Advances in CRISPR/Cas9-based research related to soybean [Glycine max (Linn.) Merr] molecular breeding

Soybean [Glycine max (Linn.) Merr] is a source of plant-based proteins and an essential oilseed crop and industrial raw material. The increase in the demand for soybeans due to societal changes has coincided with the increase in the breeding of soybean varieties with enhanced traits. Earlier gene editing technologies involved zinc finger nucleases and transcription activator-like effector nucleases, but the third-generation gene editing technology uses clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9). The rapid development of CRISPR/Cas9 technology has made it one of the most effective, straightforward, affordable, and user-friendly technologies for targeted gene editing. This review summarizes the application of CRISPR/Cas9 technology in soybean molecular breeding. More specifically, it provides an overview of the genes that have been targeted, the type of editing that occurs, the mechanism of action, and the efficiency of gene editing. Furthermore, suggestions for enhancing and accelerating the molecular breeding of novel soybean varieties with ideal traits (e.g., high yield, high quality, and durable disease resistance) are included.


Introduction
Soybean is a significant source of vegetable proteins for humans and an important oilseed crop, making it a commercially valuable plant (Zhang A, et al., 2023).More than 90% of the soybean plants cultivated in the three main soybean-producing countries (USA, Brazil, and Argentina) are genetically modified varieties generated using gene editing technology (Fang et al., 2023).In terms of sustainable food production, the demand for soybeans has continued to increase because of the scarcity of arable land.In the field of molecular breeding, clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) has emerged as a commonly used third-generation gene editing technology (Nakamori, 2023).Thus, many new and desirable soybean traits have been developed using gene editing technology, which is currently a hot topic in scientific research (Osakabe and Osakabe, 2017;Chen et al., 2022;Zhou et al., 2023a).
In recent years, CRISPR/Cas9 gene editing technology has been used by plant molecular breeders to improve various plant traits (Ma et al., 2016;Zhang et al., 2017;Rao et al., 2022).Because it can simply, effectively, and precisely edit target genes responsible for specific characteristics, CRISPR/Cas9 has replaced previously used gene editing techniques (Zheng et al., 2021;Impens et al., 2022;Liu H. et al., 2022).Several crop traits, including yield, quality, stress tolerance, disease resistance, and herbicide resistance, can be improved using CRISPR/Cas9 systems.This can lead to the development of novel germplasm with superior traits as well as significant advancements in plant molecular breeding (Yin et al., 2017;Hussain et al., 2018;Wada et al., 2020;Gan and Ling, 2022;Qi et al., 2023).
The limitations of early genome editing methods included the inability to explore the relationships between several related genes (Li et al., 2013;Nekrasov et al., 2013;Shan et al., 2013).These previous methods were mostly employed to edit individual genes.Because soybean is a paleotetraploid, it has many homologous and redundant genes, which makes the functional characterization of soybean genes challenging (Tran and Mochida, 2010;Du et al., 2023).The CRISPR/Cas9 system has recently been used to edit multiple genes in the soybean genome.This has considerably decreased the effects of redundant genes on the efficient editing of specific genes for breeding soybean varieties with desirable traits (Bao et al., 2020;Xu H. et al., 2020;Baek et al., 2022;Guan et al., 2022;Rasheed et al., 2022a).
This review describes the recent improvements in soybean traits via the application of the CRISPR/Cas9 gene editing technology.It also presents information regarding the target genes and their mechanism of action, while providing a brief overview of transformation efficiency and gene editing efficiency.Furthermore, suggestions for future CRISPR/Cas9 development and use in soybean molecular breeding programs are included.

Application of CRISPR/Cas9 gene editing technology in soybean molecular breeding
There has recently been an increase in the use of CRISPR/Cas9 to edit genes in soybean, corn, wheat, rice, cotton, and other crops (Figure 1, Table 1).The creation of new soybean germplasm with many excellent traits using various transformation methods (e.g., Agrobacterium-mediated transformation) has laid the foundation for further improving CRISPR/Cas9 gene editing technology for soybean molecular breeding (Figure 2).

