Advancements in genome editing tools for genetic studies and crop improvement

Abstract

The rapid increase in global population poses a significant challenge to food security, compounded by the adverse effects of climate change, which limit crop productivity through both biotic and abiotic stressors. Despite decades of progress in plant breeding and genetic engineering, the development of new crop varieties with desirable agronomic traits remains a time-consuming process. Traditional breeding methods often fall short of addressing the urgent need for improved crop varieties. Genome editing technologies, which enable precise modifications at specific genomic loci, have emerged as powerful tools for enhancing crop traits. These technologies, including RNA interference, Meganucleases, ZFNs, TALENs, and CRISPR/Cas systems, allow for the targeted insertion, deletion, or alteration of DNA fragments, facilitating improvements in traits such as herbicide and insect resistance, nutritional quality, and stress tolerance. Among these, CRISPR/Cas9 stands out for its simplicity, efficiency, and ability to reduce off-target effects, making it a valuable tool in both agricultural biotechnology and plant functional genomics. This review examines the functional mechanisms and applications of various genome editing technologies for crop improvement, highlighting their advantages and limitations. It also explores the ethical considerations associated with genome editing in agriculture and discusses the potential of these technologies to contribute to sustainable food production in the face of growing global challenges.

Keywords: CRISPR/Cas9; RNA interference; TALEN; ZFNs; crop improvement; genome editing; meganuclease.

Figure 1

The RNA-mediated gene silencing pathway was first described by Jinek and Doudna in 2009. In this pathway, siRNA molecules play a crucial role in silencing target genes by guiding the sequence-dependent slicing of their target mRNAs. These non-coding RNAs initially exist as long dsRNA molecules, which are then processed by the endonuclease Dicer into short, active constructs of approximately 21-25 nucleotides. Once formed, a siRNA duplex is loaded onto Argonaute (AGO2), the central component of the RNA-induced silencing complex, with the assistance of the RNA-binding protein TRBP. AGO2 then selects the siRNA guide strand, cleaves, and removes the passenger strand. The guide strand, while bound to AGO2, pairs with its complementary target mRNA, allowing AGO2 to slice the target. After slicing, the cleaved target mRNA is released, and the RNA-induced silencing complex is recycled, utilizing the same guide strand for multiple rounds of slicing.

Figure 2

Zinc finger nucleases: as highly-specific ‘genomic scissors’ (Simmons and Douglas, 2016).

Figure 3

Diagram of a TAL effector structure and sequence. (A) TALE protein visualization. The middle section of repeats (represented by blue squares) is responsible for DNA binding. Additionally, the nuclear localization signals (NLS) and acidic activation domains (AAD) are included in the illustration. The amino acid sequences for individual repeats within a typical array are displayed; repeat variable di-residues (RVDs) are highlighted in blue, while dashes indicate conserved amino acid residues. (B) Frequencies of RVD-nucleotide associations. The size of the letter in the sequence logo indicates how often RVDs are linked to specific bases (Moscou and Bogdanove, 2009).

Figure 4

A depiction of the TALEN structure. The extended perspective of the TALEN displays the sizes of different sections, including a typical TAL effector array and its corresponding nucleotide target. The alignment of a complete TALEN pair is illustrated, with the TALEN target sequence emphasized in red (Christian and Voytas, 2015).

Figure 5

(A) CRISPR loci are composed of approximately 24-47 bp palindromic repeat sequences (highlighted in red), which are interspersed with 26-72 bp spacer sequences (highlighted in blue). These spacer sequences do not share any common features in terms of their sequences. The maximum number of repeats can reach up to 249, as reported by Kim and Kim, 2014. (B) In type II CRISPR systems, tracrRNA binds to the pre-crRNA repeat to create duplex RNAs that are then cleaved by the host RNase III (PDB ID: 2EZ6), a process that may also involve Cas9. (C) Subsequent trimming of the leftover repeat sequences from the 5ʹ end is carried out by an unidentified nuclease, as described by Wiedenheft et al., 2012. The activation of Cas9 protein occurs through the binding of gRNA. This binding induces a conformational change in the Cas9 protein, leading to the activation of its nuclease activity. The RuvC and HNH domains are responsible for the specific and efficient cleavage of the target DNA when complemented with crRNA (highlighted in green), (Jinek et al., 2014).

Figure 6

Various genome editing systems used in plants can be compared based on their mechanisms. These systems include site-specific genome editing tools (GETs) such as protein-dependent DNA cleavage systems (A), RNA-dependent DNA cleavage systems (B), and RNA cleavage systems (C). Protein-dependent DNA cleavage systems, such as ZFNs and TALENs, utilize sequence-specific proteins to guide the FokI nuclease to the desired DNA site. Similarly, TALENs consist of two sequence-specific TALEN proteins that guide the FokI nuclease. On the other hand, RNA-dependent DNA cleavage systems (B), such as CRISPR/Cas9, CRISPR/Cpf1, and CRISPR/C2c1, induce double-strand breaks (DSBs) using the Cas9 nuclease and single-guide RNA. The repair of DSBs can occur through non-homologous end joining (NHEJ) or homologous recombination (HR), with NHEJ often leading to gene knock-out mutations and HR resulting in gene knock-in or replacement. In contrast, RNA-dependent RNA cleavage systems (C), like single-strand break (SSB), can cause random or targeted mutations through error-prone NHEJ or error-free HR, respectively. These genome editing approaches involve the insertion, deletion, or replacement of specific DNA sequences (Ahmad et al., 2020).

Figure 7

The analysis of different genome editing techniques, including CRISPR/Cas, TALENs, ZFNs, RNAi, and meganuclease, across published literature from 2013 to 2023 reveals that CRISPR is the most extensively studied method, with the largest number of articles. The remaining four methods are ranked below CRISPR in terms of publication frequency.