**5. Genome editing and CRISPR-Cas9**

Genome editing is a form of genetic engineering that involves the intentional alteration of DNA within living cells through the insertion, deletion, or modification of genetic material. Genome editing, previously referred to as gene targeting, is a precise method for modifying the nucleotide sequence of the genome with an exceptional level of specificity down to the individual base pair. It encompasses a range of strategies and methods designed to make deliberate and customized modifications to the genetic makeup of an [70]. The essence of this editing technology is its reliance on site-specific nucleases (SSNs), which are customizable enzymes proficient in precisely targeting specific gene sequences. By utilizing these modified nucleases, it becomes feasible to precisely remove, insert, or replace particular gene sequences, demonstrating the benefits of site-directed mutagenesis when compared to random mutagenesis methods [71]. Genome editing techniques encompass a range of approaches, including tailored homing nucleases (meganucleases), zinc-finger nucleases (ZFNs), and transcription activator-like effector nucleases (TALENs). These methods employ protein-based systems that can be tailored to possess precise DNA-binding functions, allowing them to pinpoint specific gene sequences. The most widely adopted platform in recent times is clustered regularly interspaced short palindromic repeats-CRISPR-associated 9 nucleases (CRISPR-Cas9), which relies on RNA as a targeting element directing the nuclease to a specific DNA sequence [72, 73].

#### **5.1 CRISPR-Cas9 in maize**

Maize (*Zea mays* L.) is recognized as the most widely grown grain crop globally. Its versatility in terms of applications and adaptability to diverse environmental and soil conditions have contributed to its popularity as a desirable crop across the globe [74]. The drawbacks of random mutagenesis sparked investigations into precise genome modification methods, resulting in notable progress over the last decade. These methods have significantly increased the accuracy of gene editing, enhancing fidelity by nearly a thousandfold [75, 76]. The initial generation of targeted genome editing methods, including ZFNs and TALENs, achieved partial success but exhibited specific limitations [77].

In contrast, the CRISPR/Cas9 system, a relatively recent addition, has revolutionized the field of genome editing due to its simple design, operational flexibility, and cost-effectiveness [78–80]. The system consists of a universally used Cas9 nuclease protein and a solitary guide RNA (sgRNA) that includes a 20-base pair (bp) target site sequence along with a hairpin structure. The Cas9 protein induces a double-strand break (DSB) at the 20 bp genomic locus specified by the sgRNA, occurring near the NGG sequence called Protospacer Adjacent Motif (PAM), where N can represent any nucleotide. Cas9 consists of two catalytic nuclease domains, RuvC and HNH, responsible for creating DSBs at precise target sites guided by sgRNA. These DSBs can subsequently be repaired through two main mechanisms: Non-homologous endjoining (NHEJ) or Homology-directed repair (HDR) (see **Figure 1**). It's worth noting that a mere 20 bp sequence is ample for achieving allele specificity within single-copy regions of a genome, such as in maize. The compact size of the sequence simplifies the creation of sgRNA [81, 82].

In maize protoplasts, the CRISPR/Cas9 system achieved a targeted mutation efficiency of 13.1% in the phytic acid biosynthesis gene, ZmIPK, whereas TALENs achieved 9.1% [83]. Furthermore, the CRISPR technology demonstrated a mutation frequency in maize that was 10–20 times greater than that observed with homing endonucleases [84]. In a more recent advancement, a user-friendly public sector system known as "ISU Maize CRISPR" has been established to facilitate efficient site-specific mutagenesis in maize. It employs an *Escherichia coli* cloning vector and an Agrobacterium binary vector, enabling the incorporation of as many as four single guide RNAs (sgRNAs) for single or multiplex mutagenesis. This development marks

**Figure 1.**

*A visual model depicting the genome modification process of the CRISPR/Cas9 system.*

*Enhancing Maize (*Zea mays *L.) Crop through Advanced Techniques: A Comprehensive Approach DOI: http://dx.doi.org/10.5772/intechopen.114029*

a significant stride in applying CRISPR/Cas9 for multifaceted gene editing in crops, with a specific focus on maize [85].

Delivering the CRISPR/Cas9 system, comprising the sgRNA and Cas9 protein, can be accomplished through transient techniques or by employing a long-term maize transformation process into the cell. Furthermore, this method allows for multiplexing [82]. Unlike some other nucleases, CRISPR/Cas9 is capable of targeting methylated DNA, rendering it a versatile tool for editing plant genomes [86].

#### **5.2 Potential benefits of genome editing and ethical considerations**

Genome editing starts from a molecular understanding of the target gene and utilizes a targeted and precise approach based on specific molecular knowledge. This allows genome editing to achieve highly accurate and intentional modifications in a controlled manner. One of the notable concerns linked to genome editing in plants is the potential for unintended genetic changes arising from off-target mutations [87, 88]. These off-target mutations refer to unintended changes in the DNA sequence of the plant's genome that occur at sites other than the intended target. Such unintended alterations can have unpredictable effects on the plant's characteristics and may raise safety and environmental concerns. The concerns surrounding genome editing also stem from the limited understanding of its principles and applications among the general public. There is a need for clear communication and education to address the knowledge gap and foster informed discussions about the technology. Differentiating between various categories of genetically modified plants, such as transgenic plants and genome-edited plants [89, 90], is essential. This distinction aids in comprehending the precise methods employed, the scope of genetic alterations made, and the possible consequences regarding safety, regulation, and public perception. By addressing these concerns and promoting transparency, scientists, policymakers, and stakeholders can work together to ensure responsible and ethically sound use of genome editing technologies in plant research and crop improvement. The risks related to the emerging techniques in genome editing encompass various interconnected aspects, including environmental, health-related, agricultural, economic, social, and political concerns. Among these, only a limited subset of risks is directly associated with the new techniques. One notable risk is the potential for bioterrorism, although it is currently only a theoretical concern when it comes to plants. Genome editing can have both positive and negative implications for agricultural risks, particularly in relation to biodiversity. On one hand, it has the potential to contribute to a reduction in biodiversity. On the other hand, it can also be utilized to enhance diversity and address emerging threats in agriculture.
