Front. Environ. Sci. Frontiers in Environmental Science Front. Environ. Sci. 2296-665X Frontiers Media S.A. 1745396 10.3389/fenvs.2025.1745396 Review A critical review on advanced molecular tools for bioremediation Kumar et al. 10.3389/fenvs.2025.1745396 Kumar Sandeep 1 Conceptualization Formal Analysis Investigation Software Writing - original draft Dasila Gunjan 1 Conceptualization Formal Analysis Investigation Writing - review and editing Mahara Kajal 1 Conceptualization Formal Analysis Investigation Writing - review and editing Samant Mukesh 2 * Conceptualization Formal Analysis Funding acquisition Investigation Supervision Validation Writing - review and editing Cell and Molecular Biology Laboratory, Department of Zoology, Soban Singh Jeena University Campus, Almora (Uttarakhand), Almora, India Department of Zoology, D.S.B. Campus, Kumaun University, Nainital (Uttarakhand), Nainital, India Correspondence: Mukesh Samant, mukeshsamant@kunainital.ac.in 27 01 2026 2025 13 1745396 13 11 2025 12 12 2025 31 12 2025 Copyright © 2026 Kumar, Dasila, Mahara and Samant. 2026 Kumar, Dasila, Mahara and Samant https://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

Acceleration in environmental pollution driven by rapid industrialisation and urbanisation is burdening the ecosystem with various hazardous waste and effluents. Improper waste disposal practices have introduced various pollutants, including plastics, pesticides, heavy metals, polyaromatic hydrocarbons, and volatile organic compounds (VOCs), into atmospheric, terrestrial, and aquatic environments, thereby affecting agriculture, biodiversity, and human health. Acute toxicity, carcinogenicity, developmental toxicity, cardiovascular dysfunction, endocrine disruption, and nervous system damage are the major complications caused by environmental pollutants. The limitations of conventional chemical treatment methods highlight the need for biology-based alternatives. Being a cost-effective and eco-friendly solution, bioremediation utilises potential microbes to decontaminate the environment. Conventional bioremediation techniques, although efficient, have foundered in the complete elimination of pollutants, highlighting the need for a molecular approach for total mitigation. In this review, we have highlighted modern molecular techniques, such as Zinc Finger Nucleases (ZFNs), Transcription Activator-like Effector Nucleases (TALENs), and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR), that have revolutionised the process, facilitated efficient removal and promoted environmental sustainability. This review advances the field by framing bioremediation within contemporary global challenges such as heavy metal toxicity, microplastic accumulation, and pesticide persistence, and by emphasising iterative refinements through computationally derived gene delivery models that offer targeted, ecologically safer alternatives to conventional approaches. We have summarised the advancements in gene editing technology, which could be a more efficient technique for the remediation of various environmental pollutants.

 Graphical Abstract

Diagram illustrating the process of transforming hazardous pollutants like heavy metals, plastics, and pesticides using engineered microbes and molecular biology tools, such as CRISPR/Cas and zinc finger nucleases, into environmentally sustainable outcomes. The flowchart highlights waste valorization, reduced toxicity, biodiversity restoration, cost-effectiveness, and novel strategies for environmental sustainability. Key elements in the diagram include various chemical structures and icons representing pollutants and solutions.

bioremediation environmental pollutants gene editing CRISPR/Cas9 TALEN ZFN The author(s) declared that financial support was received for this work and/or its publication. This work was funded by the G.B. Pant National Institute of Himalayan Environment and Sustainable Development under the National Mission on Himalayan Studies. The support was provided through Grant No. NMHS2023-24/SC-XI/SG87/02/150, awarded to M. S. S.K’s financial support provided by UGC India in the form of JRF (Ref. no. 231620164676). section-at-acceptance Toxicology, Pollution and the Environment Introduction

Rapid industrialisation, urbanisation, and development have contributed significantly to the growing problem of environmental pollution. While technological and economic progress continue, it often comes at an environmental cost. The disposal of waste and effluents by the industries and households is accountable for various types of pollution. Despite treatment efforts, these wastes often remain inadequately neutralised, contributing to the disposal of hazardous pollutants in soil and water, for instance, heavy metals, polychlorinated biphenyls (PCBs), polyaromatic hydrocarbons (PAHs), and volatile organic compounds (VOCs). The use of contaminated inputs in agriculture results in pollutants entering the food chain, posing health risks to humans and animals (Li, 2018). Trace metals such as aluminium, copper, zinc, nickel, lead, arsenic, cadmium, chromium, and mercury are introduced through the agricultural and industrial application of fertilisers, pesticides, and herbicides, and are pernicious to aquatic and terrestrial life (Prabagar et al., 2021). The paper and pulp industry accounts for a significant share of global water pollution through pulping, bleaching, and washing, generating wastewater containing potentially harmful compounds (Mehmood et al., 2019).

Due to their persistence and limited degradability, phenolic and chlorinated compounds are among the most challenging and hazardous environmental pollutants, leading to aquatic toxicity, genotoxicity, mutagenicity, etc (Khorsandi et al., 2018). Primary contributors to water pollution include pesticides, heavy metals, and various organic contaminants. Ingestion of polluted water has been linked to serious health effects, including carcinogenesis, hepatic dysfunction, and reproductive abnormalities (Hitt et al., 2023). The significant air pollutants, such as nitrogen oxides and sulphur dioxide, cause coughs, exacerbate asthma, and irritate the respiratory system (B. Zhao et al., 2020), and also contribute to acid rain, acidification, and depletion in crop yield (Manisalidis et al., 2020). Excessive nitrogen deposition in aquatic environments leads to algal bloom, death, and dis-equilibration in fish diversity (Zuhara and Isaifan, 2018). VOCs from paints, cleaning products, and vehicle emissions cause irritation, headaches, nausea, and dizziness, and carcinogenic effects such as benzene-linked leukaemia (Bălă et al., 2021).

