Theory

Gene editing in plants is a revolutionary domain of biotechnology that facilitates exact, targeted alterations of plant genomes to augment characteristics, bolster tolerance to biotic and abiotic challenges, and expedite crop enhancement. In contrast to conventional breeding techniques, which depend on random genetic recombination and often require extensive time, gene editing enables scientists to implement targeted modifications at specific genomic locations, providing unparalleled control over plant characteristics. This technology offers significant potential for tackling global issues like food security, climate change, and sustainable agriculture. The emergence of sophisticated tools like as CRISPR-Cas systems, TALENs, and zinc finger nucleases enables researchers to modify genes with exceptional efficiency and precision, resulting in the creation of crops with enhanced yield, nutritional value, and environmental resilience. Gene editing is transforming contemporary agriculture and offering robust tools for functional genomics and the investigation of gene regulation in plant biology. With the evolution of regulatory frameworks and public acceptance, gene-edited plants are set to assume a crucial role in the future of sustainable and precision agriculture.

1. CRISPR-Cas Systems (Clustered Regularly Interspaced Short Palindromic Repeats)

The CRISPR-Cas system is the preeminent and transformative gene editing instrument in plant biotechnology, owing to its simplicity, efficiency, and adaptability. CRISPR-Cas, first sourced from the adaptive immune systems of bacteria and archaea, operates by directing a programmable nuclease (often Cas9 or Cas12a) to a specific DNA sequence via a small guide RNA (sgRNA). Upon identification of the target site via base-pair complementarity, the Cas protein induces a double-stranded break (DSB) at that specific locus. The plant cell then rectifies this break via non-homologous end joining (NHEJ)—which may result in insertions or deletions (indels), thereby inactivating the gene—or through homology-directed repair (HDR) if a repair template is available, facilitating accurate gene insertion or correction. Variants such base editors (e.g., cytosine or adenine base editors) and prime editors have enhanced the toolset, allowing for single nucleotide alterations without double-strand breaks, hence minimizing off-target effects and enhancing editing precision.

2. TALENs (Transcription Activator-Like Effector Nucleases)

TALENs are synthetic proteins that integrate a DNA-binding domain from TALEs (Transcription Activator-Like Effectors) of Xanthomonas bacteria with a FokI nuclease domain. Each TALE repeat identifies a particular nucleotide, facilitating the creation of bespoke arrays that can bind to nearly any DNA sequence. When two TALEN constructs attach to neighboring DNA sequences on opposing strands, the FokI domains dimerize, resulting in a double-stranded break at the target location. Analogous to CRISPR-Cas, this cleavage is rectified through NHEJ or HDR mechanisms. The idea of TALENs is based on the modular characteristics of TALE repeats, rendering them extremely selective and minimizing off-target effects. Nonetheless, the design and assembly of TALENs is labor-intensive in contrast to the comparatively simple gRNA design in CRISPR systems, which has constrained their broad application despite their accuracy.

3. Zinc Finger Nucleases (ZNFs)

Zinc Finger Nucleases (ZFNs) were among the initial instruments created for precise genome editing. They comprise a DNA-binding domain formed by zinc finger proteins, each recognizing three base pairs, and a FokI nuclease domain. Similar to TALENs, ZFNs operate as dimers, with two zinc finger arrays attaching to opposing DNA strands next to the targeted cleavage site, thereby facilitating the convergence of FokI domains to generate a double-strand break (DSB). Zinc Finger Nucleases (ZFNs) enable the targeting of specific sequences; yet, their design is intricate due to the context-dependent characteristics of zinc finger-DNA interactions, frequently necessitating substantial tuning. Although ZFNs were groundbreaking in gene editing, they have been mostly replaced by CRISPR and TALENs because of their superior usability, scalability, and efficiency.

4. Base Editing and Prime Editing

These are sophisticated variants of CRISPR-Cas technology, engineered to effectuate exact nucleotide alterations without causing double-strand breaks. Base editors are fusion proteins that integrate a catalytically deficient Cas enzyme (nickase) with a deaminase. Cytidine base editors (CBEs) convert C•G base pairs to T•A, whereas adenine base editors (ABEs) convert A•T to G•C. Prime editors utilize a Cas9 nickase conjugated with a reverse transcriptase and a specifically engineered prime editing guide RNA (pegRNA) that directs the target site and incorporates the intended modification. Prime editing facilitates insertion, deletion, and all twelve possible base substitutions with less off-target effects and without the necessity of a donor template. Both techniques are optimal for precision breeding and functional genomics in plants, particularly when nuanced mutations are necessary.

Each gene editing technique presents unique advantages and compromises for efficiency, specificity, design simplicity, and the nature of permissible modifications. CRISPR-Cas has emerged as the preeminent platform owing to its versatility and reduced design intricacy, but TALENs and ZFNs continue to be advantageous for applications requiring elevated specificity or intellectual property concerns. The advent of base and prime editing has introduced an enhanced level of precision, expanding the horizons of plant genetic engineering beyond conventional gene knockouts.

