Genome Editing Technologies Accelerate Innovation in Soybean Breeding

1. Introduction

Human existence is intricately intertwined with crops, which can serve as abundant sources of food, feed, and all kinds of consumable resources. Among these vital crops, soybean (Glycine max L. Merr.) holds particular significance as a crucial food and cash crop. Nevertheless, with the ever-expanding global population and the dwindling availability of arable land worldwide, there is an urgent imperative to promote innovation in soybean breeding through cutting-edge biotechnology. In the realm of genome editing technologies, three classic types have emerged: Zinc Finger Nucleases (ZFNs), Transcription Activator-Like Effector Nucleases (TALENs), and Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)/CRISPR-associated 9 (Cas9); these have been skillfully applied in numerous important crops. It is worth noting that CRISPR/Cas9 has proven to be a game-changing breakthrough in soybean breeding over the past few years, which can achieve site-specific mutations to accurately introduce target traits to soybean.

Soybean holds a crucial position as a significant source of vegetable protein and oil worldwide. Meeting the increasing demand for high-quality vegetable protein and grade oils in soybean seeds has become a priority. Over the last few years, one of the key strategies to enhance soybean oil quality to meet both nutritional needs and industrial standards is the alteration of fatty acid profiles. A particular focus has been on increasing the content of oleic acid in soybean oil, as it not only boosts the oil’s nutritional value but also enhances its stability. To achieve this, researchers have identified two crucial regulatory genes, FAD2-1A and FAD2-1B, which are delta-twelve fatty acid desaturase genes and play pivotal roles in controlling the oleic acid content. Through the successful application of genome editing tools such as TALENs and CRISPR/Cas9, soybean varieties with elevated levels of oleic acid and reduced levels of linoleic and linolenic acids have been skillfully developed [1,2]. These advancements have paved the way for the commercial production of high-oleic-acid soybean cultivars, representing a significant breakthrough in the soybean breeding landscape. Besides, the versatility of the genome editing tool CRISPR/Cas9 has proven instrumental in improving various agronomic traits of soybean, encompassing growth period, plant architecture, and taste.

2. Genome Editing Technologies Promote the Improvement of Soybean Agronomic Traits

2.1. Broaden the Geographical Versatility of Soybean Adaptation

As a short-day plant, soybean is significantly impacted by the variations in day length experienced at different latitudes. This photoperiodic response plays a critical role in defining the regions where soybean can thrive and its capacity for widespread cultivation. For the past few years, genome editing technologies have played important roles in soybean regional introduction and domestication. Using genome editing to knock out some genes, such as GmFT2a, GmFT5a, GmFT2b or GmAP1, to delay flowering time can increase the suitability of soybean varieties for lower latitude regions [3,4,5]. In contrast, knocking out E1 or GmPRR37 can promote flowering and enhance the adaptability of soybean varieties to higher latitude regions [6,7].

2.2. Optimize the Structural Design of Soybean Architecture

Plant architecture plays a significant role in determining the grain yield of many crops. We hold the opinion that the ideal plant architecture of soybean should have the following characteristics: semi-dwarf, sufficient number of determined nodes, and short internode. For the past few years, the genome editing tool CRISPR/Cas9 has been used to improve soybean plant architecture. For instance, the application of CRISPR/Cas9 system has led to varied degrees of increase in the number of nodes on the main stem and branches in Gmspl9 mutants [8]. Additionally, GmLHY, a gene encoding a MYB transcription factor, plays a crucial role in regulating plant height through the gibberellic acid (GA) pathway, has been targeted using CRISPR/Cas9 to create soybean quadruple lhy mutants. These mutants exhibit reduced plant height and shortened internodes when compared to wild-type plants [9]. These results indicate that genome editing technologies may hold tremendous potential for enhancing soybean plant architecture.