The presence of hazardous environmental pollutants necessitates efficient removal techniques, with bioremediation emerging as a promising solution. Conventional microbial and physicochemical methods frequently produce partial degradation and persistent toxic intermediates, exhibit slow reaction kinetics, and perform poorly under variable or extreme environmental conditions (Krishna Raj et al., 2026). With pollution outpacing conventional mitigation techniques, harnessing innovative bioremediation approaches is essential. Recent reviews emphasise that while microbial consortia and enzyme-mediated degradations are effective, they lack precision in targeting pollutant-specific pathways and fail to address off-target ecological effects (Sanjana et al., 2024). Advancements in molecular techniques have resulted in better bioremediation (Krink et al., 2024). With engineered microbial communities, simultaneous remediation of multiple wastes and transformation of hazardous waste into monomer units can be achieved (Franden et al., 2018). Potent microbes efficient in pollution degradation are genetically modified, enhanced, and aligned with other microorganisms for efficient mitigation of pollutants. Moreover, the absence of robust delivery systems and biosafety frameworks has restricted the translation of laboratory successes to field applications. This gap highlights the need for advanced molecular tools such as CRISPR, TALENs, and ZFNs that can rewire metabolic pathways, enhance enzyme activity, and enable multi-kingdom remediation strategies, thereby overcoming the shortcomings of conventional bioremediation (Blessing and Olateru, 2025). In this review, we aim to critically evaluate the limitations of conventional remediation strategies and to highlight how advanced molecular tools, specifically CRISPR, TALENs, and ZFNs, can be harnessed to overcome these barriers. By synthesising recent advances in enzyme engineering, microbial pathway rewiring and multi-kingdom remediation approaches involving algae, fungi and plants, this review aims to provide a comprehensive framework for precision bioremediation. By proposing a roadmap that integrates computationally designed gene delivery systems, iterative experimental feedback, and biosafety/regulatory considerations, here we seek to consolidate state-of-the-art molecular interventions across pollutant classes, identify research gaps in field validation and ecological safety, and advance sustainable, scalable strategies for environmental restoration. We have conceptualised a shift from conventional bioremediation approaches, which rely on naturally occurring microbial and plant processes, to advanced molecular strategies such as CRISPR/Cas-mediated gene editing, TALEN/ZFN applications, and engineered enzymes, highlighting the enhanced precision, efficiency, and pollutant-specific targeting achieved through these novel tools (Figure 1).

Comparative schematic representation of conventional and molecular bioremediation approaches, inputs, processes, and limitations. The Conventional Bioremediation includes ecological and established strategies such as natural microbial degradation, bioaugmentation, biostimulation, phytoremediation, along with their inputs (indigenous microbes, nutrients) and processes (enzymatic breakdown, metabolite formation, targeted pathways). The Molecular Bioremediation highlights advanced interventions, including genetic engineering, CRISPR-based pathways, synthetic consortia, omics-guided and real-time monitoring, supported by inputs (engineered enzymes, biosensors, nanoparticles) and processes enabling enhanced pollutant specificity. Both Conventional and Molecular Bioremediation techniques conclude with their respective limitations, emphasising the comparative challenges faced by conventional versus molecular paradigms.

Comparison infographic illustrating conventional versus molecular bioremediation. Conventional methods include natural microbial degradation and phytoremediation with inputs like indigenous microbes and nutrients. Molecular methods involve CRISPR pathways and synthetic consortia using engineered enzymes and biosensors. Processes include enzymatic pollutant breakdown and enhanced specificity. Limitations cover slow degradation for conventional, and biosafety concerns for molecular methods. Methodology

Given the fragmented evidence and rapid advancements in molecular editing tools for bioremediation, this systematic review was necessary to consolidate current knowledge, critically evaluate pollutant-specific applications, and identify emerging strategies that can guide future research and deployment. This review was conducted following principles adapted from the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) framework to ensure transparency and rigour. A structured search strategy was applied across PubMed, Scopus, Web of Science, Google Scholar, and major publisher platforms (Frontiers, Elsevier, Springer Nature, Wiley), covering literature published between January 2000 and December 2025, with seminal pre-2000 studies included selectively for foundational mechanisms. Keywords combined terms related to bioremediation (plastics, pesticides, hydrocarbons, heavy metals) with molecular tools (CRISPR, TALENs, ZFNs, genome editing) and mechanistic enrichers (delivery systems, HDR, NHEJ, sgRNA design, PETase, MHETase, laccase, peroxidase). Inclusion criteria required peer-reviewed studies that linked tools, organisms, genes, and pollutant outcomes, while exclusion criteria eliminated non-peer-reviewed sources, purely clinical CRISPR applications, and articles lacking pollutant-specific data. Data extraction followed a structured template capturing study details, organism and pollutant type, molecular tool design, gene targets, delivery systems, repair pathway modulation, and remediation outcomes. Quality appraisal considered methodological transparency, reproducibility, pollutant quantification accuracy, and clarity of genetic interventions, while risk of bias was assessed for selection, performance, and reporting biases. Narrative synthesis was organised by pollutant class and tool type, supported by comparative frameworks including a schematic contrasting conventional versus molecular bioremediation and a summary table comparing CRISPR, TALENs, and ZFNs in terms of mechanisms, specificity, delivery compatibility, and exemplar applications. Limitations of this review include potential publication bias toward positive outcomes, heterogeneity of assay conditions, and limited long-term field validation data. Although the review was not pre-registered, the protocol was internally documented and consistently followed, ensuring comprehensive coverage of relevant literature and minimising bias in topic selection.

 Environmental pollutants and their hazardous effects

Industrialisation has effectively addressed the needs of the human population. While the endless demands of society are met, the environment is exposed to a variety of contaminants, including heavy metals, which negatively affect both the ecosystems and living organisms (Aluko et al., 2021). Arsenic, cadmium, chromium, lead, and mercury are the chief heavy metal contaminants, often occurring at concentrations above critical thresholds in aerial, aquatic, and terrestrial systems. Heavy metals, originating from both natural and anthropogenic sources, are widely distributed across environmental matrices, including air, soil, water, and biota (Yin et al., 2021). Fertilisers, galvanising factories, metallurgical plants, pharmaceuticals, pesticide factories, tanneries, textiles, and varnishes represent the principal anthropogenic sources of heavy metals (Chabukdhara and Nema, 2012). Mineral deposits, oceanic evaporation, pathogenic processes, and diverse rock types are some natural sources of heavy metals (Zeng et al., 2014). Although the concentration of heavy metals in biological systems is regulated by homeostatic mechanisms, their toxicity depends on factors such as concentration, duration, and route of exposure. The ill-effects of heavy metals include destabilising molecules, mimicking vital elements, hindering biological activities, and generating reactive oxygen species (ROS) (Prabhakaran et al., 2016). Table 1 provides a comprehensive summary of principal environmental pollutants and their corresponding ecological and health impacts.

Impact of major environmental pollutants.

