T/C---health internal – biotech!*microsomething biotech

6 CRISPR-CAS SYSTEMS: A PRECISION GENOME EDITING TOOL FOR MEDICINE

The CRISPR Cas systems offer a novel approach to both RNA interference and genome editing (Mahato et al., 2024). When genetically engineered, CRISPR-Cas systems serve as a valuable tool for controlling gene expression in both prokaryotic and eukaryotic cells. Approximately 90% of archaea and 40% of bacteria possess CRISPR-Cas systems to provide resistance to foreign DNA elements (Barrangou et al., 2007). Some of these systems have been adapted as genetic engineering tools for medically important human cell lines (Mahato et al., 2024). In particular, the Class 2 Type II CRISPR-Cas9 system from Streptococcus pyogenes has been utilized for a variety of genetic purposes in a wide range of organisms, from bacteria to humans (Cho et al., 2013). The wild-type system contains a nuclease protein (Cas9) that cuts specific DNA sequences based on a single guide RNA (sgRNA) that specifically interacts with the Cas9 proteins and the DNA sequence it targets. Conversely, nuclease-deactivated CRISPR–Cas (dCas) systems, which do not cut DNA, can be used to modulate transcription in cells and organisms (Qi et al., 2013). An alternative approach to bypass the limitations of double-stranded break DNA knock-out-based protocols is to induce mRNA knockdown at the target site (i.e., silencing the expression of the target gene instead of knocking out the gene). Several Cas12 and Cas13 proteins have been shown to possess RNA-guided RNase activity in a manner analogous to RNA-guided DNA targeting CRISPR-Cas9 effectors (Nie et al., 2023). Performing mRNA knockdowns instead of gene knockouts (in which the gene is irreversibly deleted) is very useful for genes that cannot be completely removed because they are essential for cell viability. In any case, because the CRISPR-Cas platform is concise and self-contained (and the sgRNA can be easily customized), it has the potential to be adapted for different purposes, such as performing gene knockouts, knock-ins, the addition of tags, or controlling the expression of a gene of interest (Barrangou et al., 2007). The emergence of CRISPR-Cas technology in the biomedical field marked an important milestone in the challenging goal of finding targeted therapies for complex diseases such as cancer or other genetic disorders (Mahato et al., 2024). A further advancement in the CRISPR-Cas systems is the use of CRISPR-associated transposase systems (CAST) found in certain specific microorganisms, such as certain cyanobacteria. For example, the natural CAST system from cyanobacteria Scytonema hofmanni (ShCAST) consists of Tn7-like transposase subunits (TnsB, TnsC, and TniQ), a CRISPR array trans-activating RNA, and a Cas12k protein. ShCAST catalyzes RNA-guided DNA transposition by unidirectionally inserting segments of DNA 60 bp downstream of the protospacer adjacent motif (PAM) (Strecker et al., 2019). The PAM motif is a specific and variable (depending on the respective CRISPR-Cas system) 2–6 bp DNA sequence immediately following the DNA sequence targeted by the sgRNA-Cas nuclease complex (Barrangou et al., 2007). When the natural ShCAST system was heterologously expressed in E. coli, the genetically developed system was shown to integrate DNA into specific targeted sites of the E. coli genome with frequencies of up to 80% (without positive selection markers or additional requirements such as DNA repairing templates) (Strecker et al., 2019). Therefore, the CAST system constitutes an excellent tool for performing precision DNA insertions (“knock-ins”) in targeted genes with few additional requirements. In summary, CRISPR-Cas and its derivative systems have emerged as revolutionary platforms in medicine, providing an unparalleled, finely-tuned gene editing tool for both DNA and RNA targeting, functional genomics, gene therapy, disease modeling, and cancer research, among other applications (Zhou et al., 2023). The key applications of CRISPR-Cas systems in medicine are outlined below.

6.1 Gene editing and therapeutics

CRISPR-Cas systems enable precise genome modification by targeting specific DNA sequences. By rectifying disease-causing mutations or introducing therapeutic genes, CRISPR-Cas-based gene therapies present promising pathways for developing effective treatments for genetic diseases such as cystic fibrosis, sickle cell disease, and Duchenne muscular dystrophy. Beyond genome editing, CRISPR-Cas systems can also be employed for programmable epigenetic modifications, facilitating targeted DNA methylation or demethylation (Yahsi et al., 2024). Furthermore, using CRISPR–Cas-based transactivation approaches (CRISPRa) with dCas proteins or antibody-mediated recruitments, transcriptional activators can be recruited to genomic regulatory elements, enabling potent and versatile synthetic transcriptional control on genes of interest, such as dysregulated genes causing disease (Mahata et al., 2023). Overall, these capabilities of the CRISPR-Cas systems hold immense promise for advancing precision medicine initiatives, allowing the development of personalized therapies tailored to individual patients' genetic profiles.

