Insertional mutagenesis, as a functional genomics approach in plant genetics, has been a powerful tool for studying gene function (Bouchez and Höfte, 1998; Przybyla and Gilbert, 2022; Ayliffe and Pryor, 2009). Traditionally, this has involved the random insertion of genetic elements, such as T-DNA or the maize transposon system (AC/DS), into the plant genome. When these elements carry strong enhancers or promoters, their insertion near a gene can lead to overexpression of that gene (Weigel et al., 2000; Fladung, 2016). An example of this change in transcription pattern due to the insertion of enhancers is presented in Figure 2. This method, known as activation tagging, generates GOF mutants, which are instrumental in uncovering the roles of genes that might otherwise remain functionally hidden, due to redundancy or whose precise role in development may be obscured by the lethality of LOF mutations. Activation tagging has enabled the identification of numerous genes involved in various plant processes, including stress responses, growth regulation, and developmental pathways (Ayliffe and Pryor, 2009; Fladung, 2016; Dutta et al., 2021; Mahendranath et al., 2023; Wan et al., 2009). Examples of successful activation tagging applications include the identification of leaf and fruit color mutants in tomato, such as the anthocyanin color1 (ant1) mutant, which exhibits a significant accumulation of anthocyanins due to the overexpression of a MYB transcription factor regulating anthocyanidin biosynthesis (Mathews et al., 2003). The development of the Purple tomato, a cherry tomato brought to market in 2024 by Norfolk Plant Sciences, traces its success back to activation tagging and the identification of transcription factors that control anthocyanidin biosynthesis (Zhi et al., 2020). Disease resistance genes have also been identified using this technique, with notable discoveries including genes conferring resistance to downy mildew in A. thaliana (Gao et al., 2014; Grant et al., 2003), bacterial blight and sheath blight resistance in rice (Gandikota et al., 2024; Vo et al., 2018; Mori et al., 2007) and trichothecene and FHB resistance in Arabidopsis and wheat (McLaughlin et al., 2015; McLaughlin et al., 2021).

In addition to its use in transcriptional activation, the dCas9 protein has also been repurposed for other applications such as CRISPR interference (CRISPRi) and epigenetic modification by dCas9 epi-editors (Kampmann, 2018; Enright et al., 2024; Fal and Carles, 2024; Cai et al., 2023; Selma, 2024). CRISPRi uses dCas9 fused to transcriptional repressors to inhibit gene expression by sterically blocking transcription or by recruiting repressive chromatin-modifying complexes. dCas9 epi-editors precisely target the genome to modify epigenetic marks such as H3K9me3, CpG methylation, and deacetylation (Selma, 2024; Pan et al., 2021b).

CRISPRi offers a complementary approach to epigenetic editing by effectively “turning off” gene expression without altering the underlying DNA sequence. This is achieved by fusing dCas9 with repressive effector domains, or by simply leveraging dCas9’s ability to physically block transcription when guided to a gene’s promoter or coding region. For example, CRISPRi has been widely used to silence specific genes to study their function in various biological pathways (Khan et al., 2025). This targeted gene knockdown allows researchers to efficiently investigate gene essentiality and complex regulatory networks. For example, the dCas9-SALL1-SDS3 repressor construct effectively blocks the transcription of target genes without introducing double-stranded breaks (Mohamad Zamberi et al., 2024; Han, 2023). The ability to precisely and reversibly repress gene activity makes CRISPRi an invaluable tool for functional genomics, analogous to the use of conditional mutants in conventional genetic studies.

The versatility of dCas9 extends to targeted epigenetic modifications, offering a powerful tool for gene regulation beyond simple transcriptional activation or repression. These dCas9 epi-editors have been used to develop stable transgenics with enhanced gene expression (Liu et al., 2022). For instance, the combination of dCas9 can effect targeted DNA demethylation at a specific locus. In Arabidopsis, the human TET1 catalytic domain (TET1cd) and the improved SunTag system (further detail is shown in Figure 5) were used to alter the methylation state and subsequent activation of the FLOWERING WAGENINGEN (FWA) or CACTA1 transposon within a heterochromatic locus (Fal and Carles, 2024; Shin et al., 2022; Gallego-Bartolome et al., 2018). These findings provide a foundation using CRISPR to study the roles of specific epigenetic modifications in gene regulation (Pan et al., 2021b; Shin et al., 2022).

