Application progress and biosafety challenges of gene editing and synthetic biotechnology in diagnosis, treatment and prevention of infectious diseases

Graphical abstract

Highlights

• •CRISPR/Cas diagnostics pose dual-use risks for potential pathogen enhancement.
• •mRNA vaccines face challenges of delivery toxicity, immune evasion, and instability.
• •Engineered probiotics carry risks of environmental release and horizontal gene transfer.
• •Lack of unified regulation increases risks of ethical lapses, inadequate risk assessment, and biosafety failures.

Abstract

Global infectious disease prevention faces escalating challenges due to the continual emergence of novel pathogens and rapid viral mutations. Synthetic biology has revolutionized this field by enabling precise diagnostics, innovative vaccine platforms, and targeted therapeutics, yet it simultaneously raises concerns regarding dual-use potential, biosafety, and ethical governance. This systematic review (2015–2025, PubMed, Web of Science, Scopus) focuses on CRISPR-based diagnostics, synthetic vaccines, and engineered probiotics. CRISPR/Cas systems such as DETECTR (Cas12a) and SHERLOCK (Cas13a) demonstrate high sensitivity and rapid pathogen detection (e.g., SARS-CoV-2, Ebola), but their misuse could enhance pathogen virulence or enable bioweapon development. mRNA and viral vector vaccines offer flexible and rapid responses to emerging infections but encounter limitations in molecular stability, delivery system toxicity, and ecological safety. Engineered probiotics, designed as “living therapeutics,” can detect pathogens and modulate immune responses, yet pose potential risks of horizontal gene transfer and host-specific variability. Overall, while synthetic biology provides transformative tools for infectious disease control, it necessitates robust global regulatory frameworks, standardized biosafety practices, and ethical oversight to ensure responsible and sustainable application.

1. Introduction

Global infectious disease prevention and control are facing severe challenges such as frequent emergence of new pathogens and accelerated virus mutation, which pose unprecedented pressure on public health security [1]. Synthetic biology provides innovative tools for precise diagnosis, vaccine development, and targeted therapy of infectious diseases through the engineering design of life systems [2]. However, its rapid development is also accompanied by biosafety and ethical risks [3], requiring a balance between innovation and regulation. The application scope of the clustered regularly interspaced short palindromic repeats (CRISPR) / CRISPR associated proteins (Cas) system has extended beyond gene editing, finding new utility in the detection of infectious pathogens. Detection technologies based on Cas12a and Cas13a, such as DETETR/SHERLOCK, have the advantages of high sensitivity and no need for complex instruments, enabling rapid screening in epidemics such as COVID-19 and Ebola [4,5]. However, the dual potential of synthetic biology, such as pathogen enhancement or gene weaponization, highlights the urgency of biosafety regulation [6] (Fig. 1).

Fig. 1.

Mind map of the dual potential of synthetic biology This figure is generated by Figdraw. Abbreviations: CRISPR / Cas, clustered regularly interspaced short palindromic repeats / CRISPR associated proteins; DNA, deoxyribonucleic acid; RNA, ribonucleic acid; mRNA, messenger RNA; WHO, World Health Oorganization.

In the field of vaccine development, messenger ribonucleic acid (mRNA) technology has become a key technology for responding to public health emergencies due to its efficient design process and flexible production capacity. However, mRNA vaccines still face some challenges, including insufficient vaccine stability, potential toxicity of delivery systems, and the risk of viral immune evasion, which urgently need further optimization [7]. At the same time, virus vector vaccines may pose potential safety hazards during genetic recombination, which requires synthetic vaccine platforms to strengthen standardization and management in quality control [8].

Synthetic biology endows engineering probiotics with the ability to accurately identify and treat pathogens through programmable gene circuits. Researchers have designed sensor modules and multi input logic gate circuits to enable engineered bacteria to release antibacterial molecules under specific infection signals, enhancing treatment specificity [9]. In addition, by utilizing microecological regulation and “synthetic immunology” strategies, engineered bacteria can not only target and eliminate pathogens, but also reprogram the immune microenvironment, offering novel insights into the early detection and therapeutic strategies for infectious illnesses [10]. In recent years, natural bioactive compounds and biomaterials have shown great potential in the prevention and control of infectious diseases. Examples include okra-derived nanoparticles, isorhamnetin, and chitosan, which exhibit antibacterial and antifungal properties. These substances can also be used in agricultural disease management and immune enhancement. Integrating these resources and strengthening biosafety evaluation represent key directions for future development [[11], [12], [13], [14], [15]].