Source Pollutant Complications References
Paper and Pulp 2,3′,4,4′,5-Pentachlorobiphenyl Carcinogenic, endocrine-disrupting compounds (EDCs) Huang et al. (2019)
2,3,6,7-tetrachloronaphtalene Developmental toxicity, EDCs, mutagenicity, genotoxicity Ohura (2007)
2,3,7,8-Tetrachlorodibenzo-p-dioxin Acute toxicity, environmentally hazardous Malhotra et al. (2013)
4-chloro-3-methylphenol Aquatic toxicity, mutagenic, and skin irritation Gadupudi et al. (2019)
5-Chlorovanillin Respiratory irritant, Skin irritation, target organ toxicity Kaur et al. (2019)
Industrial Activities Aluminium Alteration of Calcium homeostasis, cell wall loosening, plasma membrane destruction, and retardation of cell division Rehman et al. (2021)
Arsenic Cardiovascular dysfunction, CNS injury, Skin and Hair infection Balali-Mood et al. (2021)
Cadmium Bone degeneration, cancer, lung and liver diseases Fay et al. (2018)
Chromium Cancer, Kidney and Skin diseases Pavesi and Moreira (2020)
Mercury CNS injury, hepatotoxicity and renal dysfunction Zhang et al. (2020)
Agriculture Pesticides DNA damage and morphology hepatocytes De Souza et al. (2020)
Anthropogenic sources Microplastics (Polystyrene <3 µm) Increased rate of phagocytosis and cytokine production by macrophages Hwang et al. (2020)
Microplastics (Polystyrene <5 µm) Cytotoxicity and haemolysis of red blood cells Hwang et al. (2020)
Micro (nano) plastics Inflammatory lesions, infections, cardiovascular, pulmonary and intestinal disorders Gaur et al. (2025)
Low molecular weight PAH (2–3 rings) Disorientation, diarrhoea, nausea and vomiting Montano et al. (2025)
Benzo(a)-pyrene (PAH) Enhance HPV-mediated carcinogenesis, cervical cancer, and accelerated tumour growth Feng et al. (2025)
Multiple exposure pathways (PCB) Higher-chlorinated congeners Elevated serum PCB levels, reduced birth weight, and neurodevelopmental deficits Zhang et al. (2024)
Dioxin and non-dioxin-like congeners Chloracne, melanoma, reproductive toxicity Moon and Waqar (2025)

Plastic products, owing to their low cost and convenience, are used globally on a daily basis. The most widely consumed polyester plastic, Polyethylene terephthalate (PET), exceeds 300 million tons annually, accounting for 13% of global plastic pollution (Huang et al., 2019). PET is cost-effective, durable, and easy to process, with good abrasion resistance, and is therefore produced in large quantities. Alongside PET, other plastic varieties, for instance, polyethylene (PE), and polyvinylchloride (PVC), are extensively used in day-to-day life (Liu and Wu, 2024). Properties such as high hydrophobicity and chemical inertness hinder the natural degradation of plastic; meanwhile, their large-scale accumulation in the environment results in “White Pollution”. Although physical and chemical methods for plastic degradation are increasingly employed, these approaches often result in secondary environmental and human health hazards through microplastic accumulation (Liu et al., 2021a). Figure 2 provides a systematic overview of the sources and resultant impacts of environmental pollutants, emphasising their interlinked cause-and-effect dynamics.

Causes and Effects of Environmental Pollutants. The waste and effluents emitted from various sources serve as potential environmental pollutants, contaminating the ecosystem. Generated effluents have an adverse impact on the environment, leading to land degradation, aquatic toxicity, target organ toxicity, and ecosystem damage.

Illustration depicting environmental and health impacts of pollutants. Heavy metals, industrial waste, produced water, organophosphates, and pesticides contribute to issues like air pollution, ecotoxicity, organ damage, and endocrine disruption. Effects include hypertension, renal dysfunction, respiratory irritants, and aquatic toxicity. PET and microplastics lead to bioaccumulation and contamination.

The paper and pulp industries generate wastewater enriched with chlorinated, phenolic, sulfonated, and complex lignin-derived compounds (Mandeep et al., 2019). Industrial processes such as bleaching, pulping, and washing generate large volumes of effluents containing potentially hazardous substances. These pollutants exert adverse environmental impacts and are associated with mutagenic and endocrine-disrupting effects. To promote sustainability and mitigate pollution from the paper industry, current research emphasises bio-catalysis-mediated remediation strategies employing enzymes with promising potential (Ashrafi et al., 2015).

Pesticides are chemical agents designed to inhibit or eliminate pests such as microorganisms (bacteria, fungi, and algae), herbs, weeds, rodents, nematodes, and insects (Bottrell and Schoenly, 2018). Classified based on their target, pesticides not only reduce agricultural losses but also enhance crop yields (Allmaras et al., 2018). Among them, organophosphorus compounds represent the most extensively used group, with diazinon, chlorpyrifos, malathion, and parathion widely applied in agricultural and residential settings (Karalliedde and Senanayake, 2004). However, their intensive use has resulted in elevated toxicity across trophic levels and biomagnification in aquatic systems through agricultural runoff (Plattner et al., 2018). The ill effects of pesticides include organ damage, damage to DNA, causing neurological diseases, and cancer (Fatima et al., 2018). Mitigating the growth and persistence of such pollutants is essential for safeguarding health and environmental sustainability. The suboptimality of conventional remediation underscores the need for advanced methodologies, particularly those harnessing the biodegradation potential of microbes.

 Bioremediation of pollutants utilising advanced molecular tools

Enhancing microbial potential for waste decontamination can be achieved through the manipulation of biodegradative pathways. Engineering regulatory and genetic elements, for instance, promoters, terminators, and binding sequences, can modulate microbial metabolism to optimise gene expression and protein production (Carr et al., 2020). Genetically modified bacteria, recognised for their environmental compatibility, global acceptance, and reduced health hazards, have gained increasing attention in the field of environmental restructuring (Saxena et al., 2020). The complexity and optimisation of microbial genetic factors can be advanced through genome editing technologies (Basu et al., 2018). These technologies have evolved significantly, with ZFNs, TALENs, and the CRISPR/Cas9 system emerging as the most widely utilised tools for precise and programmable genetic modifications across diverse biological systems.

Zinc finger nucleases (ZFNs): the pioneer of molecular techniques

Zinc Finger Nucleases (ZFNs) are specifically designed engineered restriction enzymes capable of cleaving stretches of double-stranded DNA (Porteus and Carroll, 2005). Each ZFNs have distinct DNA-binding and cleavage domains, with the FokI from Flavobacterium okeanokoites serving as the nucleotide cleavage domain (Jaiswal et al., 2019). The recognition helix (α-helix) is structurally conserved, and alteration in DNA binding specificity of a single zinc finger can be achieved by transposing one or more of the six critical residues positioned within or adjacent to the α-helix (Jantz et al., 2004). Efficient DNA cleavage requires dimerisation of the FokI nuclease domain. As the dimer interface is weak, for effective cleavage, the neighbouring sequences are targeted by two sets of fingers, which subsequently align to facilitate cleavage at the recognition site. A short linker between the protein domain and a 5-6 bp spacer’s inverted binding site is crucial for optimum configuration (Händel et al., 2009). Because the cleavage domain itself lacks sequence specificity, DNA cutting can be redirected by substituting the natural recognition domain with an alternative one. Non-homologous end joining (NHEJ) and homologous recombination (HR) are two potential repair pathways for double-strand breaks induced by ZFNs. NHEJ, being error-prone, often results in insertions or deletions (indels), which can disrupt target genes by introducing frameshift mutations that yield truncated or dysfunctional proteins (Lieber et al., 2003).