Further examples highlight the breadth of using dCas9-based epigenetic modulation of genes. By fusing dCas9 with Arabidopsis histone acetyltransferase 1 (HAT1), researchers successfully improved the expression of the AREB1/ABF2 gene (Paixão et al., 2019; de Melo et al., 2020). This epigenetic remodeling of chromatin states at specific loci led to enhanced drought tolerance in plants. In another example, researchers successfully blocked pathogen-induced gene activation in cassava by directing a zinc finger (ZF) to the effector binding elements (EBEs) within the promoter of the host susceptibility gene MeSWEET10a. This precise methylation prevented the activation normally triggered by exposure to Xanthomonas phaseoli pv. manihotis, the causal agent of cassava bacterial blight (CBB), leading to decreased disease symptoms and demonstrating a novel epigenome editing strategy for enhancing plant disease resistance (Veley et al., 2023). Due to the success of targeting a host susceptibility gene in casava and to combat cassava brown streak disease (CBSD), researchers used a dCas9-DMRcd-SunTag system to simultaneously target and methylate the promoters of two host susceptibility genes, nCBP-1 and nCBP-2, which are required by the causal Ipomoviruses (CBSV and UCBSV). While the initial results showed reduced gene expression and decreased susceptibility to CBSD, control experiments suggest that steric CRISPR interference, rather than methylation alone, was primarily responsible for the observed effect (Lin et al., 2025).

These diverse applications showcase how CRISPRa can be harnessed to achieve targeted transcriptional activation through precise chromatin remodeling, paving the way for comprehensive functional genomics studies and the development of next-generation crops with enhanced resilience. Despite its potential, CRISPRa in plants still faces challenges including developing methods for tissue-specific activation, overcoming barriers to DNA or nucleoprotein delivery, and off-target transcriptional effects. This lack of absolute specificity can result in pleiotropic effects, where activating a gene in one tissue may beneficially impact a desired trait but inadvertently cause negative effects on growth or development in another. Developing more refined, truly orthogonal inducible or tissue-specific promoters is crucial for fine-tuning CRISPRa applications. In addition, CRISPRa in plants also faces significant hurdles in overcoming delivery barriers, such as efficiently introducing CRISPRa components into diverse plant cell types and achieving stable, heritable expression. Additionally, off-target transcriptional effects can also occur, resulting from the dCas9-sgRNA complex binding to and activating (or repressing) non-target genes, potentially confounding the interpretation of experiments and possibly also leading to undesirable phenotypes.

Although CRISPRa remains relatively underexplored in plant systems, its integration with genome editing technologies offers immense potential for developing disease resistant, high yielding crops. Targeted epigenome editing via dCas9 epi-editors presents a promising transgene-free strategy to activate defense genes, paving the way for durable crop resistance. The GMO-free method would rely on CRISPR-editing components delivered in a transient fashion (e.g., via viral vectors that do not integrate into the genome or as delivery of nanoparticles containing purified proteins/RNAs), akin to Spray-Induced Gene Silencing (SIGS). In SIGS, double-stranded RNA (dsRNA) designed to target specific genes is delivered by spraying it directly onto plant surfaces (Koch et al., 2016). As more research focuses on optimizing delivery systems and improving activation efficiency, CRISPRa is poised to become an indispensable tool for sustainable agriculture and enhancing food security (Yıldırım et al., 2023; Park et al., 2024).

High-throughput CRISPRa screens have been used to systematically identify genes associated with human diseases (Chardon et al., 2024; Jones et al., 2022; Li et al., 2024). By using gRNA libraries combined with CRISPRa and CRISPRi, researchers can screen populations of plants for the activation of defense-related genes and uncover novel targets for crop improvement (Langner et al., 2018; Zaidi et al., 2020).

The full potential of CRISPRa for developing durable crop resistance is unlocked when combined with systematic gene discovery. Methods like STING-seq and beeSTING-seq, which integrate GWAS data with massively parallel CRISPR screens and single-cell sequencing, have been used to systematically discover target genes in mammalian systems (Morris et al., 2023). These high-throughput functional genomics strategies, combining both GWAS and CRISPR, can also be applied to uncover genes, reveal novel mechanisms, and identify breeding targets important for the expression of plant disease resistance (Clark et al., 2024).

