interactions with other yield genes. This will further optimize breeding methods and help to breed new high-yield and stress-resistant wheat varieties. CRISPR/Cas9 targeted editing of TaGW2 is a precise, powerful, and scalable method. It not only increases grain weight and yield, but is also suitable for integration into modern breeding systems, and has great potential.

Acknowledgments

I appreciate Dr Xu from the Hainan Institution of Biotechnology for her assistance in references collection and discussion for this work completion.

Conflict of Interest Disclosure

The author affirms that this research was conducted without any commercial or financial relationships that could be construed as a potential conflict of interest.

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