Gene Editing in Allergic Diseases: Identification of Novel Pathways and Impact of Deleting Allergen Genes

Abstract

Gene editing technology has emerged as a powerful tool in all aspects of health research and continues to advance our understanding of critical and essential elements in disease pathophysiology. The clustered regularly interspaced short palindromic repeats (CRISPR) gene editing technology has been used with precision to generate gene knockouts, alter genes, and to identify genes that cause disease. The full spectrum of allergic/atopic diseases, in part because of shared pathophysiology, is ripe for studies with this technology. In this way, novel culprit genes are being identified and allow for manipulation of triggering allergens to reduce allergenicity and disease. Notwithstanding current limitations on precision and potential off-target effects, newer approaches are rapidly being introduced to more fully understand specific gene functions as well as the consequences of genetic manipulation. In this review, we examine the impact of editing technologies of novel genes relevant to peanut allergy and asthma as well as how gene modification of common allergens may lead to the deletion of allergenic proteins.

INTRODUCTION

The pathophysiology of the allergic diseases is complex. Understanding of causal molecular pathways has advanced considerably by introducing specific insertions, deletions, base pair changes or other modifications to gene sequences of presumed culprit target genes in cell or animal models (1). These data have been the impetus for a number of the recently introduced targeted therapies (2, 3). One of the technologies most widely used today to facilitate genome editing is clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR associated protein9 (Cas9) due to benefits such as simple design, specificity, reproducibility, and cost efficiency (1). Before the discovery of CRISPR-Cas9, other gene editing tools including zinc finger nuclease (ZFN) and transcription activator-like effectors nucleases (TALEN) were used (4). The customized nuclease based approach CRISPR-Cas9 includes two key components, a single guide RNA (sgRNA) and the Cas9 enzyme (5). The Cas9 is an RNA-guided endonuclease used for site-specific genome editing in a variety of organisms (6, 7). A sgRNA is designed with a length of approximately 100 nucleotides comprising a 17–20 nucleotide long guide sequence (protospacer) that is complementary to the target DNA sequence while the remaining sequences bind to Cas9 placing the enzyme at the correct position on the DNA (8). The binding of the sgRNA with its target DNA activates the Cas9 leading to a double strand break (DSB) in the genome (Figure 1A), which can subsequently be repaired by two highly conserved DNA mechanisms: non-homologous end joining (NHEJ) or homology-directed repair (HDR) (9). If not properly repaired, a DSB is lethal to cells. The break is introduced upstream of the protospacer-adjacent motif (PAM) (10), a short DNA sequence (2–6 nucleotides) needed for Cas9 to cut. The NHEJ repair mechanism is more often used to achieve a gene knockout by creating frame shifts via random insertions or deletions in contrast to directional modifications of a defined sequence (11). The HDR repair mechanism is commonly applied when sequence-specific alterations at a target region of interest are introduced. In this process, the donor DNA comprises two flanking homology arms on either side of the target site, with the sequence between these arms designed based on the experimental goals (9). The HDR system is activated and an efficient repair occurs through recombination at the DSB site between the homologous DNA sequence on the donor template and the genomic region of interest (12, 13). Consequently, the target gene can be edited at the break site (1). Design and development of CRISPR-Cas9 for sequence-specific modification and gene knockout have revolutionized drug discovery, disease modeling, and gene therapy as witnessed recently in the application to treatment of sickle cell anemia, B-thalassemia, and other diseases (2, 3). This was achieved by base editing which is a technology that introduces point mutations in the DNA without generating DSBs (14, 15). Two major classes of base editors have been developed (Figure 1B): cytidine base editors allowing C to T conversions and adenine base editors allowing A to G conversions (14). Prime editing is a ‘search-and-replace’ genome-editing technology that introduces the desired base-to-base conversions and small insertions and deletions (15).

Figure 1.

A. Traditional CRISPR-Cas9 system. Single guide RNA (sgRNA) and Cas9 form a complex directing Cas9 to a specific target site which creates a double-stranded break (DSB) upstream of the protospacer-adjacent motif (PAM). The DSB is repaired through non-homologous end joining (NHEJ) or homology-directed repair (HDR). NHEJ can introduce random insertions or deletions at a DSB site resulting in gene mutation(s) or gene knockout. HDR can introduce precise genomic insertions or modifications at a DSB site by adding a donor homologous DNA template. B. CRISPR-Cas9 base editing system. This systems can directly produce a point mutation without creating a DSB and without a donor template. The cytosine base editor (CBE) is composed of cytidine deaminase fused with nickase Cas9 (nCas-9) converting C to U. Following DNA repair or replication, the U:G heteroduplex is converted to a T:A base pair. The uracil glycosylase inhibitor (UGI) fused to CBE prevents U from reverting to C, thus favoring C to T conversion. Adenine base editor (ABE) consists of adenosine deaminase fused with nCas9 or dead Cas9 (dCas9), ABE deaminates A to form inosine (I). Following DNA repair or replication, the I:T heteroduplex is converted to G:C base pair. Created with BioRender.com.

