**Abstract**

Biotechnology has made significant advances in recent years, and the area of genetic engineering is progressing day by day, generating several advantages. Through the new ability to precisely change and modify the genomes of living organisms, genome editing technology has transformed genetic and biological research. Genome editing technology first appeared in the 1990s, and different approaches for targeted gene editing have subsequently been created. The fields of functional genomics and crop improvement have been transformed by advances in genome editing tools. CRISPR/Cas9 (clustered regularly interspaced short palindromic repeat)-Cas9 is a versatile genetic engineering tool based on the complementarity of the guide RNA (gRNA) to a specific sequence and the endonuclease activity of the Cas9 endonuclease. This RNA-guided genome editing tool has produced variations in plant biology fields. CRISPR technology is continually improving, allowing for more genetic manipulations such as creating knockouts, precise changes, and targeted gene activation and repression. Soybean is a leguminous crop, high in protein and oil contents that are used for poultry and livestock feed industry. In this chapter, we focus on the recent advances in CRISPR/Cas9-based gene editing technology and discuss the challenges and opportunities to harnessing this innovative technology for targeted improvement of traits in soybean and other crops.

**Keywords:** clustered regularly interspaced short palindromic repeats, genome editing (GE), guide RNA (gRNA), nonhomologous end joining (NHEJ), homology-directed repair (HDR), Cas9

### **1. Introduction**

Nowadays, almost one billion people suffer from malnourishment due to increasing population, and our agricultural system is degrading by the loss of biodiversity and climate change [1]. To overcome the malnourishment, there is a need to improve the crop plants. To achieve this goal, conventional breeding approach is labor-intensive, and it takes several years to form the commercial varieties. Genome editing tools are advanced biotechnological techniques to modify an organism's genome efficiently and precisely. Although recently developed genome editing technologies, such as zinc finger nucleases (ZFN) and transcription activator-like effector nucleases (TALENs),

have many advantages but also has some drawbacks too. CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 technology has site-specific genome editing with precision, efficiency, and ease of use.

The type II CRISPR/Cas system is a prokaryotic adaptive immune response system that guides the Cas9 nuclease to induce site-specific DNA cleavage using noncoding RNAs as a template. The CRISPR/Cas type II system is a flexible genome editing tool for crop improvement [2]. It is a simple, effective, and cost-effective approach that can target several genes. Many plants have advantage from the CRISPR/Cas9 system, including rice, maize, wheat, soybean, sorghum, and barley [3].

The CRISPR/Cas9 system has been utilized for genome editing in all mammalian cells, which may be used to make gene knockouts (through insertion/deletion). A single-guided RNA (sgRNA) is used to guide the Cas9 nuclease to a specific genomic region in order to disrupt genes (**Figure 2**). Double-strand breaks caused by Cas9 are repaired by the NHEJ DNA repair mechanism. Because the repair is prone to errors, insertions and deletions (INDELs) might occur, causing gene function to be disrupted. Cellular DNA repair processes, either the nonhomologous end joining DNA repair pathway (NHEJ) or the homology-directed repair (HDR) pathway, fix the DNA damage or DNA repair pathway (i.e., HDR).

Mechanism of CRISPR/Cas9-mediated gene disruption is as follows: (1) A single-guide RNA (sgRNA) binds to a recombinant form of Cas9 protein with DNA endonuclease activity, consisting of a crRNA sequence specific to the DNA target and a tracrRNA sequence that interacts with the Cas9 protein. (2) The resultant complex will cleave double-stranded DNA that is particular to the target. (3) Then, cleavage efficiency of sgRNAs will be tested.

Crop development techniques should enable to increase production, biotic and abiotic stress resistance, as well as quality and nutritional value. Over several decades, innovative agricultural technology has considerably enhanced crop productivity. Consumers are more concerned about crop quality since it provides many nutrients such as proteins, fiber, vitamins, minerals, and bioactive substances, all of which are directly linked to human health [4]. In addition, scientists and breeders have switched their focus from increasing production to enhancing quality. Traditional crossing breeding, chemical and radiation-mediated mutation breeding, molecular markerassisted breeding (MAB), and genetic engineering breeding have all proven successful in improving various crop qualities [5–8]. Traditional mutagenesis-based breeding techniques are time-consuming and labor-intensive, especially for polyploid crop production [9]. Recently, crop breeding has advantage from genome editing (GE) technology, which alters plant genomes in a precise and predictable manner [10].

Genome editing can produce predictable and inheritable mutations in specified regions of the genome, with minimal off-target effects and no external gene sequence integration. Deletions, insertions, and single-nucleotide substitutions (SNPs) are all examples of GE-mediated alterations. There are four SDN (site-directed nuclease) families in a nucleotide excision process, i.e., homing endonucleases or mega-nucleases (HEs) [11], zinc-finger nucleases (ZFNs) [12], transcription activator-like effector nucleases (TALENs) [13], and CRISPR-associated protein (Cas) [14]. The majority of SDNs can precisely target double-stranded template DNA and produce a double-strand break (DSB). The DSBs are naturally repaired by a plant's endogenous repair system, which uses one of two major DNA damage repair mechanisms: nonhomologous end joining (NHEJ) or homologous-directed recombination (HDR). A FokI cleavage domain and a particular DNA-binding domain from TALE proteins make up TALENs. TALENs technology has a greater target binding specificity and decreased off-target effects when

### *Role of CRISPR/Cas9 in Soybean (*Glycine max *L.) Quality Improvement DOI: http://dx.doi.org/10.5772/intechopen.102812*

compared with ZFNs [15]. In rice [16], wheat [17], maize [16], and tomato [18], it was widely used as a gene-editing method. However, ZFN and TALENs have long construction procedure, which has limited their use in plants on a wide scale. CRISPR (clustered regularly interspaced short palindromic repeat) was first discovered in *Escherichia coli* in 1987 and described as an immunological response to viral and plasmid DNA invasion [19]. CRISPR/Cas systems have become the most popular GE technology in recent years. Because the specificity of editing is governed by nucleotide complementarity of the guided RNA to a specific sequence without protein engineering, the CRISPR/Cas systems are more efficient for genome editing than other SDNs [20].

Soybean is a leguminous crop, has a great economic value, and is high in protein and oil. With the growing demand for soybeans around the world, it is more important to understand gene function and speed up functional gene research and breeding to increase yield and improve quality. Traditional soybean breeding procedures are insufficient to meet the growing demand for soybean products and the problems posed by the agricultural environment. As a result, it is critical to implement quick, precise, and effective breeding procedures in order to develop improved varieties, particularly with improved yield, quality, and stress tolerance or resistance [21, 22]. Genome editing technology is a highly desired technology given the advantages listed above, and it is also an excellent tool for improving soybean genetics. The number of crops engineered by genome editing has increased day by day. Crop quality is one of the most important objectives among the different target traits for crop improvement. Here is a brief description of different quality traits improvement through CRISPR/Cas9-mediated tool.
