Allergy cross-hypersensitivity reactions occur when the body’s immune system mistakenly identifies harmless substances (allergens) as similar to one another, triggering an immune response [35]. Figure 1 illustrates Immunoglobulin E (Inge)- mediated molecular allergic reactions that occur after accidental exposure to certain foods. Macrophages and dendritic cells are particularly important antigen-presenting immune cells that phagocytose foreign food antigens and present antigens to T lymphocytes via Major histocompatibility complex class II (MHC II) molecules [36,37]. T cells that recognize and respond to food antigens differentiate into T helper (Th2) cells and secrete interleukins (IL), which contribute to allergies. These cytokines, which are released by Th2 T cells in response to an antigen, incite B cells to produce Inge. Allergen-bound Igga interacts with its receptors on mast cells, ultimately releasing large amounts of mediators such as histamine. Mast cell histamine causes allergic symptoms such as edema, swelling, itching around the mouth, diarrhea, breathing difficulty, coronary artery spasm, and anaphylaxis, sometimes leading to death.

Food allergies are powered by a complex interplay between environmental factors and genetic predisposition, with heritability estimates ranging from 15% to over 80%. Previous studies [38,39] confirmed that family history is a crucial risk factor for food allergy. For example, a child whose parent or sibling has a peanut allergy has a higher likelihood of being peanut allergic than a child of parents without a history of this type of allergy. Furthermore, a twin study of peanut allergy found that concordance was substantially higher among identical twins than among dizygotic twins (64.3% versus 6.8%, respectively) [12]. No single specific “food allergy” gene is known; likely, an interplay between genes and the environment is involved. Meaning that allergy is not inherited as a single, Mendelian trait, but often linked to genes that regulate immune functions (e.g., HLA, IL4) and epithelial barrier integrity (e.g., FLG, SERPINB7). To date, only a few allergy-initiating genes have been recognized and edited (Table 1).

Total egg white protein accounts for approximately 11% of the egg’s weight. The ovomucoid (Gal d1) and ovalbumen (Gal d2), conalbumin (Gal d3), and lysozyme (Gal d4) account for most of the allergenic proteins in the egg white [99]. While Gal d2 is the most abundant allergenic protein, Gal d1 is the dominant egg white allergen and is responsible for most allergic reactions. Unlike other egg allergens, Gal d1 retains its allergenicity even after extensive heating. Nevertheless, protein allergenicity varies with factors such as the protein’s biochemical properties, the matrix, application conditions, processing techniques, and individual patient sensitivity. It has been found that the IgE/IgG binding ability of Gal d1 was reduced after the Maillard reaction with maltose (a non-enzymatic browning that occurs when amino acids and reducing sugars in food are heated), typically at 140-165°C [101]. Therefore, it is essential to select appropriate food processing procedures to reduce the allergenicity of Gal d1.

In the cultured chicken primordial germ cells, Gal d1 and Gal d2 were knocked out using the CRISPR/Cas9 system [86]. The achieved sgRNA editing efficiency exceeded 90%. In this study, primordial germ cells (PGCs) lacking Gal d1 were transferred into chicken embryos. Homozygous Gal d1 KOs were observed among the offspring of the second generation. However, the allergenicity of eggs yielded by ovomucoid-deleted chickens was not examined. The results provide proof of principle for applying CRISPR/Cas9 to eliminate the main egg allergen proteins and, eventually, to create hypoallergenic eggs.

In a more recent study [64], the achievements and genomic safety of CRISPR/Cas9 and CRISPRi were systematically evaluated in PGCs from fertilized Jinhua chicken (Gallus gallus). While CRISPR/Cas9 accomplished high editing efficacy, it also caused significant DNA damage, apoptosis, and sex-specific cell-cycle arrest, demonstrating the marked genotoxic sensitivity of PGCs. In comparison, CRISPRi was satisfactorily tolerated but failed to execute efficient gene repression in chicken cells. Comparative tests indicated that CRISPRi was efficient in human 293T cells but not in chicken PGCs or somatic DF-1 cells, indicating species-dependent limitations of mammalian-optimized repression systems.

Milk mainly contains two casein proteins (α-s and κ-casein), and two whey proteins (α-lactalbumin and β-lactoglobulin) [88]. Beta-lactoglobulin (BLG), the principal protein in milk whey, is a significant allergen because it is absent from human milk [102]. The traditional milk treatment methods: fermentation, pasteurization, or ultra-high temperature, not only proved costly, but also couldn’t decrease or withdraw BLG from milk [103]. In addition, these procedures affect nutritional quality and may sustain allergenicity by generating new epitopes [104]. Alternatively, recently developed GE techniques provide a precise and targeted approach to modify gene(s) responsible for milk allergenicity without compromising milk qualities.