Out of all these gene editing techniques, CRISPR-Cas system is the most trending and most important among all. Here are a few examples of gene editing in crop plants for enhanced stress tolerance by CRISPR-Cas system.

Figure 1 Schematic display of mechanistic insights of CRISPR/Cas9-based genome editing in plants. The Cas9 protein is guided by a desired single guide RNA (sgRNA) and creates a double-strand break (DSB). Subsequently, DNA repair occurs through non-homologous end-joining (NHEJ) or homology-directed repair (HDR) pathways (Source: Kumar et al., 2023).

Drought Stress:

The expression of drought-sensitive (S) genes increases plant vulnerability to drought by causing hormonal imbalance, reducing antioxidant activity, and elevating reactive oxygen species (ROS) generation. The overexpression of AREB1 has demonstrated enhanced tolerance to drought stress, while the AREB1 knock-out mutant exhibited increased vulnerability to drought stress (Singh and Laxmi, 2015). CRISPR/Cas9-mediated targeted mutagenesis of SlLBD40, a transcription factor associated with lateral organ boundaries that improves drought tolerance in tomatoes, demonstrated enhanced drought resilience compared to both overexpressing transgenic and wild-type tomato plants; the knockout of SlLBD40 via CRISPR/Cas9 augmented the drought tolerance of tomatoes (Liu et al., 2020).

Salinity Stress:

In plants, salt stress induces numerous physiological and morphological alterations due to modifications in gene expression and signaling pathways (Prusty et al., 2018). The primary adverse impacts of salt stress include necrosis, premature senescence of older plants, and severe disruption of cellular ions (Julkowska and Testerink, 2015). Numerous genes have been found and described via CRISPR/Cas-based genome editing to enhance plant salt tolerance. The knockout of AtWRKY3 and AtWRKY4 genes in A. thaliana via CRISPR/Cas9 resulted in substantial up-regulation of genes in response to salt and methyl jasmonate stressors. These double mutant plants exhibited susceptibility to salt and methyl jasmonate, characterized by increased ion leakage and decreased antioxidant activities, including peroxidase (POD), catalase (CAT), and superoxide dismutase (SOD) (Chen et al., 2021). CRISPR/Cas9-mediated knock-out mutants of abscisic acid (ABA)-induced transcription repressors (AITRs) genes enhanced salt stress tolerance in soybean (Glycine max) (Wang et al., 2021b). These mutant plants exhibited heightened ABA sensitivity and generated longer roots and shoots compared to wild-type plants. Likewise, knock-out mutants of the GmDrb2a and GmDrb2b genes exhibited increased salt stress tolerance in G. max (Curtin et al., 2018). The regulatory function of the GT-1 element in rice prompted CRISPR/Cas9-mediated editing of OsRAV2 gene expression, resulting in enhanced tolerance to salt stress (Duan et al., 2016). CRISPR mutants exhibiting loss of function in SnRK2 and osmotic stress/ABA-activated protein kinases SAPK-1 and SAPK-2 genes shown resistance to salt stress in rice (Lou et al., 2017).

Heavy metal stress:

Heavy metal stress is a significant issue that negatively impacts the agricultural productivity of plants (Jha and Bohra, 2016). Plants undergo oxidative stress when exposed to heavy metals, resulting in cellular damage (Yadav, 2010). Moreover, the buildup of metal ions in plants disrupts cellular ionic equilibrium. Consequently, plants have evolved detoxifying mechanisms to mitigate the detrimental effects and buildup of heavy metal exposure. These mechanisms encompass the regulated removal of harmful ions from roots, metal absorption, effective neutralization of metal ions within the protoplast, and the allocation or transfer to distant organs (Sruthi et al., 2017). Multiple genes regulate these pathways to improve tolerance to heavy metal stress (Hasanuzzaman et al., 2019). The loss-of-function mutant of γ-glutamylcyclotransferase exhibited protective traits against heavy metal toxicity, indicating that the loss-of-function mutants of OXP1 and γ-glutamylcyclotransferase facilitate detoxification of heavy metals and xenobiotics through enhanced glutathione (GSH) accumulation (Paulose et al., 2013). Consequently, generating CRISPR/Cas9-mediated mutants would be advantageous in combating heavy metal stress in plants. Recently, Baeg et al. (2021) engineered oxp1/CRISPR mutant Arabidopsis plants exhibiting resistance to cadmium, indicating an enhanced capacity for heavy metal detoxification in the mutants relative to wild-type Col0 plants. This study demonstrated a mechanism to impart tolerance to xenobiotics and heavy metals in plants through indel mutations via gene-editing techniques (Baeg et al., 2021).