This article systematically reviews the technological progress of CRISPR diagnosis, synthetic vaccines, and engineered probiotics, analyzes their biosafety challenges, and proposes a path for building a global collaborative regulatory system. By integrating technological innovation and risk management strategies, we aim to promote the safe and sustainable development of synthetic biology in infectious disease prevention and control, and lay the groundwork for evidence-based approaches to confronting global health emergencies.

2. Exploration of CRISPR/Cas system applications in infectious disease diagnostics and evaluation of related biosafety risks

The CRISPR/Cas system, originally discovered in bacteria and archaea, functions as an adaptive immune mechanism that enables the host to recognize and cleave invading genetic elements such as bacteriophage deoxyribonucleic acid (DNA). It is composed of CRISPR and associated Cas proteins [16]. Upon foreign DNA invasion, short fragments of the exogenous sequence are incorporated into the CRISPR array as spacers. This array is subsequently transcribed into CRISPR RNAs (crRNAs), which guide Cas nucleases to complementary target sequences for precise cleavage. Through molecular engineering, CRISPR/Cas systems—particularly CRISPR/Cas9—have been optimized for programmable, sequence-specific applications and are now extensively utilized in genome editing and nucleic acid-based diagnostics [17].

2.1. Field-deployable detection methods enabled by CRISPR/Cas mechanisms

CRISPR/Cas technology, a core tool in synthetic biology, enables precise and efficient genome editing and has shown great potential in virus diagnostics [18,19]. Originating from the bacterial immune system, it can specifically recognize and cleave foreign nucleic acids [[20], [21], [22]]. Engineered Cas proteins such as Cas12 and Cas13 activate nuclease activity upon target recognition, generating detectable signals for rapid and sensitive nucleic acid detection [23]. Although Cas9 lacks non-specific cleavage activity, it can be combined with fluorescent probes or nanomaterials to enhance detection sensitivity [[24], [25], [26]], particularly for DNA viruses and mutation sites (Table 1).

Table 1.

Comparative assessment of three CRISPR/Cas systems.

System	Advantages	Limitations
CRISPR/Cas12a	A heightened ability to detect mismatches plays a key role in increasing target accuracy while limiting unintended interactions.	The presence of PAM sequence prerequisites imposes constraints on which gene regions can be targeted.
	Reduced molecular size facilitates cellular and tissue delivery.	Demonstrates a modest decrease in cutting efficiency.
	The resulting overhangs promote accurate incorporation of donor DNA fragments.	Exhibits heightened sensitivity to temperature variations.
	The ability of trans-cleavage to act on multiple substrates facilitates a range of versatile detection techniques.	Trans-cleavage's lack of specificity could result in undesired cleavage activities.
CRISPR/Cas13a	Targets RNAs.
	Allows gene expression knockdown without permanent genomic alteration, reducing off-target risks.	The sizable molecular dimensions create obstacles to achieving efficient intracellular delivery.
	Only a single base, either with or without a PFS, is required, allowing for enhanced flexibility in target gene selection.	Incompatible with the knock-in of RNA sequences.
	Trans-cleavage activity underpins multiple versatile detection techniques.	Trans-cleavage's lack of specificity could lead to undesired cleavage events in RNA transcripts.
CRISPR/Cas9	Methodical studies focus on maximizing both the accuracy and efficiency of applications in genetic engineering.
	Simple to operate; can concurrently address numerous genes across many different organisms and types of cells.	Off-target effects.
	Engineered Cas9 such as dCas9 enables gene regulation and epigenetic editing.	The relatively bulky nature poses challenges for cellular uptake.
	Supports exact genetic edits encompassing deletions, insertions, and point mutations.	The requirement for PAM constrains target gene selection.

Abbreviations: PAM, protospacer adjacent motif; CRISPR, clustered regularly interspaced short palindromic repeats; Cas, CRISPR associated proteins; dCas9, deactivated Cas9; DNA, deoxyribonucleic acid; RNA, ribonucleic acid.

Compared to traditional methods, CRISPR-based diagnostics require no thermal cycling, offer fast results, and are easy to operate, making them suitable for low-resource settings and outbreak response [27]. Integrated with microfluidics and portable devices, CRISPR platforms are evolving into “plug-and-play” systems and are expected to become vital tools in point-of-care infectious disease control [28,29].

2.1.1. Cas12a (DETECTR system) and Cas13a (SHERLOCK system)

Cas12a is a type V CRISPR related protein that has the ability to activate non-specific single stranded DNA (ssDNA) cleavage activity upon recognition of specific double stranded DNA (dsDNA) targets [30]. The DETETR system utilizes this feature by introducing ssDNA probes with fluorescent labels to generate fluorescent signals in the presence of target DNA, thereby achieving detection of specific DNA sequences [23,31]. For example, the system has been successfully applied to the detection of pathogens such as human papillomavirus (HPV), demonstrating high sensitivity and specificity [32].