Knocking out the BLG gene was attained in goat fibroblasts by co-injecting three BLG-specific CRISPR sgRNAs with Cas9 mRNA into goat embryos [105]. This protocol not only significantly reduced BLG expression in the mammary glands of the engineered goats but also lowered BLG levels in their milk, laying the groundwork for improving the composition of goat milk. Among the progeny of BLG-KO goats, genome-targeting efficacies ranged from 12.5% to 28.6% after injection of 1 or 2 sgRNAs, respectively.

As for cow milk, the BLG gene was KO using CRISPR/Cas9 to produce hypoallergenic cow milk [90]. These hypoallergenic milk studies underscore the importance and therapeutic potential of applying the GE strategy to livestock for human health.

CRISPR/Cas9 technology was used to edit the primary allergen gene, BLG, in buffalo milk [88]. These researchers cloned lines of BLG-edited fibroblast cells. Three sgRNAs were constructed and evaluated for electroporation-mediated CRISPR editing of the BLG gene. Then, the somatic cell nuclear transfer technique was used to generate blastocyst-stage embryos that were developmentally similar to wild-type embryos. They reported an overall success rate of approximately 50% in CRISPR-based editing of the BLG gene in buffalo. This is the first report that established the generation of BLG-modified embryos in buffalo. Thus, the described CRISPR strategy opens the door to producing BLG-free milk in buffaloes.

Wheat (Triticum aestivum) allergies are principally triggered by gluten proteins. These proteins are mainly responsible for the development of gluten sensitivity and celiac disease [106]. The α-gliadin genes harbor several conserved activating peptides, including an immunodominant 33-mer peptide. Because of the large number of gluten genes and the complexity of the wheat genome, traditional breeding alone cannot produce coeliac-safe wheat that retains its baking quality. Researchers [92,107] successfully targeted and knocked out the α-gliadin-encoding genes using CRISPR/Cas9 with gliadin-specific gRNAs in polyploid wheat cultivars (bread and durum [pasta]). In these studies, the content of gluten proteins in the grains of the edited wheat lines was significantly reduced by up to 85%, thereby lowering the allergenicity of the wheat by reducing the presence of immunogenic gluten epitopes [107]. No OTEs were detected at predicted likely off-target sites. This opens the door to developing wheat varieties that are safer for individuals with celiac disease or wheat allergies.

Another example of successful application of CRISPR in durum wheat cultivar Svevo to simultaneously knock down two of the α-amylase/trypsin inhibitors (ATI) subunits, leading to decreased expression of both subunits [9]. These subunits, particularly the 0.28, CM3, and CM16 subunits, are implicated in Celiac disease [108] and baker’s asthma [9] because they elicit a strong IgE response and contribute to the progression of wheat allergies [109]. The sgRNA targets were tailored to the coding region of the CM16 and CM3 genes. These sgRNAs were constructed and cloned into several vectors. Then, the plasmid vectors were co-bombed with the durum wheat cultivar. The regenerating plantlets were transferred to a regeneration medium and allowed to mature. Evaluation by sequencing and biochemical analyses showed that 14 of 97 regenerated plants carried CRISPR edits [108]. No off-targets were identified by in silico analysis, and the ELISA revealed no reactivity to ATI CM3, demonstrating that the mutations caused a gene KO.

Taken together, the outcomes of these studies confirm the importance of high-efficiency CRISPR editing for advancing the development of new hypoallergenic wheat varieties with reduced immunogenicity. Although the polyploid nature of wheat poses an additional challenge to targeting multiple gene copies or alleles simultaneously to achieve a functional KO, the enhanced efficacy and versatility of CRISPR technology will no doubt advance targeted modification of polyploid genomes compared with conventional breeding approaches.

In Peanut (Arachis hypogea) seeds, 16 allergenic glycoproteins can induce IgE antibody production in sensitized individuals. Of these allergens, Ara h1, Ara h2, and Ara h3 are the best known for triggering allergic reactions in individuals with peanut allergy [110]. Despite the intense hypersensitivity response, there was no specific cure for peanut allergy until CRISPR was developed. This occurred when researchers [111] suggested using CRISPR to deactivate specific genes encoding the major peanut allergens. Alternatively, knocking down gene expression with RNAi to efficiently delete Ara h2 and other key peanut allergens (Ara h1, Ara h3, Ara h6) using CRISPR. Later, multiplex CRISPR/Cas9 genome modification was applied to KO the Ara h2 gene in peanut protoplasts [112]. These studies provided evidence that these strategies could cultivate peanut varieties that maintain their nutritional value while reducing or eliminating allergenic risk [34].