Enhancement of soybean resistance to abiotic stresses
During different soybean developmental stages, many genetic and biochemical processes control how soybean perceives and responds to abiotic stresses, including salinity and drought.One of the primary objectives of molecular breeding research is improving stress tolerance (Deshmukh et al., 2014;Amoanimaa-Dede et al., 2022;Cadavid et al., 2023).Osmotic stress in plant cells is typically caused by abiotic factors (e.g., drought or excessive  salinity).Analyses of the sequences of the related genes revealed the regulatory effects of various plant cellular components, such as sensors, receptors, phytohormones, transcription factors, kinases, phosphatases, and microRNAs, on abiotic stress response-related pathways (Ramesh et al., 2019;Mangena, 2020;Staniak et al., 2023).
Water deficiency substantially restricts soybean growth and development, which can decrease the soybean yield by up to 40% (Khan, 2018).Thus, there is a critical need for exploring the mechanism underlying soybean drought resistance and generating new drought-resistant soybean germplasm (Ramlal et al., 2022).By deleting miR398c in soybean, Zhou et al. (2020) increased the expression of GmCSD1a/b, GmCSD2a/b/c, and GmCCS (relative to the corresponding levels in over-expression strains), thereby increasing the capacity to scavenge O 2− (Zhou et al., 2020).In 2021, Xiao et al. identified 112 GmPLA family genes in the soybean genome and used CRISPR/Cas9 technology to knock out two homologous genes (GmpPLA-II epsilon and zeta).Knocking out one or both genes affected the root response to phosphorus deficiency, with some mutant lines exhibiting increased resistance to flooding and drought conditions (compared with the control) (Xiao et al., 2021).Additionally, in 2021, Yu et al. reported that the GmNF-YC14 deletion mutant created using CRISPR/Cas9 technology is more susceptible to drought stress than wild-type soybean, implying GmNF-YC14 may be useful for increasing soybean drought tolerance (Yu et al., 2021).By comparing the agronomic features of soybean plants over-expressing sHSP26 with those of soybean plants in which sHSP26 had been edited, Liu S. Y., et al. (2022) revealed that sHSP26 may considerably increase soybean drought tolerance and yield (Liu S. Y., et al., 2022).In 2022, Yang et al. edited the soybean transcription factor gene GmNAC12, which decreased the survival of the transgenic plants exposed to drought stress by at least 12%.They concluded that GmNAC12 is a key gene that positively regulates soybean tolerance to drought conditions (Yang C.F., et al., 2022).
Salinity can severely decrease the seed yield and quality of soybean, which is a salt-sensitive crop species (Phang et al., 2008;Cai et al., 2022;Feng et al., 2023).In addition to accelerating the development of salt-tolerant soybean varieties to increase grain yield, research on salt stress tolerance can also optimize the use of saline farmland (Chen et al., 2018;Jin et al., 2021).In 2021, Niu et al. clarified the effects of knocking down and over-expressing lncRNA77580 on the expression of nearby protein-coding genes linked to the soybean response to salt stress.Additionally, increases in the length of the DNA fragment deleted from lncRNA77580 via the application of CRISPR/Cas9 technology increased the changes in the expression of lncRNA77580 and nearby genes (Niu et al.,FIGURE 2 General soybean genetic transformation process.The following steps are generally included in the genetic transformation of soybean: sprouting, cotyledon treatment, infestation, induction of healing tissue, indeterminate shoot induction, elongation, and rooting.A schematic diagram is provided to show how the CRISPR/Cas9 system cleaves the target genomic segment.

2021)
. By simultaneously targeting six GmAITR genes using a CRISPR/Cas9 system, Wang et al. (2021) produced a Cas9-free GmAITR3 and GmAITR6 double mutant and a GmAITR2 GmAITR3 GmAITR4 GmAITR5 GmAITR6 quintuple mutant.They determined that salt tolerance was more pronounced in the higher-order mutants, suggesting that mutating GmAITR genes can enhance soybean salt tolerance (Wang et al., 2021).Zhang M.H. et al. ( 2022) produced three soybean mutants in which GmSOS1 was edited and observed that Na + accumulated significantly more in the mutants than in the control.Accordingly, this gene is essential for soybean salt tolerance because it helps maintain Na + homeostasis (Zhang M.H., et al., 2022).