As a type VI CRISPR effector, Cas13a is designed to recognize and bind specific RNA targets, subsequently initiating non-specific cleavage of surrounding ssRNA. The SHERLOCK system combines isothermal amplification techniques (such as RPA) with the characteristics of Cas13a to achieve rapid detection of RNA viruses by detecting fluorescence signals (as shown in Fig. 2) [33]. Applications of this platform include the detection of Zika and dengue viruses, facilitated by its straightforward procedures and minimal cost requirements [34].

Fig. 2.

Schematic diagram of the SHERLOCK system working (By Figdraw). Abbreviations: RPA, recombinase polymerase amplification; RT-RPA, reverse transcriptase RPA; CRISPR, clustered regularly interspaced short palindromic repeats; Cas, CRISPR associated proteins; dCas9, deactivated Cas9; DNA, deoxyribonucleic acid; dsDNA, double stranded DNA; RNA, ribonucleic acid; crRNA, CRISPR RNA.

2.1.2. Application of CRISPR/Cas tools in viral genome recognition

CRISPR/Cas system has been widely used in nucleic acid detection of many viruses, especially in detection of novel coronavirus, influenza virus, Ebola virus, Lassa fever virus, monkeypox virus and other infectious viruses [[35], [36], [37]]. Many innovative tools such as DEETECTR and SHERLOCK systems have been rapidly developed and put into use, particularly in nucleic acid testing for COVID-19 [38]. These systems can complete sample analysis within 30 to 60 min and have extremely high sensitivity, achieving sensitivity sufficient to recognize viral RNA at levels down to a few dozen copies per microliter. This feature makes the CRISPR/Cas system have great potential for application during epidemics, especially in rapid screening and community monitoring, which can provide accurate diagnostic results conveniently and efficiently without complex instruments [39,40].

In addition, CRISPR/Cas can also be used to detect variants of viruses, such as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus and its variants [41], influenza virus, etc. [42]. It has served as a vital component in facilitating effective measures for epidemic containment and prevention [25,43]. By detecting virus variants, public health departments can gain real-time insights into the spread and mutation dynamics of the virus, enabling the advancement of more targeted and efficient control and prevention methods. This CRISPR based technology can provide real-time monitoring for the rapid mutation of viruses, facilitating improved precision and effectiveness in international epidemic response initiatives [44]. In summary, the CRISPR/Cas system not only performs well in conventional virus detection, but also demonstrates great potential for application in virus variant detection and real-time monitoring of epidemics. The widespread application of these innovative tools enables us to identify the epidemic trends of infectious diseases earlier and more accurately, promote the development of global public health, aiding in the resolution of the progressively intricate issues surrounding infectious disease control and prevention [21].

2.2. Advantages, risks, and dual-use challenges of CRISPR/Cas gene editing

The CRISPR/Cas system, owing to its programmability and high specificity, enables facile target design and can be applied to virtually any genomic locus [45]. Compared with zinc-finger nucleases (ZFN) and transcription activator-like effector nucleases (TALEN), it offers greater operational accessibility and lower economic cost, significantly enhancing the universality and efficiency of genome editing [46]. Moreover, the system supports multiplexed genomic modifications, enabling precise regulation of complex traits, and demonstrates revolutionary advantages in disease model construction, functional gene screening, and potential gene therapy applications. Nevertheless, its use faces multiple challenges, including off-target effects that may cause unintended pathogenic consequences, variable editing efficiency depending on cell type and repair pathway preference, and delivery systems that may elicit immune responses or nonspecific toxicity [47,48].

Importantly, CRISPR/Cas exhibits typical dual-use research of concern (DURC) characteristics. Its ease of use and low cost not only facilitate advances in disease treatment, cancer immunotherapy, and vaccine development [49,50], but also raise the risk of misuse, such as enhancing pathogen virulence, transmissibility, or drug resistance, and even synthesizing or reconstructing novel or extinct pathogens, posing serious biosafety threats [51,52]. Further risks involve non-therapeutic human genome editing, such as enhancement of cognitive or physical traits, which could exacerbate social inequality and raise ethical dilemmas [53,54]; embryonic genome editing carries unpredictable heritable consequences and ethical concerns [[55], [56], [57]].

In agricultural applications, CRISPR also presents risks related to off-target effects and gene flow, potentially disrupting ecological balance. To mitigate such risks, researchers have developed CRISPR-based “kill switch” systems that induce microbial self-destruction under specific conditions, ensuring biological containment [58]. Therefore, although CRISPR provides new impetus for synthetic biology [49], its potential risks necessitate careful governance. Future strategies should include global shared databases and transparent review mechanisms to prevent the dissemination of sensitive technologies due to regulatory gaps [59], and a balanced integration of innovation and governance is required to ensure the long-term safety, ethical compliance, and sustainable development of CRISPR/Cas applications in medicine, agriculture, and ecological management [60,61].