On the other hand, the Cytochrome P450 family 11 subfamily A member 1 (CYP11A1) gene was directly modified using the CRISPR/Cas9 tool in the human CD4⁺ T-cell line SUP-T1 [113]. Targeting CYP11A1 decreased the expression of the CYP11A1 gene by >50%, and IL-13 generation was considerably reduced. These data indicate that the CYP11A1-CD4⁺ T cell-IL-13 axis might be linked to the development of peanut allergy in children. Consequently, the GE of CYP11A1 may represent a new therapeutic target in children with peanut allergy.

Using tools such as CRISPR-dCas9 systems, researchers epigenetically modified DNA by altering histone marks at specific loci to regulate allergen expression in peanut [114], thereby reducing peanut allergenicity. Epigenetic modification of the expression or silencing of the Ara h6 and Ara h8 genes, without altering the peanut genome, may produce allergen-low peanuts that are safer for patients with specific sensitivities. Although research on the epigenetic control of Ara h6 and Ara h8 is in its infancy, initial results are promising. One of the main challenges in generating allergen-free peanuts through epigenetic approaches is achieving stable, consistent modifications [115].

Soybean (Glycine max) has high plant-based nutritional value because it is rich in protein, which is increasingly used in food processing. The glycoproteins: Gly m Bd 28 k, Gly m5, Gly m6, and Gly m8 are the main allergenic seed storage proteins [116]. Research has focused on targeting the Gly m5 and Gly m6 genes, which encode known soy allergens. The genes that encode these were simultaneously knocked out in two soybean varieties using two sgRNAs [116]. The soybean seeds from both the second and third generations showed indels at both target loci, as well as several deletions that induced frame-shift mutations. Subsequently, these changes decreased protein expression and storage in the seeds [117]. The outcomes of this study confirmed that GE using CRISPR/Cas9, coupled with Agrobacterium tumefaciens-mediated transformation, provides a direct solution for producing hypoallergenic soybean products. Thus, CRISPR-based down-regulation of such allergen genes may pave the way for hypoallergenic soy varieties [61]. Today, genome editing is not restricted to a few model cultivars. Researchers [118] targeted the two subunit genes of β-conglycinin (Glyma. 20G146200, Glyma. 20G148200 ) using the CRISPR/Cas9 technique. The generated soybean lines have stable, inherited seed protein phenotypes characterized by reduced recognition of β-conglycinin-specific IgE. These results underscore the potential of targeted genome editing to enable accurate genome modification in crop development, leading to safer soy-based foods. Another group of scientists [119] targeted GsDELLA and successfully generated homozygous mutants in wild soybean (Glycine soja), with reduced plant height and branch number. An adaptable transformation platform may accelerate genetic improvement in both wild and cultivated soybeans.

Gene editing of the main brown mustard (Brassica juncea) allergen has emerged as a key agricultural technique, with recent developments emphasizing improvements in seed quality (particularly taste) and modifications to pollen development to enable effective hybrid generation. Whole-genome sequencing technology has enabled the identification of crucial structural variants, contributing to allelic diversity in breeding populations. CRISPR’s potential to reduce allergenicity in brown mustard involves modifying genes encoding the main allergenic proteins, such as Bra j1. Research shows that CRISPR could decrease allergenic potential without affecting agronomic traits [98]. Significant biotechnological advancements, including the application of CRISPR/Cas9, have enabled the production of high-quality mustard hybrids [120,121,122]. These researchers demonstrated the feasibility and potential of CRISPR/Cas9 for genetic transformation in Brassica spp.

The Bra j protein belongs to a family of seed storage proteins known as 2S albumins. In addition, Sin a1 and Sin a2 (11S globulins) have been found and identified in the seeds of two Canadian mustard varieties (the white and the brown mustard; Sinapis alba and B. juncea, respectively) [25]. These proteins have been found to bind IgE, initiating the allergic cascade responses in patients’ sera [123]. cDNAs encoding Sin a3 and Sin a4 have been amplified by polymerase chain reaction, cloned, and sequenced [26]. Knocking out the Bra j1 gene using CRISPR/Cas9 resulted in hypoallergenic or nonallergenic proteins in mustard-derived products [98]. The generation of allergen-free mustard plants is now possible, potentially advancing safety for people with mustard allergy.