Furthermore, multiomics (integrating genomics, transcriptomics, proteomics, and metabolomics) provides a comprehensive molecular context that can enhance CRISPRa-mediated gene discovery and validation. Transcriptomic data can highlight gene expression patterns linked to resistance, while proteomic analysis can identify defense-related signaling proteins. Integrating these datasets can help prioritize candidate genes for subsequent CRISPRa pertubation, focusing on those genes implicated in the expression plant immunity. Advances in gRNA structure design can also be used in combinatorial CRISPRa approaches, accelerating the identification of gene networks involved in plant defense and biosynthesis pathways (Fontana et al., 2024).

Recent advancements, such as synthetic promoter engineering and enhancer knock-ins, are revolutionizing plant biotechnology by offering unprecedented and precise control over gene expression (Yao et al., 2024; Tang and Zhang, 2023). These methods move beyond traditional random transgene integration, allowing targeted manipulation of a plant’s natural regulatory machinery. For instance, researchers successfully engineered MFH17, a strong, highly constitutive synthetic promoter derived from pararetroviral elements, which effectively drives high-level gene expression across both monocot and dicot plant species (Sherpa and Dey, 2024). The ability to precisely knock-in these elements into the plant genome offers enhanced control over gene expression. Recently, researchers demonstrated a powerful strategy for improving plant abiotic stress tolerance through precise knock-ins. In this approach, CRISPR-Cas9-mediated gene targeting was used to precisely insert stress-responsive cis-acting regulatory elements (SRCEs) into the promoter regions of candidate genes (Ke et al., 2025). This resulted in Arabidopsis plants with enhanced tolerance to drought, salt, and osmotic stress, notably without hindering normal growth, showcasing a significant step towards improved crop resilience.

Innovations such as the CRISPR-Combo system are further streamlining plant genetic engineering (Pan et al., 2023). This system enables simultaneous, orthogonal genome editing and transcriptional regulation by employing a single Cas9 protein guided by two distinct RNA architectures. This is achieved by using a sgRNA to mediate double-strand breaks for mutagenesis at one locus, while a concurrently deployed, engineered scaffold sgRNA recruits transcriptional activator complexes to a separate promoter, thereby upregulating gene expression without inducing DNA cleavage. This system enables speed breeding of transgene-free, genome-edited Arabidopsis plants and also substantially enhances hormone-free rice regeneration, hence increasing the pool of regenerated plantlets available for screening heritable, targeted mutations (Pan et al., 2023; Haber et al., 2024). This innovative approach allows for the simultaneous modulation of both DNA and RNA, accelerating trait stacking and improving crop resilience by streamlining the enhancement of desired traits while suppressing unwanted pathways (Gardner et al., 2025; Jaegle et al., 2025). The comprehensive all-in-one CRISPR toolbox (Cheng et al., 2025) further simplifies guide RNA library cloning, making large-scale genetic screens more time and cost-efficient. This toolbox has been successfully used to engineer herbicide resistance in rice by employing PAM-less CRISPR-Cas9 base editors to target the acetolactate synthase (OsALS) gene, enabling comprehensive coverage of known resistance-associated regions and the discovery of novel herbicide-resistant alleles (Clark et al., 2024). Similar experiments conducted in protoplasts hold promise for discovering and evaluating genes related to disease resistance, particularly for testing PTAs in plant immunity (Casas-Mollano et al., 2023; Mukundan et al., 2025; Sychla et al., 2022).

Integrating CRISPRa with GWAS and multiomics, combined with advancements in precise gene editing, holds immense potential for crop improvement. Numerous GWAS studies have identified disease-resistant regions in the genomes of various crop plants (Gangurde et al., 2022; Gardner et al., 2025; Jaegle et al., 2025; Sahito et al., 2024; Zhu et al., 2023). These integrated approaches can now accelerate the discovery, characterization, and modification of genes within these identified regions. A recent study proposed using high-quality GWAS data for grain total weight traits and applying CRISPR to modulate expression (Jamil et al., 2025). As applied to disease resistance, this approach could mean accelerating the development of broad-spectrum resistance in wheat to devastating fungal diseases like Fusarium head blight, rust and powdery mildew, significantly reducing yield losses. Similarly, in rice, the precise knock-in of regulatory elements could enhance tolerance to environmental stressors, such as specific soil pathogens or extreme temperatures, leading to more stable and higher yields in vulnerable regions. By identifying and precisely modulating the expression of key defense genes or stress response pathways, these sophisticated molecular tools can pave the way for developing more resilient and productive crops.