3. Synthetic vaccine innovation and new approaches to prevention and control

3.1. The application advantages of synthetic biology in the vaccine field

The application of synthetic biology technology in vaccine platforms provides more refined and customized solutions for vaccine development. Synthetic biology can accurately design vaccine antigens through methods such as gene synthesis and protein engineering, which not only accelerates vaccine development but also enables more effective immune responses against novel pathogens [62]. The development of mRNA and DNA vaccines has become an innovative breakthrough in the field of vaccine development in recent years. mRNA vaccines are synthesized in vitro and delivered to cells using lipid nanoparticles (LNPs) carriers, inducing the synthesis of target antigens and triggering immune responses. DNA vaccines induce immune responses by constructing plasmid DNA containing the target gene and using gene transfection techniques to deliver it into cells [63].

In addition to lipid nanoparticles (LNPs), polymeric nanoparticles [such as poly(lactide-co-glycolide)], inorganic nanomaterials (such as gold nanoparticles and graphene oxide), and self-assembling nanostructures (such as virus-like particles, VLPs) have been used to encapsulate vaccine antigens or nucleic acid fragments, enhancing their bioavailability and sustained release in vivo [64]. Certain nanomaterials can also be surface-modified with targeting ligands to enable active delivery to specific immune cells, such as dendritic cells or macrophages, thereby improving the efficiency of immune responses. Moreover, nano-adjuvants (such as nano-alum and nano-emulsions) can significantly activate innate immune pathways, promote antigen presentation, and facilitate the establishment of immune memory, offering new directions for next-generation vaccine development [65].

The application of synthetic biology in vaccine platforms has greatly improved the speed and efficiency of vaccine development, enabling rapid response to emerging infectious diseases. The traditional processes involved in vaccine development typically entail significant financial investment and extended production durations, while the application of synthetic biology can produce vaccine raw materials through engineered microorganisms such as yeast or Escherichia coli, reducing production costs and improving efficiency. Vaccine formulations may exhibit improved stability and biocompatibility through this method, enabling them to be efficiently distributed globally, especially in resource limited areas [66]. In addition, the advancement of synthetic biology has facilitated the development of vaccines with diverse functionalities, such as developing vaccines that can simultaneously produce immune responses to multiple pathogens to improve immune efficacy and coverage. In the future, with further technological development, synthetic biology is expected to play a more important role in the vaccine industry and public health prevention and control [67].

3.2. mRNA vaccine platform

3.2.1. mRNA vaccine synthesis process

The mRNA vaccine refers to a form of immunization that utilizes messenger RNA as its core component. The underlying mechanism involves the delivery of mRNA encoding pathogenic antigens into human cells, prompting these cells to synthesize pathogen antigens and activate immune responses [68]. Unlike traditional vaccines that generate immunity through the direct use of inactivated viruses or viral proteins, mRNA vaccines induce immune responses against pathogens by producing antigens in the body [7]. mRNA vaccines, as a revolutionary technology, have shown great potential in controlling infectious diseases. It can not only quickly respond to sudden outbreaks of infectious diseases, but also has flexibility and efficiency, and is expected to become an important component of future global vaccination and prevention strategies. However, in the face of technological challenges such as storage and transportation, as well as virus mutations, the development of mRNA vaccines still needs continuous improvement and optimization [69].

The synthesis process of mRNA vaccines includes three key steps: antigen selection, structural design, and mRNA synthesis. The initial and essential step is the identification of a suitable antigen, often a protein or a defined segment of the pathogen, capable of eliciting a robust immune response. Antigen selection directly affects the immune efficacy and protective efficacy of vaccines [7]. For example, in the design of influenza vaccines, selection of the hemagglutinin (HA) protein derived from the influenza virus was carried out for use as the target antigen. Choosing appropriate antigens can ensure that vaccines can stimulate specific immune responses, thereby providing effective protection [70]. Secondly, during the design stage of mRNA vaccines, the genetic code corresponding to the desired antigen is translated into messenger RNA sequences. In this phase, it is necessary to optimize the structure of mRNA to ensure its stability and efficient expression in vivo. Meanwhile, mRNA sequence design must ensure the complete or accurate encoding of key regions of the target antigen. The final step involves the generation of mRNA through cell-free transcription methods. During synthesis, template DNA (usually obtained through PCR technology) is used to transcribe mRNA, and auxiliary components such as modified nucleotides and cofactors are added to improve the stability and delivery efficiency of mRNA [7]. This process ensures that mRNA can be transmitted and effectively expressed in the body, therefore stimulating immune recognition and the subsequent targeting of antigens.