Description and functional analysis of genes preferentially expressed in late pollen may help clarify the processes vital to pollen germination and tube growth. However, examining the biological functions of prospective candidate genes using mutant studies may be both expensive and time-consuming [60]. Developing a strategy for direct genome editing in pollen would overcome the problem of targeted mutagenesis in plants, which is useful for determining gene functions and rapidly generating new crop varieties.

In plants, the vacuolar protein sorting (VPS) genes control vacuolar transport and vesicle fusion, which are required for pollen wall development and nutrient transport during seed germination. CRISPR-based GE of pollen is an advanced technology for introducing intended genetic changes, such as modifying traits or preventing the dispersal of transgenic pollen [69,124]. It enables accurate, transgene-free adjustments in plants by modifying reproductive cells, using haploid cells to modify plant genomes without continually transferring undesired transgenic material. Genome editing has several valuable uses for which the Cas transgene needs to be retained in the plants, including RNA-guided Cas9 as an in vivo desired-target mutator [125] and haploid induction-coupled editing [126] through the paternal haploid, with a null mutant as the female gametophyte. Partial sterility indicates that, although mutant pollen grains are less competitive than wild-type pollen, they preserve their fertilization capacity [60].

In allergy therapy, CRISPR/Cas9-engineered dendritic cells were used to treat allergic rhinitis [127]. Targeted KO of the VPS-associated protein 37A (VPS37A) and VPS37B genes in human blood dendritic cells lessened Th2 cytokine assembly after being co-cultured with allergic rhinitis patients-derived CD4⁺ T cells. This innovative modality, which uses genetically engineered dendritic cells, can provide an efficient therapeutic and preventive approach for allergic diseases.

Despite their widespread consumption, tomatoes (Lycopersicon esculentum) are a known source of food allergens, particularly for people sensitized to birch pollen due to cross-reactivity [100]. Therefore, decreasing the allergenic potential of these crops is a primary objective for food safety and customer health. At a minimum, seven allergenic proteins have been identified in tomato, including Bet v1-homologous PR-10 proteins, β-fructofuranosidase, cyclophilin, nonspecific lipid transfer proteins 1 and 2, and profilin [128]. Among these, profilin is a key allergen encoded by two closely related genes, Lyc e1.01 (Solyc08g066110.3.1) and Lyc e1.02 (Solyc11g070130.2.1). Earlier investigations using RNAi to silence Lyc e1 and Lyc e3 reported a partial decrease in profilin expression and lessened allergic responses [100], indicating the potential for genetic interference targeting these loci.

Despite the identification of profilin as a major allergen in tomato fruits [100], no efficient methods have been established to abolish its expression through precise genome editing. generated transgenic plants via Agrobacterium-mediated transformation, in which homozygous Cas9-free lines without profilin proteins were generated in post-generations. A framework using the CRISPR/Cas9 system to KO Lyc e1.01 and Lyc e1.02, and eventually to evolve hypoallergenic tomato cultivars, has been successfully applied to Solanaceae crop improvement [100]. More recently, innovative procedures, such as [ultra-efficient prime editing (UtPE)], are being utilized to simultaneously edit multiple targets for better nutrition and allergen reduction [129]. The mutant lines retained their reproductive ability, and although plant height was reduced, fruit growth was unaffected. Future research should focus on comprehensive allergenicity testing and on assessing the field performance and safety of the edited lines.

Previous sections highlighted the value of the CRISPR approach for engineering hypoallergenic food, developing allergen-free plant models, or determining the biological functions of allergen proteins. They also outlined existing clinical applications of CRISPR technology to edit genes associated with food allergies. The next sections will discuss its advantages and limitations.

In this review, a few studies involving animal models were included. This may be attributed to differences between small-sized animals (mice and rats), medium-sized animals (rabbits and cats), and large-sized animals (sheep, pigs, cattle, and buffaloes). More specifically, reproduction physiology (number of eggs produced and length of the gestation period) as well as the size and the complexity of the genome. Difficulties in editing the cat genome, such as the limited availability of cat embryos and the invisibility of the pronucleus in embryos, have been reported previously [140]. Microinjection into the pronucleus is preferred for CRISPR/Cas9 GE owing to its direct access to the target DNA, thereby promoting efficacy and reducing OTEs [141]. However, zygote microinjection frequently yields mosaic embryos and small GE rates. This poses difficulties in producing homozygous animals due to the prolonged gestation time of large animals. In the case of the cat embryonic cells, the presence of dark lipids in their cytoplasm makes it extremely hard to microinject the Cas9 and sgRNA construct directly into the pronucleus [141].