Pollutants treated using ZFNs- pesticides and organophosphates

Bioremediation supported by gene editing tools offers significant potential for the elimination of xenobiotics through the degradation of toxic compounds and pesticides (Basu et al., 2018). Microorganisms, for instance, Enterobacter, degrade Chloropyrifos (Niti et al., 2013), Bacillus subtilis degrades Cypermethrin, while DDT has been degraded by Fomitopsis pinicola (Purnomo et al., 2020). By altering genes involved in pesticide metabolism or resistance, ZFNs can enhance the bioremediation capacity of these organisms (Jaiswal et al., 2019). ZFN-based molecular techniques have also been applied in Chlamydomonas reinhardtii to target the COP3 gene, demonstrating their utility in precise genetic manipulation for environmental applications (Sizova et al., 2013).

 TALENs: a cornerstone in molecular biology

Transcription activator-like effector proteins (TALEs) are secreted by the Xanthomonas spp., a pathogenic bacterium affecting pepper, rice, and tomato plants (Wright et al., 2014). Their versatility in genetic engineering arises from the ability to associate with diverse functional domains, including endonucleases, repressors, or transcriptional activators, thereby enabling genome editing (Thakore and Gersbach, 2016). Fusion of TALE with FokI generates Transcription activator-like effector nucleases (TALENs). The crucial TALE components are: (i) Signal for secretion and transmission (N-terminal T3S signal), (ii) Central tandem repeat domain, (iii) C-terminal nuclear ocalization signal (NLS), (iv) Acidic transcriptional activation domain (AD) (Pelletier, 2016). Repeatable variable di-residues (RVDs) are the repetition of 33–35 conserved amino acids of the central tandem repeat domain, varying only at positions 12 and 13. The binding between a DNA nucleotide and RVD is specific (Bogdanove and Booher, 2016). More than 25 types of RVDs have been identified, each contributing to nucleotide recognition efficiency (Richter et al., 2016). RVDs (NN, HD, NI, NK, NH, and HD) are frequently used for TALEs as they exhibit variable precision and binding affinity. Robust correlations have been observed in HD and NG (Streubel et al., 2012). The RVDs NI, ND, NS, and NN exhibit optimal recognition specificity for the nucleotides A, C, T, and G/A, respectively (Sun and Zhao, 2013). The codon degeneracy property of NS and NN aids them to bind with any nucleotide and A/G, hence, targeting multiple DNA sequences. Structurally, the left-handed helix-turn-helix configuration of RVDs interacts with the major groove of DNA (Pruett-Miller, 2014). For optimal TALEN activity, inclusion of 3–4 RVDs capable of forming strong hydrogen bonds is critical (Richter et al., 2016).

 CRISPR: the revolution in molecular techniques

Clustered regularly interspaced short palindromic repeats (CRISPR) represent a unique organisation of short, partially repeated DNA sequences within the prokaryotic genomes. Functioning as an adaptive immune system, CRISPR and its associated protein (Cas-9) defend prokaryotes against viruses and bacteriophages (Hille and Charpentier, 2016). The groundbreaking discovery by Doudna and Charpentier that CRISPR/Cas9 can be programmed with a template to edit virtually any DNA sequence revolutionised genome editing (Jinek et al., 2012). The CRISPR/Cas9 editing process comprises three fundamental steps: precise recognition of the target DNA sequence, induction of a double-strand break (DSBs), and cellular repair of the cleaved DNA through endogenous cellular mechanisms (Shao et al., 2016). The Cas9 enzyme is guided by a synthetic guide RNA (sgRNA), which identifies the target gene by binding to a complementary sequence within the 5′crRNA region. In the absence of sgRNA, the Cas-9 protein remains inactive. At a site 3 bp upstream of the Protospacer Adjacent Motif (PAM), the Cas-9 nuclease produces double-strand breaks (Ceasar et al., 2016). The protein products of Cas genes (CRISPR-associated) exhibit helicase and nuclease activity, and are positioned adjacent to the CRISPR cassette (Haft et al., 2005). The protospacer adjacent motif (PAM), typically represented as 5′-NGG-3′ (where N denotes any nucleotide), is the most frequently recognised sequence by Cas9 nucleases. This short, conserved DNA segment—usually spanning 2 to 5 base pairs—is located downstream of the cleavage site, having varying length across different bacterial species. The RNA-DNA hybrid forms when Cas-9 and PAM combine, and the Cas-9 protein gets activated for DNA cleavage. Cas9 induces DSBs with blunt ends by employing two distinct nuclease domains: the HNH domain cleaves the DNA strand complementary to the guide RNA, while the RuvC domain targets the non-complementary strand. The cellular machinery of the host repairs DSBs (Chen et al., 2019). Blunt-end double-strand breaks form upon cleavage by SpCas9, while staggered-end DSBs form when Cas12a cleaves DNA after identifying the T-rich PAM site via crRNA (Zetsche et al., 2015). To repair DSBs created by Cas-9 protein, the two pathways are Homology-directed repair (HDR) and Non-homologous end joining (NHEJ) (Liu, et al., 2019a). Two additional error-prone repair pathways are Single-strand annealing (SSA) and microhomology-mediated end joining (MMEJ) (J. Huang and Cook, 2022). NHEJ, being active in all phases of the cell cycle, utilises an enzymatic process independent of exogenous homologous DNA to repair the DSBs. However, it can generate frameshift mutations or premature stop codons by causing random small indels (insertion or deletion) at the cleavage site. HDR, mostly active in late S and G2 phases of the cell cycle, is highly precise and uses a homologous DNA template. By adding a donor DNA template having sequence homology at the predicted DSB site, HDR executes precise gene insertion or replacement (Rehman et al., 2021). Table 2 summarises the comparative features of ZFNs, TALENs, and CRISPR/Cas9 systems in bioremediation. CRISPR emerges as the most efficient tool, though off-target risks remain a concern. TALENs provide superior precision but at higher design complexity and cost. ZFNs, while historically important, are less efficient and more expensive due to protein engineering requirements. Representative bioremediation applications are included to highlight pollutant-specific successes.

Comparative overview of ZFNs, TALENs, and CRISPR in bioremediation applications.