2.3. Improve the Quality and Taste of Soybean Seeds

The beany flavor poses a drawback to the taste of soybeans and is under the regulation of three lipoxygenases (LOXs), namely LOX1, LOX2, and LOX3. By using CRISPR/Cas9 technology, researchers have successfully created gmlox1 gmlox2 gmlox3 triple mutants and gmlox1 gmlox2 double mutants, both of which exhibit the loss of corresponding lipoxygenase activities [10]. These results indicate that gene editing has great potential in improving soybean quality and taste.

3. Perspectives for Future Applications of Genome Editing in Soybean

3.1. Precise Targeting of Soybean Genes Accelerates Functional Analysis

Genome editing systems enable researchers to target specific genes within the soybean genome with high precision. This precision ensures that only the intended genes are targeted, reducing the risk of off-target effects and promoting the safety and predictability of the process, and then accelerates gene functional analysis. Using genome editing, the process of gene function validation in soybean can be significantly accelerated. Researchers can efficiently create targeted mutations in candidate genes and study the resulting phenotype, which expedites the identification of genes responsible for crucial traits.

3.2. Expand the Delivery Systems of Genome Editing in Soybean

So far, genome editing systems used in soybean have been delivered mainly by Agrobacterium tumefaciens, but its delivery efficiency remains relatively low. In a cutting-edge development, researchers have introduced the use of nanoparticles for genome editing in wheat. In this innovative study, they employed naturally occurring carbon dots, characterized by their quasi-spherical shape and nanometer-scale size (<10 nm), to efficiently deliver CRISPR/Cas9 vectors into mature plant cells. This remarkable technique achieved transient plant transformation and successful gene editing [11]. In addition, developing various suitable small Cas9 orthologs and using viruses to deliver them is a promising strategy in soybean [12].

3.3. Enhance the Diversity of Optional Sequences for Genome Editing

At present, the commonly used Cas9 in soybean is derived from Streptococcus pyogenes (SpCas9), which targets a 5′-NGG-3′ protospacer adjacent motif (PAM). Additionally, researchers have identified a smaller endonuclease, Staphylococcus aureus Cas9 (SaCas9), recognizing a 5′-NNGRRT-3′ PAM (where R represents a purine, A or G) in soybean [13]. Furthermore, the successful implementation of a type V CRISPR-Cas system, Cpf1 (Cas12a), has allowed for targeting a thymidine-rich 5′-TTTN-3′ PAM in FAD2 paralogues in soybean [14]. However, many other genome editing systems, which recognize NAC, NTT, NTG, NCG, or NG PAM sequences, have limited application in soybean research. Hence, incorporating more effective systems into future gene editing research in soybean holds promise for advancing the field.

3.4. Improving the Efficiency of HDR-Based Genome Editing in Soybean

An ideal genome-editing tool should offer greater flexibility in modifying genes as desired. While CRISPR/Cas9 has demonstrated the ability to delete large genomic fragments [15] and achieve target base editing in soybean [16], HDR-mediated gene replacement has proven more challenging. Enhancing the operational reliability of HDR is currently the focal point to address, along with the need to increase the availability of donor DNA templates. To tackle this, we propose the use of nanoparticles or suitable viruses as carriers for delivering Cas9-gRNA vectors and donor DNA templates, thus facilitating efficient HDR-based genome editing in soybean.

3.5. Rapid Aggregation of Various Good Agronomic Traits by Genome Editing

We hold the opinion that the future advancement of soybean agriculture will be driven by the simultaneous incorporation of desirable agronomic traits, necessitating the concurrent editing of multiple genes. However, the current application of genome editing in soybean primarily focuses on improving individual traits. Achieving simultaneous editing of multiple genes can be accomplished through two approaches: either by simultaneously knocking out multiple homologous genes using one sgRNA or by connecting multiple sgRNA expression cassettes in series within a single vector. In addition, this strategy needs higher efficiency of genetic transformation. Therefore, we propose the optimization of platforms for sgRNA design and selection, vector assessment, high-throughput genetic transformation, and mutation type identification, specifically tailored for soybean, to facilitate the rapid integration of diverse agronomic traits through genome editing.