3.2.2. Biological safety assessment of mRNA vaccines

In the development process of mRNA vaccines, biosafety assessment is a critical step. Initially, particular focus must be placed on maintaining the stability of mRNA molecules. Due to the natural susceptibility of mRNA to degradation by nucleases both in vivo and in vitro, insufficient stability may lead to a decrease in vaccine efficacy. Therefore, researchers must evaluate the stability of mRNA under different environmental conditions and improve its in vitro and in vivo survival time through chemical modifications, structural optimization, and other means [7]. Secondly, the safety of the delivery system is also an important aspect of biosafety assessment. Currently, mRNA vaccines widely use LNPs as delivery carriers, but LNPs may trigger local or systemic inflammatory responses. A systematic investigation into the biocompatibility and in vivo metabolism of the delivery vehicle is crucial to decrease toxicity risks while preserving efficient delivery [71]. The risk of immune system evasion requires significant attention in the third step. Due to the susceptibility of viruses to mutation, existing vaccines may have reduced protective efficacy due to changes in antigenic epitopes. Therefore, vaccine design needs to comprehensively consider virus mutation trends, select highly conserved antigen regions, or enhance broad-spectrum immune protection through multi epitope design [69]. Fourthly, mRNA vaccines also pose a risk of immune overactivation. mRNA itself and its delivery system may activate overly strong innate immune responses, such as abnormal activation of interferon pathways or excessive inflammatory reactions, leading to side effects. Therefore, in the early stages of vaccine development, it is necessary to evaluate its immunogenicity, optimize mRNA sequences and delivery strategies to control unnecessary innate immune activation [72]. Finally, ensuring biosafety relies heavily on standardized production processes and effective quality management.

The synthesis, modification, and delivery system preparation of mRNA vaccines need to be standardized to prevent safety or efficacy fluctuations caused by batch differences. By establishing a strict production and quality control system, potential biosafety hazards can be effectively reduced [73].

3.2.3. Comprehensive strategy for addressing the biosafety issues of mRNA vaccines

Multiple effective solutions have been proposed in current research to address the biosafety challenges in the development of mRNA vaccines. Firstly, in terms of mRNA molecular design optimization, scientists have chemically modified mRNA sequences by introducing modified bases such as pseudouridine and 5-methylcytosine to reduce the mRNA's ability to activate innate immune responses, while improving its stability and translation efficiency. This strategy can effectively reduce excessive inflammatory reactions, improve the overall safety and expression level of vaccines [7]. Secondly, in terms of delivery system optimization, researchers have made compositional and structural improvements to LNPs. For example, using ionized lipids instead of traditional cationic lipids to enhance the encapsulation and release efficiency of mRNA, while reducing toxic reactions during delivery. Reasonable adjustment of particle size and surface charge has also been proven to improve delivery efficiency and reduce inflammation [70]. Faced with the problem of virus immune escape, developers have proposed a multi antigen, multi epitope vaccine design strategy. By encoding multiple antigen fragments or epitopes in mRNA vaccines, multiple specific immune responses can be induced simultaneously, thereby enhancing broad-spectrum protection against viral variants and improving vaccine adaptability and persistence [69].

In terms of production and quality control, to ensure the safety of large-scale vaccine applications, enterprises and research institutions have established standardized production processes, strictly screened raw materials, and introduced in vitro mRNA activity detection systems to ensure the quality consistency and biological safety of each batch of products. In addition, through dose optimization and immune regulation strategies, researchers can adjust the intensity of immune response reasonably according to the delivery dose and vaccination program, avoiding side effects caused by excessive immune activation, thereby reducing the risk of adverse reactions while ensuring protective effects [71].

3.3. Biosafety considerations for vaccine synthesis platforms

In the development and application of vaccine synthesis platforms, issues such as sample contamination, toxic residues, and vaccine recombination constitute major biosafety challenges.

Firstly, sample contamination not only compromises vaccine quality control but also highlights the vulnerabilities of current production systems in terms of source management and risk response. The complexity of contamination sources—including raw materials, culture media, environmental factors, and operational procedures—means that conventional aseptic techniques and GMP standards may not be fully effective in high-throughput production environments [74]. Therefore, relying solely on physical isolation and disinfection measures is insufficient to eliminate contamination risks at their root. Future vaccine platform design urgently requires the integration of automation, real-time biosensor monitoring, and AI-assisted contamination detection systems, shifting from a “detection-response” model to a “prediction-prevention” dynamic prevention and control mechanism [75].