The adaptation of soybean to severe drought and salt stresses involves the activation of overlapping pathways at the morphological, physiological, and molecular levels.Drought tolerance and salt tolerance are polygenic traits (Chen et al., 2018;Kofsky et al., 2018;Mammadov et al., 2018).Additionally, the perception of stress and its effects on soybean growth or development are similar among the abiotic stress factors.In an earlier study by Du et al. (2018), soybean plants in which the transcription factor gene GmMYB118 was silenced were more susceptible to drought and saline conditions than soybean plants over-expressing GmMYB118.Moreover, the decreased production of minor heat shock proteins increased the resistance of plants to drought, cold, and salt stresses (Du et al., 2018).However, when Zhang M.H. et al. (2022) knocked out GmHsps_p23, which encodes a minor heat shock protein in soybean, the transgenic plants were highly susceptible to salt and drought conditions.Future research will need to focus on the use of several gene editors to simultaneously target and regulate the expression of functional genes mediating drought and salinity tolerance to produce novel soybean genotypes with superior traits (Zhang Y.Z., et al., 2022).

Enhance disease and insect resistance in soybean
Tobacco ringspot virus, soybean dwarf virus, soybean vein necrosis virus, soybean mosaic virus (SMV), bean pod mottle virus, and alfalfa mosaic virus are only a few of the viruses that can infect soybean (Liu et al., 2016;Widyasari et al., 2020;Lin et al., 2022).Multiple viruses can simultaneously infect soybean plants, causing more harm than an infection by a single virus.Hence, the use of gene editing tools to target genes that control soybean disease resistance and improve disease resistance-related traits has become a major objective in soybean molecular breeding programs (Chang et al., 2016;Chandra et al., 2022;Zhao et al., 2023).
Several non-homologous end-joining and homology directed repair-mediated gene replacement mutants were produced by Fang et al. (2015), who targeted the soybean blast fungal pathogenicity gene Avr4/6.These mutants were more resistant to diseases caused by oomycetes than the controls (Fang and Tyler, 2016).Ochola et al. (2020) edited the usual effector genes of the soybean root pathogen Phytophthora sojae.They observed that disease resistance was affected by the Avr gene expression level in soybean (Ochola et al., 2020).In 2020, Ma et al. confirmed that GmLMM2 deficiencies increased the resistance to P. sojae by increasing tetrapyrrole biosynthesis, but decreased the chlorophyll content by disrupting tetrapyrrole biosynthesis.The elimination of GmLMM2 expression resulted in the appearance of necrotic regions in the growing leaves of the CRISPR/Cas9-edited mutants (Ma et al., 2020).Zhang P.P, et al. (2020) targeted GmF3H1, GmF3H2, and GmFNSII-1 in soybean plants (including the hairy roots) using a CRISPR/Cas9-mediated multiple gene editing system.They detected a significant increase in the isoflavone content and a significant decrease in the SMV coat protein content (approximately 33% decrease) in the mutants, indicating that the increased isoflavone content enhanced the leaf resistance to SMV (Zhang P.P., et al., 2020).Three crucial genes in the soybean Rsc4 gene family (Rsc4-1, Rsc4-2, and Rsc4-3) were modified by CRISPR/Cas9 in 2021 to alter soybean resistance to SMV (Yin et al., 2021).To investigate the effector gene Avr1b-1 in the soybean pathogen Blastomyces in terms of its function as well as the underlying mechanism.Gu et al. (2021) created target locusspecific knockout and knock-in mutants.All selected knockout mutants were virulent on plants expressing Rps1b, whereas the infection of plants lacking Rps1b was unaffected.When a sgRNAresistant variant of Avr1b-1 was re-introduced into the Avr1b-1 locus of the mutants in which Avr1b was knocked out, the resulting knockin transformants expressing Avr1b-1 were unable to infect soybean plants carrying Rps1b (Gu et al., 2021).Compared with the RNAi and over-expression strains, the soybean plants in which GmDRR1 was knocked down (in 2022) were considerably less resistant to Blastomyces infections (Yu et al., 2022).By altering the coding region of the soybean transcription factor gene GmTCP19L, Fan et al. (2022) produced a mutant with a 2 bp deletion.This mutant soybean germplasm resource exhibited increased susceptibility to blast molds (Fan et al., 2022).