Feature ZFNs TALENs CRISPR References
Mechanism Zinc finger arrays bind DNA; FokI dimer induces double-stranded breaks TALE repeats bind DNA; FokI dimer induces double-stranded breaks RNA-guided nucleases (Cas9, Cas12a; Cas13 for RNA) introduce site specific breaks Hille and Charpentier (2016), Jinek et al. (2012), Porteus and Carroll (2005), Zetsche et al. (2015)
Targeting specificity Moderate; depends on zinc finger design and context Very high; RVDs provide programmable nucleotide recognition High; improved with high-fidelity variants and PAM-tuned enzymes Ceasar et al. (2016), Jaiswal et al. (2019), Richter et al. (2016)
Ease of Design Protein engineering intensive; limited automation Complex repeat assembly; design kits streamline but are still laborious Simple sgRNA design; PAM constraint; growing design tools Basu et al. (2018), Thakore and Gersbach (2016)
Delivery compatibility Plasmids and viral vectors; construct complexity limits breadth Plasmids and viral vectors; construct size can constrain delivery Plasmids, phage/viral vectors, nanoparticles; broad host range (microbes, algae, fungi, plants) Hille and Charpentier (2016), Sizova et al. (2013)
Repair pathway control NHEJ and HDR; indel-prone at the cut site NHEJ and HDR; precise but less flexible than CRISPR variants NHEJ, HDR, MMEJ; base/prime editing expand precision without DSBs Rehman et al. (2021), Zetsche et al. (2015)
Efficiency Moderate; efficiency depends on zinc finger design; prone to indels High precision; efficient in diverse organisms, but labor-intensive High efficiency; rapid editing; prime/base editing improves precision Jaiswal et al. (2019), Jinek et al. (2012), Porteus and Carroll (2005), Sizova et al. (2013)
Cost Expensive; requires custom protein engineering Moderate; repeat assembly kits reduce cost but are still complex Low; sgRNA design is inexpensive; widely accessible Basu et al. (2018), Hille and Charpentier (2016), Thakore and Gersbach (2016)
Off-target effects Higher risk; specificity limited by zinc finger design Very low; RVDs provide strong site recognition Moderate; PAM dependency; improved with engineered variants (SpCas9-HF, eSpCas9) Ceasar et al. (2016), Richter et al. (2016), Zetsche et al. (2015)
Advantages Established protocols; useful for targeted knockouts in defined loci Very high precision; strong in plant systems; complementary in fusion designs Programmable, scalable, adaptable to non-model organisms; rapid iteration across pollutant targets Bogdanove and Booher (2016), Porteus and Carroll (2005), Thakore and Gersbach (2016)
Limitations Complex protein design; lower efficiency; potential off-target binding Labour-intensive assembly; large constructs Off-target risk, PAM dependency, biosafety/regulatory hurdles Basu et al. (2018), Jaiswal et al. (2019)
Bioremediation exemplars Chlorpyrifos and DDT degradation via engineered microbes and co-cultures; ZFN utility in algal editing TALEN targeting in Chlamydomonas (COP3); organophosphate/carbonate degradation frameworks PETase/MHETase engineering; heavy metal redox optimisation in Shewanella; CRISPR knockout of opdB in Cupriavidus for pesticide remediation Niti et al. (2013), Purnomo et al. (2020), Sizova et al. (2013), Zhao et al. (2023)
Pollutants removed utilising the CRISPR/Cas technique Lignin bioremediation

Lignin-modifying enzymes such as laccases and peroxidases, produced by bacteria, fungi, and plants, catalyse the oxidation of phenol and lignin-derived compounds (Sitarz et al., 2016). While oxidation of phenolic lignin compounds is done directly by laccases, on the other hand, peroxidases such as Lignin peroxidase (LiP), Manganese peroxidase (MnP), Versatile peroxidase (VP), and Dye-decolourising peroxidase (DyP) require a co-substrate, hydrogen peroxide (H2O2) for catalysing the oxidation reaction (Riyadi et al., 2020). LiPs facilitate the oxidative breakdown of both phenolic and non-phenolic structures (Lundell et al., 2010). DyPs, LiPs, and VPs exhibit catalytic activity toward benzene derivatives, enabling the degradation of compounds such as diols and sulfonic acid substituents. MnP, an extracellular enzyme, catalyses the oxidation of Mn2+ to Mn3+ in the presence of H2O2, contributing to the elimination of phenolic compounds (Hakala et al., 2006).

 Plastic biodegradation

PETase and MHETase are two chief polyethylene terephthalate (PET) hydrolases discovered in the bacterial strain Ideonella sakaiensis, capable of utilising PET as its sole carbon source (Yoshida et al., 2016). PETase initiates PET degradation by hydrolysing it into bis(2-hydroxyethyl) terephthalate (BHET), which is subsequently converted into mono (2-hydroxyethyl) terephthalate (MHET). MHETase then hydrolyses MHET into environmentally benign end-products: terephthalic acid (TPA) and ethylene glycol (EG) (Zhao et al., 2023). A chimeric dual-enzyme system comprising MHETase and PETase linked together by glycine-serine adapters proved efficient in PET degradation (Lai et al., 2023). Current research explores the CRISPR/Cas-9-based strategies for constructing dual-enzyme mutants of PETase and MHETase to improve PET recycling (Wang et al., 2024). Beyond I. sakaiensis, enzymatic depolymerisation of plastic by Bacillus, Escherichia, and Pseudomonas species has been enhanced through synthetic biology approaches, including computational methods, cloning, and metagenomics (Amobonye et al., 2021). Table 3 provides a summary of key genes and their associated functions in microbial plastic degradation and wastewater bioremediation. Figure 3 provides a schematic overview of the delivery systems and molecular mechanisms of ZFNs, TALENs, and CRISPR/Cas9, highlighting their application in microbial bioremediation.

Representative organisms and genes with associated effects in plastic and wastewater bioremediation.

Tool Organism Gene Effects Pollutant remediated References
CRISPR E. coli BL21 (DE3) Enzyme- DuraPETase Hydrolytic cleavage of ester bonds within PET. PET→TPA + EG** PET (Polyethylene terepthalate) Y. Liu et al. (2022)
CRISPR I. sakaiensis IsPETase Depolymerisation of PET via hydrolysis of the Ester bond. PET→ (MHET, BHET) →TPA + EG*** PET (Polyethylene terephthalate) Deng et al. (2019)
CRISPR Pichia pastoris Enzyme- Lipase cutinase Hydrolysis of the ester bond degrades PCL to ε-caprolactone PCL [poly (ε-caprolactone)] Liu et al. (2019b)
CRISPR M257E. Xiangfangensis vgrG Effector delivery that modifies or disrupts hydrocarbon structures in oil sludge and contributes to biofilm formation Oil Sludge Muneeswari et al. (2022)
CRISPR E. coli BL21 (DE3) mdeABCD Oxidative cleavage of the ether bond, hydroxylation of aromatic rings and ring fission pathways to produce less harmful metabolites 3-Methyldiphenylether Yang et al. (2016)
CRISPR Shewanella oneidensis MR-1 minCDE, SO_3166 Reductive cleavage of azo bonds (-N=N-) Azo dye Y. Chen et al. (2022)