With respect to specifically targeting reduced allergenicity in plants, considerable progress has been made using CRISPR for broader crop development goals. However, few studies have been reported in the published literature on plants used to produce hypoallergenic mutants. A review of the literature on hands-on allergy management notes a significant, persistent problem: the difficulty of eliminating or modifying allergenic proteins without compromising the food’s nutritional value or consumer acceptance. Another major research obstacle lies in the complexity and limited genetic variability in plants, such as peanut, soybean, and other legumes, as well as the complex profiles of allergenic proteins [142,143]. The complex, heterogeneous, and polyploid nature of plants such as wheat poses an additional challenge: the need to simultaneously target multiple alleles or gene copies to achieve a functional KO [144]. The enhanced efficiency and versatility of CRISPR systems will certainly improve targeted genome editing in polyploid genomes compared to traditional breeding approaches [145]. As mentioned before (Page 12), plant PE technology, especially when codon-optimized for plant cells, is being developed as a cutting-edge tool for plant genome engineering, enabling genetic corrections across diverse crop species, broadening its application in plant breeding [83,146].

Delivering CRISPR elements (the Cas9 enzyme and guide RNA) directly to the specific immune cells responsible for IgE generation in humans is complicated. Current research emphasizes the use of nanoparticles (NPs) to deliver gene-editing tools. A significant challenge for CRISPR technology is the effective transfer of CRISPR components into target cells for GE. Currently, strategies that efficiently carry the CRISPR construct to diseased cells in vivo are lacking [147,148]. However, there are three primary methods to achieve this: viral, non-viral, and physical, each with advantages and disadvantages [149]. Despite the outstanding efficacy and specificity of viral vectors, such as adenovirus-associated viruses (AAVs), the limited cargo capacity of the AAV genome restricts their applications. Nonviral vectors with target-identification roles, such as LNPs, may be a focus of future research. Yet, transferring CRISPR reagents via NPs may be limited by potential toxicity concerns or insufficient targeting specificity [150,151,152]. In addition, LNPs exhibit limited organ selectivity, frequently accumulating in the liver, thereby limiting their wider clinical translation [153]. Recent advances, particularly those in cationic nanocarriers, are promising means to advance this GE platform. Pathological and physiological alterations following disease onset are anticipated to act as identifying factors for selected delivery or targets for GE [151]. More recently, the use of nanosized, naturally occurring extracellular vesicles for delivering therapeutic molecules to target cells has attracted particular attention owing to their unique characteristics [147]. The combination of CRISPR technology and exosome delivery offers a remarkable prospect to develop a highly effective and individualized therapeutic strategy. Still, key obstacles to translating exosome-mediated CRISPR therapeutic strategies from bench to bedside remain [152].

Mosaicism is a critical technical challenge in GE, in which the two alleles of the edited gene are present in some cells but absent in others. Mosaicism occurs following errors during DNA duplication or repair during GE, specifically when applying techniques such as CRISPR/Cas9. This could result from different sources. First, the GE elements (Cas9 and guide RNA) may not be present or active in all cells; second, GE efficacy may vary among cells; and third, the timing of GE relative to cell division can affect the level of mosaicism [150,154,155]. Mosaicism can decrease the efficiency and safety of GE by producing a blend of edited and unedited cells. This can be particularly problematic for treatments aimed at correcting genetic diseases, where the presence of unedited cells may lead to disease resolution or relapse. Mosaicism can result in OTEs and, consequently, generate Cas-generated mutations and pleiotropic effects [155]. Furthermore, mosaicism may lead to inconsistent expression of edited genes, potentially affecting treatment outcomes and risk assessments.

Uncovering and understanding mosaicism is critical because it is a potential source of OTEs, can affect the outcomes of GE treatments, and can hinder the development of strategies to reduce it. In addition to optimizing the editing protocols, such as selecting ideal cell kinds that are less liable to mosaicism, timing of GE, and delivery system, two strategies can be followed to reduce the occurrence and impact of mosaicism during more precise GE technologies, such as BE and PE (two forms of developed GE technologies) [154]. The BE strategy enables the direct, irreversible replacement of one DNA base with another without requiring DSBs. Prime editing extends CRISPR/Cas9 to enable more accurate editing by combining Cas9’s accuracy with the efficacy of reverse transcriptase. Conventional microinjection techniques have struggled with variable delivery efficiency, often leading to mosaicism or incomplete GE, which confounds downstream analyses [155]. By systematically timing injections, this research team achieved significantly improved biallelic editing efficiency with both Cas9 and Cas12a nucleases.