Secondly, the issue of toxic residues highlights the current technical limitations in identifying and eliminating potential risk components during vaccine production [76]. For instance, residual host cell DNA, chemical reagents, and enzymes may pose carcinogenic or immunological side effects, yet their low abundance and complex backgrounds make it difficult to fully assess the effectiveness of removal. Current quality control practices primarily rely on endpoint testing and lack a mechanistic understanding of residue risks and source-level process optimization. Therefore, it is essential to strengthen “design-oriented” synthetic biology strategies to minimize the use of harmful components at the production source and to establish an integrated residue risk assessment framework that encompasses omics analysis, bioinformatics prediction, and animal toxicity evaluation [77].

An even greater challenge is the potential risk of genetic recombination in viral vector vaccines. Although existing literature has issued warnings about such risks, particularly in immunocompromised individuals, systematic response measures in practice remain underdeveloped. The essence of genetic recombination lies in risk exposure under conditions of information asymmetry, and its occurrence is often unpredictable. Therefore, vaccine design must go beyond traditional strategies employing “non-replicating” or “defective” vectors and instead adopt a dual-safeguard approach that integrates genetic stability design with biological containment systems [78]. At the same time, vaccine platforms should be encouraged to incorporate “recombinant sequence risk prediction” and “environmental gene exchange assessment” as mandatory evaluation modules early in the development process, thereby enhancing the overall system’s proactive biosafety capabilities [79].

3.4. Synthetic biology and global vaccine safety regulatory system

Synthetic biology technology has driven a profound transformation in vaccine development models, enabling rapid design and large-scale production of vaccines based on platforms such as mRNA, DNA, and viral vectors. However, these new vaccine platforms have brought unprecedented biosafety and regulatory challenges due to their complex structure, programmable components, and diverse expression systems [80]. Therefore, the global vaccine safety regulatory mechanism urgently needs to be restructured based on the development of synthetic biology.

Organizations represented by the European Medicines Agency (EMA) have established a lifecycle oriented pharmacovigilance system for the marketing and use of COVID-19 vaccines. The EudraVigilance system enhances real-time monitoring and data-driven management of adverse reactions, providing valuable experience for future vaccine safety regulation [81]. In addition, the World Health Organization (WHO) and the Global Alliance for Vaccines and Immunization (GAVI) stress that a global vaccine safety capability framework should be constructed from the perspective of the entire lifecycle of vaccines, covering the complete chain from research and development, clinical trials, approval to post market monitoring [82]. In this context, developing a unified global scientific agenda for vaccine regulation has become a necessary measure. The agenda should include elements such as platform technology identification, data model standardization, risk assessment consistency, and regulatory personnel capacity building. In particular, a dedicated regulatory pathway should be established for the specific risks of synthetic biology platforms (such as LNP vectors, modified nucleotides, and artificial gene elements) (Table 2) [83].

Table 2.

Core strategies and high-level milestones defined under the Global Vaccine Safety Blueprint initiative.

Strategic objective	High-level targets
To reinforce vaccine safety monitoring systems globally.	Aiming to build functional systems across countries that enable spontaneous adverse event following immunization reporting. Expansion of pharmacovigilance capacity in regions manufacturing vaccines and in areas where novel vaccines are being rolled out.
Aiming to improve country-level capabilities in monitoring and analyzing vaccine safety indicators.	Nations are equipped to confirm vaccine safety signals and take suitable public health actions in response. Nations possess the necessary capabilities to evaluate and examine emerging public health risks. A global cooperation framework is in place to facilitate unified public health efforts.
To create effective vaccine safety communication frameworks at the national level that foster awareness of vaccine pros and cons, gauge public risk perception, and support swift action regarding adverse events and safety matters.	Aiming to establish or improve countries’ capabilities for sustained communication with community members, medical staff, and authorities about key vaccine safety matters. Rumors concerning vaccine safety will be quickly identified and examined, with communication plans established accordingly.
To establish globally standardized tools and methods that facilitate and strengthen country-level vaccine safety activities.	Uniform protocols and methods will be accessible for examining and addressing vaccine safety concerns. Unified resources will be accessible to aid efforts in monitoring vaccine safety.
Advancing robust legal, regulatory, and management systems to secure vaccine safety within countries, regions, and internationally.	Provisions for vaccine safety monitoring, along with clear responsibilities, have been implemented across all nations. Vaccine pharmacovigilance is recognized as a global responsibility.
To enhance regional and international technical support mechanisms tailored to the specific needs articulated by countries.	Universal access to educational content aimed at building vaccine safety monitoring systems at the country level. Global access to technical support for addressing vaccine safety issues.
Offering expert guidance concerning vaccine safety across countries, regions, and worldwide.	At all governance levels—national, regional, and global—advisory bodies safeguard the global vaccine safety status and assist in investigating safety signals of international concern.
Establishing mechanisms to promote effective communication among governments, multilateral bodies, and manufacturers at the national, regional, and global scales.	Mechanisms enabling smooth communication between the public and private domains are in place, supported by consistent tools and methodologies.