**TPA- terephthalic acid, EG- ethylene glycol.

***MHET- Mono-2-hydroxyethyl terephthalate, BHET-bis(hydroxyethyl) terephthalate.

Mechanistic overview of molecular biology tools- ZFNs, TALENs, and CRISPR/Cas9 used in microbial gene editing for enhanced bioremediation of environmental pollutants. The diagram illustrates the delivery of gene editing constructs into microbial cells via plasmid and bacteriophage-based systems using both viral and non-viral methods. The lower panel details the molecular mechanisms and bioremediation applications of three major genome editing platforms: Zinc Finger Nucleases (ZFNs), Transcription Activator-Like Effector Nucleases (TALENs), and CRISPR/Cas9. ZFNs utilise modular zinc finger domains for DNA recognition and the FokI endonuclease for double-strand break (DSB) induction, enabling targeted gene insertion and overexpression of genes such as cph and ACC deaminase, which enhance degradation of chlorophenol and uptake of heavy metals like Cu and Cd. TALENs, derived from Xanthomonas spp., consist of tandem repeat domains with repeat variable di-residues (RVDs) for nucleotide-specific binding, fused to FokI for DSB generation. TALEN-mediated editing facilitates the insertion of genes such as PCSs and alkB, promoting cadmium accumulation and hydrocarbon degradation. The CRISPR/Cas9 system employs a guide RNA (crRNA/tracrRNA or sgRNA) to direct Cas9 to specific DNA sequences adjacent to a protospacer adjacent motif (PAM), where it induces blunt-end DSBs via its HNH and RuvC nuclease domains. This system enables precise editing of genes such as mer and Tars M, which encode mercury reductase and arsenite methyltransferase, respectively, contributing to detoxification of Hg2+ and arsenic. Collectively, these molecular tools offer programmable, high-efficiency strategies for engineering microbial strains capable of degrading xenobiotics and remediating contaminated environments.

Diagram illustrating gene editing methods using plasmids, bacteriophages, and bacteria. Three sections detail Zinc Finger Nucleases, TALENs, and CRISPR. Each section shows processes like dimerization, double-stranded breaks, homologous recombination, and gene insertion, leading to functions such as over-expression, transcription, and detoxification. Waste water treatment

With freshwater resources becoming increasingly scarce, greater emphasis is being placed on reclaiming, reusing, and recycling wastewater. Microalgae have demonstrated significant potential in removing contaminants from diverse effluents, including heavy metals and agricultural, industrial, and pharmaceutical wastewater streams (Singh et al., 2020). Owing to their transformable nature, microalgal cells can be genetically redesigned to enhance bioremediation capacity, for instance, methylation of inorganic arsenic by transforming Bacillus subtilis with the arsenite S-adenosylmethionine methyltransferase gene of red microalgae Cyanidiochyzon merolae. (Huang et al., 2015). Additionally, CRISPR/Cas-9 application to increase lipid content in algal cells for biofuel production has been successfully conducted, with the omega-3 fatty acid desaturase gene (fad) of Chlorella vulgaris serving as a recent example of targeted gene editing (Lin and Ng, 2020). Beyond pollutant removal, the bioremediation potential of microalgae coupled with biomass reutilisation offers opportunities for the production of biofuels, biofertilizers, and feed supplements (Shahid et al., 2020). Using the plasmid-based CRISPR/Cas9 system, insertion and expression of the arsenite S-adenosylmethionine methyltransferase gene (CmarsM) in Bacillus subtilis 168 has enabled conversion of Arsenic into dimethylarsenate and trimethylarsine oxide (Huang et al., 2015). Table 3 summarises the tools, organisms, and genes employed in plastic degradation and wastewater bioremediation, along with their functional effects.

 Pesticides biodegradation

To accelerate the biodegradation of synthetic pollutants, microbial strains of Achromobacter, Pseudomonas, Rhodococcus, Sphingomonas, and Ralstonia have been engineered using CRISPR/Cas-9 (Bilal and Iqbal, 2020). Cycloclasticus sp., capable of metabolising naphthalene, pyrene, and other xenobiotic compounds, has been enhanced through CRISPR-mediated modification (Mahas and Mahfouz, 2018). Similarly, CRISPR systems have been applied in Sphingomonas sp. and Paracoccus sp. YM3 and to facilitate carbamate degradation (Chaudhari and Solanki, 2023). Co-culture approaches, such as the combination of the brown-rot fungus Fomitopsis pinicola with Ralstonia pickettii, have demonstrated improved degradation of 1,1,1-Trichloro-2,2-bis (4-chlorophenyl) ethane (DDT) (Purnomo et al., 2020).

Chlorpyrifos degradation has been achieved by diverse microbial strains, including Bacillus pumilus strain C2A1, Bacillus aryabhattai, Streptomyces olivochromogenes, Pseudomonas resinovarans AST2.2, and P. indoloxydans (Sharma et al., 2016). A study on bacterial strain (Bacillus pumilus) showed increased degradation of 3,5,6-trichloro-2-pyridinol (TCP) (300 mg L-1) within a week of incubation in the presence of nutrient-supplemented conditions (Anwar et al., 2009). Among 30 actinobacterial strains, Streptomyces sp. AC1-6 and Streptomyces sp. ISP4, isolated from pesticide-contaminated soils, exhibited diazinon degradation within 2–3 days of incubation (Briceño et al., 2015). The bacterial strain SanPS1 from Narigram, West Bengal, India degraded half of parathion within a day (Pailan et al., 2015). Pseudomonas resinovarans AST2.2 was reported to degrade approximately 50% of chlorpyrifos (400 mg/L) within a few days (Sharma et al., 2016). In contrast, Stenotrophomonas sp., Enterobacter strain B-14, and Sphingomonas sp. achieved complete chlorpyrifos removal within 24 h of incubation. Strain Sphingomonas sp. Dsp-2, having 99% similarity to mpd gene (encoding parathion-methyl hydrolysing enzyme), degraded chlorpyrifos (X. Li et al., 2007). Within 18 h, Serratia sp. and Trichosporon sp. eliminated chlorpyrifos (Xu et al., 2007). Model organisms such as Achromobacter sp. HZ01 and Comomonas testosteroni have also proven effective in pesticide bioremediation (J. Zhang et al., 2021). Genetic modification of Cupriavidus nantongensis XIT using CRISPR/Cas-9 to delete the opdB gene, responsible for organophosphorus catabolism, underscores the potential of CRISPR/Cas-9 in advancing bioremediation of organophosphorus compounds (Y. Zhang et al., 2023). Representative tools, organisms, and genes involved in pesticide degradation and their associated effects are summarised in Table 4.

Representative genes and microbial effects contributing to pesticide degradation and bioremediation.