In the future, the overrepresentation of allergy-associated gene mosaicism in late-onset forms should be investigated in patients with adult-onset allergy symptoms.

Although CRISPR shows great promise, there are still challenges to consider. A primary concern associated with the application of GE is verifying the desired on-target mutations and avoiding unintended OTEs, including unintended gene editing that introduces random mutations into the genome and creates new risks [88,156]. The occurrence of OTEs is due to abundant homology between the exact sgRNA sequences and untargeted genomic sequences, leading to cuts that may result in adverse outcomes [157]. Ethical concerns also persist regarding the broader use of gene editing in food production, and public acceptance remains a hurdle in some regions.

This concern has driven ongoing attempts over the years to polish the CRISPR technique, particularly to make genetic modifications reversible. The first message to convey is that advances in CRISPR techniques have significantly reduced off-target editing, making it a safer option than earlier GE methods [158,159], making it a safer option than earlier gene-editing methods. Furthermore, the wild-type RNA-guided CRISPR/Cas9 nuclease can be repurposed to achieve improved on-target specificity and reduced off-target potential [158,160]. For example, Cas12 or Cas13 CRISPR systems may be used to generate staggered DNA DSBs or to directly target RNA, respectively [84]. Alternatively, adopting systems (e.g., BEs and PEs) that work independently of Cas9-mediated DSBs, which are a significant source of OTEs in CRISPR/Cas9 genome editing [83,84,158].

Detecting OTEs is difficult because their positions and numbers are unknown [161]. The potential off-target mutation rate must be assessed using Sanger sequencing during the preclinical development of CRISPR-based therapies [158]. Genome-wide analysis can also detect off-target mutations [155,162], underscoring the importance of a comprehensive sequencing-based approach to off-target assessment. The planned design bioinformatics programs predict off-targets by comparing CRISPR sgRNA sequences against the entire genome of interest. When substantial OTEs are detected, the specificity of GE can be improved by using accurate delivery of more precise sgRNAs and the Cas involved [163]. The delivery vector of Cas9, whether DNA, mRNA, or ribonucleoprotein, plays a key role in determining its exposure duration and expression, thereby impacting both editing efficacy and off-target activity [148]. It should be recalled that detecting OTEs is important for identifying their safety risks. A recent study [164] demonstrated that hybrid gRNAs can substantially reduce OTEs, including bystander edits, while preserving high on-target editing efficiency. A CHANGE-seq-BE-specific method for identifying off-target activity is being developed [165].

The recognition of specific CRISPR/Cas9 components, such as Cas9, sgRNA, and viral or non-viral delivery vectors, may trigger both innate and adaptive responses, limiting its efficacy [166,167]. The resulting complex interactions between these components and host immune reactivity play an important role in determining the safety and efficacy of CRISPR-based therapies [167]. Evidence from preclinical and clinical in vivo CRISPR trials indicates that specific immunity can fail the desired GE therapy. The challenge of immunogenicity remains a significant roadblock to the clinical utility and applications of CRISPR therapeutics [166]. Therefore, there is a need for advanced immunogenicity prediction algorithms that address current limitations to advance clinically translatable CRISPR-based therapies for universal use. Machine learning-based prediction implementations are expected to advance the development of less immunogenic CRISPR therapeutics [168].

To mitigate severe Cas9-immunogenicity-related immune reactions, researchers are developing strategies, including immunosuppressive therapies. (a) ex vivo GE strategies, where the immunotoxicity of CRISPR therapeutics might not be a significant concern [169]; (b) selecting a target tissue that has limited innate immune response [170]; (c) Optimizing CRISPR delivery systems [166,167]; (d) engineering innovative CRISPR nucleases that can minimize the OTEs and escape the immune system [167]; (e) controlling the CRISPR time of activity [170]; (f) epitope engineering [167]; and (g) monitoring the immune reaction to CRISPR therapeutics [166]. Addressing these immunological obstacles requires an integrated method that combines visions from immunology with pioneering engineering solutions. As the field advances, overcoming Cas9 immunogenicity will be a major hurdle to realizing the full therapeutic potential of the CRISPR/Cas9 system across diverse clinical applications.