Plants that are resistant to Rps gene products can perceive certain pathogen effectors encoded by Avr genes.By deleting Avr45a, Arsenault-Labrecque et al. (2022) produced novel soybean plants resistant to Rps8 (Arsenault- Labrecque et al., 2022).In 2022, Zhang et al. identified Glyma.07g110300(LOC100775351) as a quantitative trait locus (QTL)-M marker gene encoding the UDP-glycosyltransferase (UGT) primarily responsible for soybean resistance to leaf-chewing insects.Using a CRISPR/Cas9 system, they enhanced the resistance of soybean to Helicoverpa armigera and Spodoptera litura via the following two mutation types: large fragment deletion and single base insertion.Zhang Y.X., et al. (2022) confirmed that GmUGT confers resistance to leaf-chewing insects by changing the flavonoid content and the expression of genes related to flavonoid biosynthesis and defense (Zhang Y.X., et al., 2022).By editing the soybean 14-3-3 gene (Glyma05g29080) via large fragment insertions and deletions and producing transgenic plants with increased susceptibility to hard tick infestations and decreased nodulation, Zhang Y.F., et al. (2023) showed Glyma05g29080 contributes to nodulation and defense responses (Zhang Y.F., et al., 2023).Using a CRISPR/Cas9 gene editing method, Liu et al. (2023b) silenced GmTAP1 in soybean, which resulted in increased resistance to P. sojae strains P231, P233, and P234.An analysis of reactive oxygen species revealed that a loss-of-function mutation to GmTAP1 does not substantially alter plant basal immunity (Liu T.F., et al., 2023).

Improvement of seed quality in soybean
Soybean is used as a source of food for animals, including humans (Medic et al., 2014).It has the highest protein content of any crop and is a significant source of edible oils (Gupta and Manjaya, 2022;Zaaboul et al., 2022;Song et al., 2023).In the past few years, several studies have employed CRISPR/Cas9 gene editing technology to enhance the protein and oleic acid contents of soybean.
Using germinal root transformation technology, Li et al. altered the soybean seed storage protein-encoding genes Glyma.20g148400,Glyma.03g163500, and Glyma.19g164900 to increase soybean seed protein contents (Li et al., 2019a).By simultaneously modifying the soybean genes GmFAD2-1A and GmFAD2-1B, Do et al. (2019) managed to increase the oleic acid content by more than 80%, while also decreasing the linoleic acid level by 1.3%-1.7%(Do et al., 2019).Zhang et al. (2019) silenced the soybean phospholipase D1encoding gene, which increased the oil content and germination rate of the mutant seeds (compared with the wild-type seeds) at high temperatures and high humidity levels (Zhang et al., 2019).In 2021, Qu et al. analyzed the oleic acid contents of soybean plants over-expressing Gm15G117700 and soybean plants in which the gene was edited; the oleic acid content increased in the gene-edited plants by 3.49% (Qu et al., 2021).Zhou et al. (2023a) recently edited five important enzyme-encoding genes in the GmFAD2 family and analyzed the associated effects on soybean oil synthesis.Editing GmFAD2-1A increased the oleic acid content by 91.49% (Zhou et al., 2023a).In another recent study, Li et al. (2023) edited two target genes by altering the conserved PAP2 structural domainencoding sequences of GmPDCT1 and GmPDCT2.The decrease in phosphatidylcholine-derived diacylglycerol contents via the k n o c k d o w n o f G m P D C T p r e v e n t e d t h e e n t r y o f phosphatidylcholine-modified polyunsaturated fatty acids into the triacylglycerol biosynthesis pathway (Li et al., 2023b).
In addition to increasing the protein and oleic acid contents, researchers have attempted to enhance other soybean characteristics.Phytic acid (PA) is an anti-nutrient in grains that prevents humans from absorbing trace minerals (e.g., iron and zinc).In soybean, GmIPK1 encodes an enzyme that converts inositol 1,3,4,5,6-pentaphosphate to inositol 1,2,3,4,5,6hexaphosphate (Alkarawi and Zotz, 2014;Sarkhel and Roy, 2022).Using the CRISPR/Cas9 system, Song et al. (2022) edited the GmIPK1 gene and sgRNA to introduce mutations to create soybean lines with low PA levels.The decreased PA levels in the T 2 generation mutant seeds were not accompanied by defective growth or seed development (Song et al., 2022).