Therefore, the deep integration of synthetic biology technology with global vaccine safety regulatory mechanisms will provide a solid foundation for building a more scientific, reliable, and fair global vaccine immunization system. Through the combination of innovative technology and a sound regulatory framework, global vaccine safety and public health protection capabilities will be significantly enhanced [84].

4. Application of synthetic biology in engineering probiotics for Coping with infectious diseases and biological safety challenges

4.1. Synthetic biology promotes innovative application of engineering probiotics in the diagnosis and treatment of infectious diseases

Both Gram-positive and Gram-negative bacterial species in the human microbiota are amenable to engineering as probiotics for functions such as immune system modulation, pathogen inhibition, metabolic adjustment, and disease detection (as depicted in Fig. 3). Synthetic biology endows traditional probiotics with the ability to accurately identify and treat pathogens through modular and programmable genetic circuit construction [10]. By constructing sensor modules that can sense inflammatory factors (such as nitric oxide, lipopolysaccharide (LPS)), bacterial toxins, or pH changes, researchers have transformed engineered Escherichia. coli, lactobacilli, and other bacteria into “live biological probes” to achieve targeted recognition and response therapy for pathogens such as Salmonella, Clostridium difficile, and Helicobacter pylori [85,86]. Researchers have further summarized the design and optimization strategies of various sensor modules, such as lactate sensing elements and NO responsive promoters based on two-component systems, and demonstrated the high specificity recognition effect of these modules on infectious lesions in multiple animal models [87].

Fig. 3.

By exploiting the natural localization tendencies of microbiota in the body, microbial therapeutics can be tailored to address illnesses or sustain host well-being. This figure is generated by Figdraw. Abbreviations: AMP, adenosine monophosphate; GFP, green fluorescent protein; DC, dendritic cell; Tc, cytotoxic T lymphocyte.

In addition, the design of multi input logic gate circuits (such as AND/OR gates) enables engineered bacteria to release antimicrobial peptides or RNA only after sensing multiple infection signals simultaneously, thereby improving specificity [88,89]. The researchers further described the use of CRISPR interference system and RNA synthesizer to construct targeted regulatory pathways, boosting the capability of designed probiotics to counteract genetic mechanisms of pathogen resistance. These strategies have achieved good results in various animal models, providing new ideas for early diagnosis and treatment of infectious diseases [86,90,91].

Researchers have also demonstrated the important role of treatment concepts based on microbiota regulation in anti infection, such as regulating intestinal homeostasis to inhibit pathogen colonization and promote beneficial bacterial expansion [[92], [93], [94]]. It is worth noting that some studies have explored the interaction mechanism between engineered bacteria and the host immune system [9]. Researchers have pointed out that by expressing chemokines, bacterial surface antigens, or MAMPs (microbial associated molecular patterns) mimetics, engineered bacteria can not only neutralize specific pathogens, but also reprogram the local immune microenvironment, achieving a coordinated strategy of “ antigen exposure, triggering immune activation, and targeted removal”. This “synthetic immunology” model is expected to make breakthroughs in complex scenarios such as drug-resistant bacterial infections and virus carrier control [9].

4.2. Biological safety challenges and design prevention and control strategies for live therapeutic materials

Although synthetic biology provides powerful functional expansion capabilities, engineered probiotics face significant biosafety challenges when entering human or environmental systems as living materials. Firstly, engineered bacteria may survive and reproduce outside the host's body, leading to disturbances in non target ecosystems. Secondly, there is a risk of horizontal transfer of the therapeutic modules or resistance genes carried by it, which may be acquired by environmental bacteria or opportunistic pathogens. Furthermore, some engineered bacteria may cause host inflammation or immune disorders due to uncontrolled expression [95,96].

To address these risks, scientists have suggested multiple biosafety management strategies: (1) genetic insulation design, such as using non natural codons and synthetic amino acids to avoid gene drift. (2) Inducing control systems, such as using arabinose, tetracycline, etc. implementing promoter-based switches to regulate gene expression with spatiotemporal precision. (3) Biological self destruct mechanism, which introduces a self destruct protein expression system triggered when detached from the host or lacking certain environmental signals through synthetic circuits. (4) Logic gate linkage feedback regulation further enhances system robustness [95,96]. Researchers have further expanded the library of biological components, including low leakage promoters, dual negative regulators, and feedback steady-state systems, providing a foundation for the safe operation of synthetic circuits [97]. From the perspectives of host microbiota co adaptation and stability of synthetic module implantation, safety control strategies have been further enriched [9,98].