Tool Organism Gene Effects Pollutant remediated References
CRISPR E. coli Enzyme- Esterase Est816 Hydrolysis of ester linkages Pyrethroids Fan et al. (2023)
CRISPR Pseudomonas putida KT2440 cehA, gfp, mcd, mpd, pytH, vgb Enzymatic hydrolysis of ester and carbamate bonds Carbamates, pyrethroids and organophosphates Gong et al. (2018)
CRISPR Cupriavidus sp. DT-1 Mpd Hydrolysis of the phosphotriester bond Chlorpyrifos Lu et al. (2013)
CRISPR Pseudomonas aeruginosa Phd Hydrolysis of the phosphotriester bond in parathion, yielding p-nitrophenol and diethylthiophosphate Parathion Zhongli et al. (2001)
CRISPR/TALEN/ZFN Serratia marcescens MEW06 mph Enzymatic cleavage of P-O or P-S ester bonds Dimethoate, Paraoxon, Methyl parathion Wang et al. (2018)
CRISPR/TALEN/ZFN Lactobacillus brevis WCP902 opdB Hydrolysis of phosphotriester bonds Chlorpyrifos, Coumaphos, Diazinon, Methyl parathion & Parathion Islam et al. (2010)
Heavy metals bioremediation

Microorganisms represent potent contenders for the remediation of heavy metal-polluted wastewater (Ren et al., 2015). Depending on the microbial species, the type of metal, and environmental conditions, interaction occurs through direct or indirect mechanisms. Functional groups, such as amino, carboxyl, phosphate, and sulphate present on polysaccharide layers of bacterial cell walls, serve as binding sites for heavy metals, facilitating their attachment and subsequent uptake. Several microbial strains have demonstrated selective absorption and detoxification capabilities. For instance, Pseudomonas aeruginosa selectively takes up mercury ions (Yin et al., 2016), while Bacillus sp. PZ-I and Pseudomonas sp. 13 absorb Pb (II) from wastewater (Ren et al., 2015). Arthrobacter viscosus exhibits the ability to absorb hexavalent chromium Cr(VI) and enzymatically reduce it to the less toxic trivalent form Cr(III), thereby contributing to chromium bioremediation (Hlihor et al., 2017). Total absorption of mercury in the marine environment and reduction of Hg (II) to Hg (0) has been accomplished by Pseudomonas putida SP1 (Zhang et al., 2012). Detoxification of Cr (VI) via adsorption into phosphate, imidazole, hydroxyl, carboxyl, and sulfhydryl groups has been achieved by the fungi Termitomyces clypeatus (Ramrakhiani et al., 2011). Marine algae Sargassum have the potential to detoxify Cu (II) from aqueous solution (Barquilha et al., 2017). Using the CRISPR/Cas9 platform in Shewanella oneidensis, a gene network was established to optimise the electron conductive Mtr complex, flavin synthesis, and NADH generation, thereby improving extracellular electron transfer (EET). When applied to hexavalent Uranium U(VI), the edited strains achieved 3.62-fold higher bio-reduction capacity compared to control (Fan et al., 2021). Gene repression in Shewanella oneidensis also increased the riboflavin production, enhancing EET for the remediation of chromium and methyl orange (Li et al., 2020). Cupriavidus metallidurans strain MSR33, genetically engineered with mercury resistance genes merA and merB, achieved complete mercury removal from two contaminated water sources within 2 h of treatment. Additionally, much of Hg2+ removal after 3 h was observed in the absence of thioglycolate (Rojas et al., 2011). Arabidopsis thaliana phytochelatin synthase gene (PCSAt), when expressed heterogeneously in Mesorhizobium huakuii subsp. rengei B3 enhanced phytochelatin synthesis, thereby improving tolerance and sequestration capacity for heavy metals (Sriprang et al., 2003). Representative tool, organisms, and genes involved in heavy metals and hydrocarbon bioremediation are summarised in Table 5.

Representative genes and microbes involved in bioremediation of heavy metals and hydrocarbons.

Tool Organism Gene Effects Pollutant remediated References
CRISPR Shewanella oneidensis MR-1 mtrA, omcA, cymA, ribC, ribD, fdh, mdh Reductive precipitation of soluble U(VI) to insoluble U(IV) i.e., immobilisation Uranium (VI) Fan et al. (2021)
CRISPR Thermus therrmophilus HB27 Ttars M Methylation of inorganic Arsenic, i.e., As(III)→MMA (III)→DMA (III)→TMA a Arsenic Gallo et al. (2021)
CRISPR Shewanella oneidensis MR-1 dmsE Reductive transformation of mobile Chromium (VI) to immobile Chromium (III) Chromium (VI) Li et al. (2020)
CRISPR Cupriavidus metallidurans MSR33 merR1TPAGB1, merRB2D2E Regulation, uptake and enzymatic reduction (NADPH-dependent) of Hg 2+ →Hg0 Mercury Rojas et al. (2011)
CRISPR E. coli DH5A alma, xylE, p450cam Hydroxylation of long-chain alkanes Long-chain alkane Meng et al. (2018)
CRISPR Ustilago maydis Emt1, Mmc1, Mac1, Mac2 Enhanced tolerance and biodegradation Heavy metals and hydrocarbons degradation Markande et al. (2021)

MMA-monomethylarsonous acid, DMA-dimethylarsonous acid, TMA-trimethylarsine.

 Bioremediation of hydrocarbons

Hydrocarbons, for instance, aromatic dyes, hexachlorocyclohexane (HCH), polyaromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs), petroleum and its derivatives, and organohalides have been successfully bioremediated using synthetic biology approaches (Dangi et al., 2019). Strategies to enhance biodegradation of PAHs and other compounds include identification of genes encoding degradative enzymes, increasing biocatalyst activity, modulating gene expression, and introducing novel degradation pathways (Sakshi and Haritash, 2020). In E. coli, genetic manipulation has optimised aromatic pollutant bioremediation, while over-expression of nitroalkane oxidising enzyme (NOEs) in two thermophilic bacteria has further improved degradation efficiency (Zhang, 2021). Several microbial and fungal systems have demonstrated polyurethane (PU) degradation. Corynebacterium, Comomonas, Pseudomonas, and Bacillus species are prominent PU-degrading bacteria, whereas depolymerisation is most effectively achieved by fungi such as Aspergillus, Pestalotiopsis, and Penicillium (Liu et al., 2021b). Diesel biodegradation has been enhanced by Escherichia coli DH52, engineered to express alkane hydroxylase in combination with Acinetobacter and a transgenic E. coli consortium (Hassanien et al., 2023). Similarly bacterial consortium with recombinant pCom9-GPo1 alkB/E. coli DH5α increased the oil degradation rate through the heterologous expression of the GPo1 gene in Escherichia coli DH5α (Luo et al., 2015). Extracellular polymeric substances produced by marine algae Dunaliella salina and Porphyridium cruentum act as emulsifiers to metabolise oil hydrocarbons (Sukla et al., 2019). CRISPR-based systems have expanded hydrocarbon bioremediation beyond model organisms (E. coli, Pseudomonas) to non-model strains such as Rhodococcus ruber TH, Comamonas testosteroni, and Achromobacter sp. HZ01 (Jaiswal and Shukla, 2020). Co-biodegradation of petroleum hydrocarbons has been enhanced using Pseudomonas sp. B-1, Bacillus subtilis PH-1, engineered with either exoenzyme systems or the plasmid construct pET-28a (+)/almA-E (Meng et al., 2018). Overexpression of the monooxygenase gene (cph) from Arthrobacter chlorophenolicus significantly increased the removal of 4-chlorophenol (Kang et al., 2018). Furthermore, CRISPR-based genetic modification systems for extremophiles, including acidophiles, thermophiles and halophiles, offer new opportunities to advance the bioremediation process (Qin et al., 2018). CRISPR has also been applied to bio-surfactant-producing organisms to improve bio-electrokinetic remediation of hydrocarbons and pesticides (Bokade et al., 2023). Table 5 summarises key genes and associated microbial mechanisms that facilitate the detoxification and degradation of heavy metals and hydrocarbons.