Flavor is an important soybean quality-related attribute.Accordingly, CRISPR/Cas9 technology has been exploited to develop soybean germplasm with superior flavor-related traits (Fernandez-Marin et al., 2014).Because soybean proteins are allergens, decreasing the abundance of allergenic proteins will likely increase the utility of soybean as a source of protein (e.g., in processed food) (Cordle, 2004;L'Hocine and Boye, 2007;Gharibzahedi et al., 2022;Gracio et al., 2023).In 2020, Sugano et al. simultaneously targeted and edited GmBd28k and GmBd30K to eliminate two allergenic proteins in the Japanese soybean cultivars Enrei and Kariyutaka (Sugano et al., 2020).Soybean flavor and quality are influenced by three lipoxygenases (LOX1, LOX2, and LOX3).By editing three genes in the soybean GmLox family (GmLox1, GmLox2, and GmLox3), Wang J., et al. (2020) improved the edibility of soybean oil and protein products.Editing these genes decreased soybean odors (Wang J., et al., 2020).The raffinose oligosaccharide (RFO) family members are the main soluble carbohydrates in soybean seeds, but they are antinutritional seed components because they typically cause gas and indigestion, while also decreasing energy efficiency (Salvi et al., 2022).In 2021, Le et al. decreased the soybean seed RFO content by knocking down two galactinol synthase-encoding genes, namely GmGOLS1A and its homolog GmGOLS1B (Le et al., 2020).To decrease the RFO content in mature seeds, Cao et al. (2022) used a CRISPR/Cas9 multi-gene editing method to delete the RS2 and RS3 genes in soybean and cottonseed (Cao et al., 2022).Qian et al. (2022) mutated GmBADH2 and confirmed this gene contributes to soybean odors (Qian et al., 2022).In addition, Bai et al. (2022) used CRISPR/Cas9 gene editing technology to produce two multi-gene mutants, one lacking the 7S subunit and the other lacking the 11S subunit.Both of these mutations enhanced the flavor of soybean meal (Bai et al., 2022).

Improvement of phenotype in soybean
One of the key factors influencing the development of highyielding soybean cultivars is the appropriate regulation of plant structural features (e.g., plant height, number of nodes, number of pods, internode length, number of branches, and number of grains) (Hu and Wiatrak, 2012;Kuzbakova et al., 2022).In recent years, soybean phenotype-related genes have been edited using CRISPR/ Cas9 gene editing technology to produce soybean germplasm with a variety of improved features.
Using the CRISPR/Cas9 system, Bao et al. ( 2019) mutated four SPL9 family genes that encode SQUAMOSA promoter-binding protein-like (SPL) transcription factors.The higher-order mutant plants with different combinations of mutations had more nodes and branches on the main stem (compared with the control plants), resulting in varying numbers of nodes per plant (Bao et al., 2019).In 2019, Cheng et al. used four gRNAs to alter four late elongated hypocotyl (LHY)-encoding GmLHY genes in soybean.Phenotypic analyses showed that the quadruple mutant plants had relatively short internodes and exhibited dwarfism (Cheng et al., 2019).In the Tianlong 9 variety, Jia et al. (2020) knocked out two copies of the soybean DCL2 gene, which altered the color of the soybean seed coat from yellow to brown (Jia et al., 2020).To increase soybean production, Cai et al. (2021) modified the low-latitude spring soybean variety Huachun 6 using a CRISPR/Cas9 multi-gene editing technique.Specifically, they targeted GmJAG, which affects the number of seeds per pod (Cai et al., 2021).In 2022, Mu et al. targeted six GmBIC genes in soybean using CRISPR/Cas9 technology.The single, double, and quadruple mutants were shorter than normal (Mu et al., 2022).In another recent study, Zhong et al. (2022) edited the soybean GmHdz4 gene, which increased the total root length, root surface area, and number of root tips (compared with the mutant lines over-expressing GmHdz4) (Zhong et al., 2022).Furthermore, Zhang Z. et al. (2023) silenced the soybean GmNSS gene, which resulted in the production of abnormally small seeds.(Zhang Z. et al., 2023).
Abscisic acid is an essential phytohormone that controls various processes related to plant growth, development, and stress responses (Nguyen et al., 2023).Using a CRISPR/Cas9 system, Zhang Z. H. et al. ( 2022) mutated GmPYL17, GmPYL18, and GmPYL19.Compared with the wild-type plants, the mutants were taller, had more branches, and were less sensitive to abscisic acid during the seed germination stage (Zhang Z. H. et al., 2022).