The ability to enhance crop traits without inserting foreign DNA has become an important goal in plant biotechnology, reflecting both the regulatory landscape and the public trust of gene-edited foods versus genetically modified organisms (GMOs). GMOs are plants or animals modified through methods that do not occur naturally, often involving the insertion of genes from other species to introduce traits such as pest resistance or drought tolerance (Holst-Jensen et al., 2012; Waigmann et al., 2012). In 2018, the U.S. Department of Agriculture (USDA) announced that genome-edited plants indistinguishable from those developed through traditional breeding methods would not be subject to regulation, highlighting the distinction between conventional GMOs and gene-edited organisms (Grossman, 2019; Menz et al., 2020). Notably, CRISPR-based technologies can produce gene-edited plants with enhanced disease resistance that are considered transgene-free, since no foreign DNA is integrated into the final product. This has been demonstrated in studies involving basil (Zhang et al., 2021) and wheat (Li et al., 2022), using transient CRISPR-gene editing vectors.

CRISPR technology holds significant promise for enhancing crop resilience against biotic and abiotic stresses, improving yield potential, and achieving these advancements with minimal environmental impact. Despite these benefits and the scientific and regulatory distinctions between gene-edited crops and conventional GMOs, public perception and acceptance remains a significant barrier. This challenge is particularly pronounced in regions such as the European Union, where public opposition to GMOs has been persistent and regulatory frameworks remain stringent (Kato-Nitta et al., 2023).

Several CRISPR-edited crops developed using traditional CRISPR-Cas9 or similar systems for gene knockout or precise gene edits have been approved for commercialization (Table 1). Interestingly, while these crops were developed using gene-editing tools like CRISPR-Cas9, they do not contain integrated foreign genes in their genomes. This distinction is critical under certain regulatory frameworks in which gene-edited plants are not classified as GMOs, potentially easing regulatory barriers and public concerns.

However, the application of CRISPRa specifically in commercial agriculture remains largely unexplored, with current research focusing on enhancing disease resistance and other agronomically important traits (García-Murillo et al., 2023). This cautious pace of commercialization stems from several factors, including the technical challenges of efficiently delivering the large CRISPRa protein complexes into plant cells, ongoing regulatory uncertainty distinct from that for gene knockouts, and the need to validate stable and predictable gene activation across diverse field conditions. Additionally, progress will be influenced by the discovery of key regulatory elements (such as upstream regulatory regions) which can then be edited by CRISPR (Si et al., 2020; Xiang and Dong, 2025).

Public perception of gene-edited crops continues to be mixed, largely stemming from a persistent confusion with traditional GMOs (Frisio and Ventura, 2019; Thornton, 2025; Vindigni et al., 2022). This is reflected in consumer data; for example, while the cited study on willingness-to-pay for CRISPR-edited rice showed varied acceptance across countries, other consumer surveys consistently find that public support increases when the specific benefits, such as enhanced nutrition or improved disease resistance leading to lower pesticide use, are clearly explained (Vasquez et al., 2022). However, initial skepticism often remains high without this context.

This knowledge gap highlights the pivotal role of proactive science communication and educational outreach. To build public trust, it is essential for the scientific community and industry stakeholders to transparently explain the distinction between transgene-free gene editing and conventional genetic modification. Clear, accessible information about the technology’s precision, safety assessments, and tangible benefits, as well as any risks, can empower consumers to make informed decisions rather than relying on outdated perceptions of GMOs. As regulatory frameworks continue to evolve, fostering an informed public dialogue through these outreach efforts will be as critical as the scientific advancements themselves in guiding policy decisions and shaping the future of CRISPR-based crop improvements for sustainable agriculture.

While significant consumer and market resistance has historically blocked GMOs in U.S. staple crops like wheat and barley, the widespread adoption of transgenic corn and soybeans reflects a permissive regulatory trend that is mirrored in other key agricultural nations. For example, Brazil’s regulatory agency (CTNBio) established early on that crops with transgene-free edits are not considered GMOs, streamlining approvals for products like high-yield sugarcane (Segretin et al., 2025). Similarly, India’s government has exempted certain categories of gene-edited plants from its stringent GMO regulations to accelerate crop improvement (Sankaranarayanan, 2024). Most notably, China has recently shifted its policy to create a clearer, more efficient approval pathway for gene-edited organisms, granting biosafety certificates for staple crops like wheat and rice in 2024 to bolster its food security goals (Liang et al., 2025). This contrasts with more cautious regions like the European Union, creating a complex and evolving global regulatory landscape for these technologies.