6.2 Cancer research, infectious disease control, and disease modeling

CRISPR-Cas technology facilitates the study of tumor suppressor genes and oncogenes by modulating gene expression and allowing the construction of either knock-out or knock-in mutants. The resulting knowledge can be utilized to develop targeted cancer therapies and identify biomarkers for early detection and prognosis. CRISPR-Cas systems also enable the creation of cellular and animal models that closely mimic human diseases. Researchers can introduce disease-associated mutations into cells or organisms to study disease mechanisms, screen potential drugs, and develop personalized therapies (Lopes & Prasad, 2024). In addition, CRISPR-based screening platforms enable high-throughput interrogation of gene function across the entire genome, allowing researchers to identify genes involved in disease pathways, drug resistance, and therapeutic responses. Lastly, CRISPR-Cas systems offer innovative strategies for combating infectious diseases. These systems can be programmed to destroy viral and bacterial genomes, offering alternative treatments to antivirals and antibiotics (Zahedipour et al., 2024).

7 BIOSENSORS USING WHOLE BACTERIAL CELLS

Modern medicine necessitates the rapid identification of pathogenic microbial strains to choose the best possible treatment as quickly as possible. In this regard, nanotechnology enables the development of highly sensitive and specific biosensors to detect biomarkers, greatly expanding diagnostic possibilities. A biosensor is an instrument used to detect and/or measure a specific compound (i.e., “analyte”), which incorporates a biological agent to act as a “recognition agent” for the “analyte” that will be detected or measured. All biosensors consist of a detector (the component that interacts with the analyte) and a transducer (which transforms the detected interaction into a measurable analytical signal). There are different kinds of biosensors depending on the type of recognition agent in the detector, which can vary from an entire bacterial cell to a very specific component (such as an enzyme, an antibody, or an aptamer) (Li et al., 2024). Aptamers are single-stranded oligonucleotides with sizes between 70 and 100 nucleotides that form specific three-dimensional structures and are capable of recognizing various types of target molecules with high affinity (similar to antibodies). Aptamers are expected to provide superior affinity to antibodies since they can be selected and enhanced with in vitro techniques using a screening method known as SELEX (Uğurlu et al., 2024). Therefore, these molecules are being investigated as a real alternative to monoclonal antibodies in biomedical research. However, for some applications of certain biosensors, an entire cell is employed rather than a specific component, for example, an antibody. The use of bacteria as recognition agents in the detectors of biosensors offers a low-cost, compact, and robust option for the continuous detection of analytes in the context of their application. In this sense, optimal designs for the integration of biosensors into final medical contexts, such as ingestible capsules, will be crucial to ensure their efficacy and safe use in clinical therapy (Chiang & Hasty, 2023; Hoang Trung Chau et al., 2020).

8 BACTERIOPHAGE-BASED THERAPIES TO COMBAT FORMS OF RESISTANCE IN BACTERIA

Bacterial spores, including exospores and endospores, represent the most prominent forms of bacterial resistance, particularly endospores. These spores pose a risk when generated in hospitals, where they can survive most of the disinfectant compounds typically used (Dyer et al., 2019). Endospores produced by Bacillus and Clostridium species are extremely resistant to intense heat and irradiation. In addition to endospores, bacteria have evolved other natural mechanisms that protect them from antibiotics, disinfectants, and natural host defenses. These include the production of capsules, biofilms, and the so-called L-forms. Capsules are nonmetabolically active structures composed of repetitive units of polysaccharides or polypeptides. They provide important properties to bacterial cells, including adhesion to sister cells (forming colonies) and adhesion to inert or living substrates (forming biofilms) (Hall-Stoodley et al., 2004). Growth in biofilms represents the typical way bacteria grow in nature. However, when formed on inert surfaces (such as catheters and surgical prostheses) or living substrates (such as tissues of higher organisms), these structures protect bacteria from host immune responses and antibiotic treatment. Therefore, biofilm-associated infections can have a serious impact on health, making the diagnosis and treatment of biofilms challenging (Pai et al., 2023). One of the most promising therapeutic alternatives to antibiotics for treating these forms of bacterial resistance is bacteriophage-based therapy (Usman et al., 2023). Bacteriophages (Figure 4) are viruses that exclusively infect bacteria and are among the most numerous, diverse, and dynamic biological entities on this planet (Fogg et al., 2017). Bacteriophages possess a remarkable ability to infect and destroy bacteria with precision, and they can be specifically customized to attack particular strains or species, including problematic encapsulated bacteria; in fact, capsules serve as receptors for many bacteriophages (Carascal et al., 2022). Several studies have shown that bacteriophage-based therapy could be an effective treatment for biofilm infections affecting the skin, the gastrointestinal tract, and the urinary tract, among other tissues (Saeed et al., 2023). Bacteriophage-based therapy could also be highly beneficial in combating chronic diseases due to L-form bacterial infections, a term used to denote cell wall-deficient bacteria (Errington et al., 2016). The peptidoglycan cell wall is highly conserved across the domain of bacteria, suggesting its early appearance in bacterial evolution (Errington, 2013). While normally essential, under certain stress conditions, wall-deficient (or “L-form”) bacteria can be isolated. The cell wall is the target for most commonly used antibiotics, and therefore, L-forms are completely resistant to antibiotics that specifically act on cell wall synthesis, such as penicillins and cephalosporins (Errington, 2017). In many cases, the host's immune system recognizes epitopes associated with the cell wall, so the absence of this layer allows bacteria to evade their host's defenses, linking L-forms to the development of chronic diseases. Furthermore, wall-deficient cells are flexible, able to squeeze through narrow spaces, and capture exogenous DNA, which increases the risk of infections with these bacterial forms (Claessen & Errington, 2019). Therefore, new therapeutic strategies, such as bacteriophage-based therapy, are still needed to address the real consequences that L-forms have in both the containment of bacteria in certain tissues and their resistance to antibiotics and host immune defenses. However, there are still several obstacles to overcome in the production and safety of bacteriophages for the clinical use of this type of therapy (Saeed et al., 2023).