In addition, researchers are developing more sophisticated environmental response mechanisms to meet the biosafety needs in different scenarios. For example, based on photosensitive systems (such as blue light sensors) or magnetic control systems, non-invasive and remote control of the survival and activity of probiotics can be achieved, greatly improving their adaptability and safety in complex clinical environments [91,93]. At the same time, the combination of physical chimeric materials (such as hydrogel packaging) and engineering bacteria was also discussed to enhance their localization and survival control ability. Some researchers have further pointed out that the regulatory mechanism based on the overall structure induction of microecology can help achieve regional activation and inhibition of engineering bacteria, mitigating impact on ecological areas outside the intended target [99].

4.3. Practical obstacles and coping strategies for the clinical translation of engineering probiotics

From model experiments to clinical applications, the transformation of engineered probiotics in the field of infectious diseases still faces multiple obstacles. Firstly, there is uncertainty in the regulatory system. It is pointed out that there is currently no unified international approval standard for engineering live drugs, and there is a lack of systematic methods for evaluating their safety, persistence, and environmental impact [92]. Secondly, the public's acceptance of “genetically modified bacteria” is limited, and some patients or social groups may have a guarded mentality towards the ingestion or implantation of “artificial bacteria” in their bodies [100].

In addition, individual microbiota differences also pose challenges to the consistency of treatment efficacy. Researchers have emphasized that the gut microbiota backgrounds of different populations vary, and the expression and colonization of the same engineered strain differ significantly among individuals [98]. Therefore, researchers are exploring personalized treatment strategies by combining gut microbiome data to design “precise” engineered probiotic combinations that achieve more stable and efficient treatment outcomes. Although this strategy has broad prospects, it poses higher requirements for the standardized production, transportation, and storage of engineering bacteria.

To promote the clinical translation process, it is necessary to establish a multidisciplinary collaborative research and development system, including: (1) an engineering bacterial evaluation platform jointly built by bioengineers, clinical doctors, and microbiome` experts. (2) Accumulation of preclinical safety data for large-scale populations. (3) An ethical promotion mechanism jointly constructed by the government, enterprises, and the public to enhance social acceptance. (4) Promote the development of internationally unified standards for quality control and registration of engineering bacteria [92,95].

In summary, synthetic biology endows engineering probiotics with unprecedented therapeutic capabilities, opening up new paths for the prevention, diagnosis, and treatment of infectious diseases. However, its biosafety risks and transformation barriers cannot be ignored. In the future, it is necessary to strike a balance between functional enhancement and risk control, and promote the establishment of a comprehensive framework covering multiple levels such as functional assessment, safety verification, and ethical supervision, in order to achieve sustainable development and standardized application of engineered probiotics in the global public health field.

5. Prospect

CRISPR diagnosis will integrate AI with miniaturized devices to achieve rapid detection of multiple targets, while requiring global regulatory conventions to prevent abuse risks [101]. RNA vaccines focus on broad-spectrum design (such as conservative antigens) and stable delivery (freeze-drying technology) to enhance protection against mutant strains and track the long-term safety of synthetic components [102]. Engineering probiotics require the development of an intelligent regulation system (multi signal sensing + CRISPR circuit), combined with “orthogonal genome” to block gene transfer risks and reduce ecological disturbances [103]. Led by international organizations such as WHO, establish a technology research and risk warning network to share global adverse reaction data. Develop “traffic light” rules for gene editing, define prohibited, restricted, and encouraged areas, and increase public acceptance of engineered live therapies through science popularization [104]. The combination of synthetic biology, nanotechnology, and organoid models has spurred the development of targeted therapy nanorobots, organ chips to accelerate drug evaluation, and a “holistic approach to prevention and containment” infrastructure to boost outbreak response capabilities. Synthetic ecology can design microbial community interaction systems to optimize the ecological safety of engineering bacteria [105]. Promote personalized treatment of engineering probiotics, customize strain combinations based on microbial ecological data, and unify international approval standards [106]. The vaccine industry needs to strengthen the biosafety of the production chain to ensure the controllability of synthetic components.

Synthetic biology is expected to become a key technology to end pandemics, but its potential release requires a three-dimensional balance of technological innovation, ethical constraints, and global collaboration. Technological breakthroughs and regulatory improvements will reshape the prevention and control system, providing long-term protection for global health equity.