The shattering of soybean pods can significantly decrease yield.By altering the GmPDH gene family in soybean variety Huachun 6, Zhang Z. et al. (2022) showed that the PDH1 mutation dramatically increases pod shatter resistance without modifying other important agronomic parameters (Zhang Z. et al., 2022).

Regulation of nitrogen fixation by nodules
Rhizobia can produce a symbiotic nitrogen-fixation system with legumes that increases plant output without damaging the local ecosystem (Chakraborty et al., 2022;Hawkins and Oresnik, 2022).More than 65% of the nitrogen fixation is due to the symbiotic interaction between rhizobia and legumes (Fields et al., 2021;Jimenez-Guerrero et al., 2022).Soybean converts free nitrogen in the air to chemosynthetic nitrogen that can be absorbed and used by the plant via nitrogen-fixing nodules.This process yields soybean seeds with a high protein content, thereby increasing the nutritional value of soybean (Dadnia, 2011;Meng et al., 2015;Igiehon et al., 2021).Xu et al. (2021) promoted soybean nodulation by using CRISPR/Cas9 technology to knock down miR9c (Xu et al., 2021).By deleting the soybean RFG193 gene, Fan et al. (2020) generated transgenic plants with mature nitrogen-fixing nodules on purple or red roots, which produced anthocyanins, whereas nodules were undetectable on the non-transgenic roots (Fan et al., 2020).In 2021, Yang et al. reported that a loss-of-function mutation to GmHSP17.9 significantly affects soybean plant growth and seed yield through the associated changes to the number of root nodules, nodule fresh weight, nitrogenase activity, poly-hydroxybutyrate vesicles, and urea and total nitrogen contents (Yang Z.W., et al., 2022).Nguyen et al. (2021) silenced GmUOX in a soybean mutant, which exhibited nitrogen deficit atrophy and early nodule senescence as revealed by decreased nitrogenase (acetylene reduction) activities in the nodules, a greenish-white hue inside the nodules, and a decreased root protein output (Nguyen et al., 2021).Gao et al. (2021) investigated the role of the PIN protein during the nitrogen fixation by soybean nodules.More specifically, they produced a triple mutant (GmPIN1-abc family) (Gao et al., 2021).The modification of the soybean Rfg1 allele by Fan et al. (2022) revealed Rfg1 mediates the resistance to Sinorhizobium fredii and Bradyrhizobium japonicum strains, leading to broad-spectrum resistance to nodulation in transgenic plants (Fan et al., 2017).After knocking down GmNN1, Li et al. (2022) detected yellowing leaves as well as decreased nitrogen contents and decreased nodulation (compared with the wild-type control plants) (Li et al., 2022).By silencing GmNAC039 and GmNAC018 as well as the four target genes GmCYP35, GmCYP37, GmCYP39, and GmCYP4, Yu et al. (2023) showed that the transcription factors encoded by GmNAC039 and GmNAC018 directly increase the expression of GmCYP genes to induce root tumor senescence (Yu et al., 2023).

Regulation of flowering time in soybean
Because soybean is a short-day (SD) plant, it blooms more quickly during SD conditions than during long-day (LD) conditions (Weller and Ortega, 2015;Lin et al., 2021;Xia et al., 2021).Modulating the blooming time and minimizing the sensitivity to sunshine duration through molecular breeding can increase soybean adaptability and production by mitigating photoperiodic responses (Zhang L.X. et al., 2020;Zhang M. et al., 2022;Du et al., 2023).Cai et al. (2018a) edited the soybean genes GmFT2a and GmFT9a and discovered that both mutants in the T 2 generation exhibited a late-blooming phenotype (Cai et al., 2018a).Using a double sgRNA design and CRISPR/Cas9 technology, Cai et al. (2018b) deleted specific DNA fragments in GmFT2a (Glyma16g26660) and GmFT5a (Glyma16g04830).The homozygous GmFT2a mutants (1,618 bp deletion) in the T 2 generation flowered late (Cai et al., 2018b).Two QTL regions that respectively included GmFT2a and GmFT5a were identified by Cai et al. (2020b).They were linked to various genetic effects on flowering during various photoperiods.Under LD and SD conditions, the flowering times of transgenic plants overexpressing GmFT2a or GmFT5a, GmFT2a mutants, GmFT5a mutants, and GmFT2a and GmFT5a double mutants were examined.There was