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Figure 4

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Bacteriophage-based therapies to combat forms of resistance in bacteria. Bacteriophages, viruses that infect bacterial cells, are emerging as promising tools to address challenging infections. These include those stemming from “superbugs” (multidrug-resistant bacteria), “biofilms” (communities of bacteria shielded by polysaccharides), and “L-forms” (cell wall-deficient bacteria).

9 BIOPLASTICS OF MICROBIAL ORIGIN FOR DEVELOPING MEDICAL DEVICES

Plastics have revolutionized human lives, finding a wide range of applications from packaging to medical devices (García-Depraect et al., 2021). The introduction of bioplastics to the market has opened a new perspective in the field of medicine. Due to their bioresorbability, these bioplastics can pass through biological barriers and concentrate in specific tissues (Silva et al., 2023). Among all existing bioplastics, only polyhydroxyalkanoates (PHAs) are fully synthesized by prokaryotic microorganisms (Bordel et al., 2021). Owing to their excellent biodegradability and biocompatibility properties, PHAs have emerged as one of the most promising biomaterials for the development of medical devices, including applications in tissue engineering, drug delivery, and orthopedics. Among the different PHAs, poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) has garnered significant interest in the field of biomedicine. The encapsulation of various drugs (i.e., anticancer, antibiotic, and anti-inflammatory compounds) in PHBV microspheres is currently being extensively investigated. Overall, PHBV is a highly intriguing biomaterial for drug delivery, as it exhibits prolonged release kinetics with high drug stability, significantly reduced side effects, and greater bioavailability compared to other materials (Rodríguez-Cendal et al., 2023). Additionally, PHBV has been tested as a support material for tissue engineering applications, such as bone, cartilage, and skin regeneration. The incorporation of PHBV into scaffolds appears to improve mechanical properties, biocompatibility, and cellular interactions, making them suitable for tissue engineering constructs (Wei & Tan, 2023). However, PHAs are not yet among the biomaterials approved by the FDA for use in clinics. Indeed, the two main bioplastics used as devices for drug delivery systems approved by the FDA are polylactic acid (PLA) and poly-lactic-co-glycolic acid (PLGA). In particular, the use of PLGA nanoparticles as drug-delivery systems to treat brain diseases has been widely investigated in recent years (Lamparelli et al., 2023). For example, two different preclinical studies have obtained promising results with PLGA nanoparticles loaded with either doxorubicin or chlorotoxin-conjugated morusin for the chemotherapy of glioblastoma (Agarwal et al., 2019; Maksimenko et al., 2019). Nonetheless, PHAs are driving the growth of the biodegradable bioplastics market, and the production capacity is expected to triple in the next 5 years. Therefore, it is anticipated that similar to PLA and PLGA, natural PHA biopolymers will soon find various applications in the biomedical field, offering innovative approaches to numerous treatments and contributing to the advancement of modern medicine (Bhatia et al., 2021; Silva et al., 2023).

10 CONCLUSION

Advances in genetic engineering and microbial genetics are crucial for the development of various biotechnology processes, which are currently categorized as red (medical processes), white (industrial processes), yellow (food production processes), and green (agricultural processes) biotechnologies. Consequently, it is possible to address the challenges of today's society through research and innovation focused on the simplest forms of life, namely, microorganisms. Microbial biotechnology is the discipline most frequently employed by biotechnologists, microbiologists, biochemists, environmentalists, and genetic engineers. However, a significant amount of research is still needed to spark a true biotechnological revolution in most of society's productive sectors, including the pharmaceutical and medical fields. The findings presented in this review serve as an example of what scientists and clinicians can contribute to medicine. By working together, we can navigate the complex landscape of human disease, enhance global health, and make diseases curable.