 Future aspects and conclusion

Environmental contamination remains a critical challenge, driven by unsustainable agricultural practices and the persistence of pesticides, plastics, hydrocarbons, and heavy metals across terrestrial and aquatic ecosystems. Cutting-edge molecular technologies offer bioremediation as an environmentally responsible and sustainable alternative to conventional treatment methods. This review emphasises the significance of gene editing tools, such as CRISPR, ZFNs, and TALENs, in enhancing the efficiency, precision, and scope of microbial remediation. These technologies enable the development of genetically engineered microbes with improved pollutant degradation capabilities, increased resistance, and large-scale application. The acceleration in biodegradation of otherwise recalcitrant compounds has been demonstrated through enzymatic systems such as DuraPETase, Esterase Est816, and PETase-MHETase chimaeras.

Although molecular techniques have been widely implemented in microbial systems, other organisms, for instance, algae, cyanobacteria, and fungi, have also been extensively tested. Current molecular biology tools occasionally exhibit off-target site binding, which can compromise the gene knock-in accuracy; therefore, emphasis must be placed on precise target recognition. TALENs, with their strong site-specific recognition, open possibilities for fusion systems combining CRISPR and TALEN to achieve greater precision. Integrating plants and microbes provides additional opportunities for remediation, as weeds can be engineered to uptake pollutants from contaminated soils, while phytoremediation strategies can be applied to plants with high hydrocarbon content for biofuel production. Further advancements in molecular tools, coupled with expansion to a broader range of model organisms, will continue to elevate bioremediation processes. Figure 4 illustrates a proposed comprehensive framework for achieving precise genome editing through the integration of molecular, cellular, and computational strategies. Adaptive and resilient bioengineered organisms will be essential for maintaining ecological balance and safeguarding human health.

Integrated framework for precise genome editing: molecular strategies, delivery systems, repair modulation, and computational optimisation. This schematic illustrates the interconnected components of a precision genome editing workflow, encompassing molecular design, cellular repair modulation, delivery technologies, and in silico validation. The diagram is organised into five major domains—Advanced DNA binding strategies, Modulation of repair pathways, Enhanced delivery systems, Computational design and validation, and Target cell experimentation, each contributing to the iterative refinement of genome editing outcomes. Arrows labelled (a) through (f) represent directional flow and feedback between modules, highlighting the dynamic and cyclical nature of genome engineering. Advanced DNA Binding Strategies: This domain includes molecular tools for accurate DNA targeting, such as designer zinc finger arrays, context-independent TALE molecules, large fragment integrases, and high-fidelity nucleases. Epigenetic modifiers and optimised Cas variants with sgRNA design further enhance specificity. These components feed into the design pipeline via arrow (a), which connects both DNA-binding strategies and delivery systems to the Design & Validation module. Modulation of Repair Pathway: To improve editing precision, this module focuses on enhancing homology-directed repair (HDR) using activators like RPA and RAD51, inhibiting competing pathways (e.g., NHEJ) with small molecules, and modulating cell cycle regulators to favour HDR-active phases. These strategies also contribute to construct refinement via arrow (b), which links repair modulation to the Design & Validation process. Enhanced Delivery System: Efficient delivery is achieved through nanoparticle carriers, ligand-targeted systems, minimised immunogenicity capsids, and inducible expression platforms. These systems are optimised for payload size and controlled release. Like DNA binding tools, delivery systems are routed into the design pipeline via arrow (a) and receive iterative updates from it via arrow (c), forming a bidirectional exchange between molecular design and delivery optimisation. In-Silico Design and Validation: This computational core integrates genomic and epigenomic data with genomic databases to inform target selection. It employs in silico screening, machine learning models, and design refinement algorithms to validate constructs before experimental deployment. Outputs from this module are directed to the Target Cell via arrow (d), initiating experimental validation. Target Cell and Feedback Loop: Edited constructs are introduced into the target cell, where experimentation and detection confirm editing success. This leads to precise genome editing, as shown by arrow (e). Post-editing data is fed back into the design module via arrow (f), forming a feedback loop that enables continuous refinement based on empirical outcomes.

Diagram illustrating a genomic editing pathway. It includes advanced DNA binding strategies with elements like protein-based targeting and epigenetic modifiers. Inhibition, enhancement of repair pathways via small molecules, and cell cycle regulation are shown as gears. Enhanced delivery systems are depicted, emphasizing increased packaging and nanoparticle delivery. Centralized learning models and in-silico screening interact with genomic and epigenomic data for design validation. A feedback loop connects to experimentation and detection in target cells, leading to precise genome editing. Labels (a) to (f) indicate different stages of the process.

Nevertheless, several challenges must be addressed. Biosafety concerns remain paramount, as genetically engineered microbes and plants may pose risks of unintended ecological interactions, horizontal gene transfer, or persistence in non-target environments. Regulatory hurdles also limit large-scale application, with approval processes for genetically modified organisms (GMOs) varying across countries and often requiring lengthy evaluations. Public acceptance of GMOs is another critical issue, necessitating transparent communication, clear risk–benefit analyses, and demonstration of environmental safety to build societal trust.

Additional future aspects include ensuring scalability and cost-effectiveness to translate laboratory successes into field-scale applications, implementing long-term ecological monitoring to evaluate the stability and persistence of engineered organisms, integrating bioremediation with circular bioeconomy models to link pollutant removal with resource recovery (e.g., biofuels, biofertilizers, feed supplements), and expanding molecular tools to extremophiles and fusion systems (such as CRISPR–TALEN hybrids) for improved precision.

 Author contributions

SK: Conceptualization, Formal Analysis, Investigation, Software, Writing – original draft. GD: Conceptualization, Formal Analysis, Investigation, Writing – review and editing. KM: Conceptualization, Formal Analysis, Investigation, Writing – review and editing. MS: Conceptualization, Formal Analysis, Funding acquisition, Investigation, Supervision, Validation, Writing – review and editing.

 Conflict of interest

The author(s) declared that this work was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

 Generative AI statement

The author(s) declared that generative AI was not used in the creation of this manuscript.

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Edited by: Yalçın Tepe, Giresun University, Türkiye

Reviewed by: José Belisario Leyva Morales, Autonomous University of the State of Hidalgo, Mexico

Shahnoush Nayeri, The Pennsylvania State University (PSU), United States

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