no overlap between GmFT2a and GmFT5a, which cooperatively control the blooming time, but GmFT2a has a greater effect than GmFT5a under SD conditions, while GmFT5a has a greater effect than GmFT2a under LD conditions (Cai et al., 2020a).Wang L. W. et al. (2020) mapped QTLs and identified GmPRR37 as a functional gene encoding a regulator of soybean flowering.A natural mutation to GmPRR37 results in early flowering, thereby enabling the cultivation of soybean plants at high latitudes (Wang L. W., et al., 2020).Li et al. (2020) used CRISPR/Cas9 technology to knock out GmPRR3b.The resulting soybean mutant exhibited retarded growth and a delayed transition to the flowering stage (Li et al., 2020).In 2020, Chen et al. modified the soybean GmAP1 gene in a quadruple mutant.The observed increase in plant height was associated with delayed flowering, altered flower shapes, and increases in the number of nodes and the internode length.In contrast, under SD conditions, the overexpression of GmAP1 led to early flowering and dwarfism (Chen et al., 2020).Li et al. (2021) edited four LNK2 genes using a CRISPR/ Cas9 system to produce a quadruple mutant lacking transgenes.This mutant flowered earlier than the wild-type control under LD conditions.In addition, the LNK2 transcript level was lower in the quadruple mutant than in the wild-type plants (Li et al., 2021).Zhao et al. (2022) mutated GmPHYA or GmPHYB using CRISPR/Cas9 technology.The phenotypic changes due to the mutations to GmPHYA2 and GmPHYA3, which have redundant and additive roles in seedling responses to daylight, indicated GmPHYB1 is primarily responsible for daylight-induced photomorphogenesis (Zhao et al., 2022).In 2022, Zhai et al. suggested that GmMDE and GmFT2a/GmFT5a contribute to a positive feedback regulatory loop that promotes flowering in soybean.Knocking down the soybean E1 gene induces GmMDE expression.Moreover, the over-expression of GmMDE06 increases the expression of GmFT2a and GmFT5a, which regulate flowering (Zhai et al., 2022).In 2023, Wan et al. investigated the relationship between the dominant E1 gene and photoperiodic regulation via the CRISPR/Cas9-mediated targeted mutation of E1 in soybean variety Tianlong 1. Four mutations were introduced into the E1 coding region.The significant structural changes in the generated mutants included the commencement of terminal flowering, the creation of distinct stems, and a decrease in the number of branches (Wan et al., 2022).

Creation of male sterile soybean germplasm resources
Because soybean is a self-pollinated plant that has small flower organs, artificial cross-breeding is both difficult and ineffective (Li et al., 2019b;Chen G. M., et al., 2021).Furthermore, differences in flowering times among varieties originating from various geographical regions frequently further restrict the exchange of genes, resulting in a limited genetic base for soybean breeding and genetic modifications (Li et al., 2019b).Accordingly, methods for increasing the genetic diversity of soybean varieties are needed (Bohra et al., 2016).In particular, for sexually reproducing crops, male sterility is a crucial precondition for hybrid seed generation and crop reproduction (Jiang et al., 2011;Yang et al., 2014).Male sterile lines can increase the quality of hybrids, lower the cost of hybrid seed production, and even broaden the utility of hybrids.The scarcity of adequate male sterile lines has limited the commercial use of soybean accessions (Li et al., 2016;Ramlal et al., 2022).
To create stable male sterile soybean lines, Chen et al. ( 2021) targeted AMS homologs using CRISPR/Cas9 technology.Although editing GmAMS2 failed to produce a male sterile line, editing GmAMS1 yielded plants with a male sterile phenotype.GmAMS1 contributes to the development of pollen walls as well as the regulation of soybean tapetum degeneration (Chen et al., 2021a).Jiang et al. (2021) modified Glyma.13G114200 using a CRISPR/ Cas9 system; the phenotypes of two gene-edited lines were consistent with the male sterility of the MS1 mutant (Jiang et al., 2021).By eliminating GmSPL2b, Ding et al. (2023) decreased the heat tolerance of a soybean cytoplasmic male sterility-based recovery line during flowering (Ding et al., 2023).

Application of other CRISPR gene editing technology in soybean
Compared with Cas9, the CRISPR family member Cas12a is more practical and effective.Hence, CRISPR/Cas12a can effectively edit multiple genes because of the specific way that CRISPR RNA (crRNA) functions (Bandyopadhyay et al., 2020;Paul and Montoya, 2020;Zhou et al., 2023b).In 2017, Jiang et al. used CRISPR/Cas12a to achieve editing in the soybean FAD2 gene for the first time (Jiang et al., 2017).In addition, large chromosomal segments of the target genome were deleted by Duan et al. (2021) using CRISPR/Cas12a, with an editing efficiency of 91.7% (Duan et al., 2021).In 2023, Liang et al. produced CRISPR/Cas12a-edited soybeans in just 45 days, with transformation and gene editing efficiencies of 30% and 50%, respectively (Liang et al., 2023).To produce gene-edited soybeans with better traits, CRISPR/Cas12a-based multi-gene editing methods will increasingly be used to modify the soybean genome.
Because they enable the replacement of a single base via RNA editing without introducing DNA double-strand breaks or requiring donor templates, base editor tools created using the CRISPR/Cas9 system are especially useful for plant molecular breeding (Molla et al., 2021;Yang et al., 2021;Hua et al., 2022).A CRISPR/Cas9-mediated base editing tool was designed by Cai et al. (2020a) to alter individual bases in the soybean genome.A base editor was developed by combining Cas9n (D10A), rat cytosine deaminase (APOBEC1), and a uracil glycosylase inhibitor.This base editor was then cloned into the pTF101.1 vector.The targeted genes were GmFT2a and GmFT4a, which were under the control of the 2× CaMV 35S promoter.There were two types of base substitutions (C to T and C to G), both of which occurred within the target sequence (Cai et al., 2020a).Single nucleotide polymorphisms, which influence phenotypic diversity and are linked to many significant agronomic parameters, are abundant in the soybean genome.Future genetic improvement and breeding of soybean can greatly benefit from the application of base editing technology (Bharat et al., 2020;Xu R. F., et al., 2020).

Discussion and prospect
Because of increases in the global population and living standards, CRISPR/Cas9 technology must be exploited to quickly develop high-yielding, high-quality soybean varieties (Khan et al., 2018;Zhang and Showalter, 2020).Field tests of high-oleic soybean varieties produced using CRISPR/Cas9 gene editing technology in the US have produced positive results, with potential implications for soybean molecular breeding.There have been considerable advances in the molecular breeding of soybean since the development of CRISPR/Cas9 gene editing technology, which has decreased concerns about the safety of products made from genetically modified soybeans, leading to the gradual acceptance of genetically modified crops.The CRISPR/Cas9 system, which continues to be refined and enhanced, has largely outperformed the older technologies involving zinc finger nucleases and transcription activator-like effector nucleases in terms of gene editing efficiency and convenience (Samanta et al., 2016;Demirci et al., 2018;Farooq et al., 2018).Researchers will use CRISPR/Cas9 gene editing systems to develop soybean lines with improved features as more functional soybean genes are identified and characterized.
However, there are certain limitations to the utility of CRISPR/ Cas9 for soybean breeding.Unanswered questions include the following: (i) How can genome editing tools be efficiently delivered to soybean plants?(ii) How can the functional redundancy in gene families be rapidly and precisely determined?(iii) How can the editing of multiple genes be exploited to modify various traits?(iv) How can base editing, prime editing, and government regulations regarding genome-edited crops further increase the effectiveness of gene editing?Despite encouraging results, many obstacles must be overcome before CRISPR/Cas9 can be widely used for soybean breeding.
Additionally, numerous sgRNAs for different plant genomes have been assembled into CRISPR editing vectors.Moreover, sgRNA pooling techniques have made it possible to mutate multiple genes.The diversity in the sequences that PAM can detect has increased, leading to improved gene editing, because of the creation of Cas9 homologs, such as StCas9 and SaCas9, for plant molecular breeding.The highly efficient editing of plant genomes has been achieved using the nCas9-mediated single-base editing system, while the saturation mutagenesis of plant genomes and optimal gene editing efficiencies have been attained via the two-base editing method.The CRISPR/Cas9 gene editing method will be applied to soybean molecular breeding more effectively, conveniently, and broadly in the future, thereby facilitating increasingly precise molecular breeding and accelerating soybean molecular breeding.