Although important advances have been made over the last few years, a decade of CRISPR-Cas9 is only the beginning with challenges and limitations (16). Major concerns associated with the use of genome editing include confirmation of the desired on-target mutation(s) and the avoidance of undesired off-target effects through introduction of random mutations in the genome (17, 18). These occur when Cas9 acts on untargeted genomic sites and creates cleavages that may lead to adverse outcomes. The detection of off-target effects are challenging as the number and position is unknown (18). To some extent, the likelihood of an off-target mutation at a given site can be predicted (19). The CRISPR-Cas9 system is more prone to off-target effects because Cas9 works as a monomer recognizing a shorter target sequence and the sgRNA can tolerate a certain number of mismatches. Newer approaches are being rapidly developed and introduced to reduce off-target edits (20–22). To achieve successful in vivo administration of CRISPR-Cas9, precise delivery of the sgRNA and the enzyme is prerequisite. Different systems have been introduced including adenovirus-associated vectors (23, 24) or lipid nanoparticles (25). Although the delivery of the CRISPR-Cas9 system remains challenging, recent advances specifically of cationic nanocarriers and the development of non-viral vectors are promising tools to enhance this gene-editing platform (26). Despite these limitations and challenges, the ease of applying DNA targeting technologies to understanding the full array of allergic diseases is just now beginning and most of the work in allergy has been to determine the role of specific genes. In this review, we highlight the application of gene editing to the identification and novel role of a specific gene, cytochrome P450, family 11, subfamily A, polypeptide 1 (CYP11A1, Table 1), in the development of allergic disease as well as the applicability of gene-editing technologies to introduce deletions and modifications of genes in known allergens.

Table 1:

For each species (mouse, chicken, cow, goat, human), the international gene/protein nomenclature guidelines and requirements were followed.

Species	Genes (in alphabetical order)	Proteins (in alphabetical order)
Mouse	Gene, mRNA, and cDNA symbols from rodents are italicized; the first letter is written in upper case while all the rest in lower case: Cyp11a1, Il-13, Il-17a, Gata3, Rorγt, T-bet, Vps37a, Vps37b	For protein, the same gene symbol is used but not italicized and all in upper case: CYP11A1, GATA3, IFN-γ, IL-4, IL-13, VDR
Chicken, cow, goat, human	Gene, mRNA, and cDNA symbols are italicized and all letters are in upper case: BLG, CYP11A1, JAK3, MUC18, OVA, OVM, VDR, VPS37A, VPS37B	For protein, the same gene symbol is used but not italicized and all in upper case: ALA, BLG, CYP11A1, IL-4, IL-5, IL-13, JAK3, MUC18, OVA, OVM, STAT, TLR2, TLR3, TLR4

GENOME EDITING AND IDENTIFICATION OF CYP11A1 IN PEANUT ALLERGY

Peanut allergy is one of the most common food allergies which often persists throughout life (27). Similar to other allergic diseases, peanut allergy results from the activation of T helper 2 (Th2) cells and production of allergen-specific immunoglobulin E (IgE). Type 2 cytokines, interleukin 4 (IL-4) and IL-13, play critical roles in IgE-mediated intestinal peanut allergy in humans and mice (28–30). In a mouse model, we identified CYP11A1 for the first time as an essential component influencing the development of peanut-induced intestinal anaphylaxis (28). CYP11A1 is the first and rate-limiting enzyme in the steroidogenic pathway, converting cholesterol to pregnenolone and impacting steroid hormone production including glucocorticoid production (31) and is primarily expressed in the adrenal cortex, thymus, and intestine (32). Cyp11a1 gene expression was increased in activated T cells in the small intestine in mice sensitized to peanut and developed anaphylaxis on subsequent exposure to peanut (28). In addition, elevated Il-13 and Il-17a mRNA levels as well as their lineage-specific transcription factors (Gata3, Rorγt) were detected in the small intestine. In vivo treatment of peanut-sensitized and challenged mice with aminoglutethamide (AMG), an inhibitor blocking the enzymatic activity of CYP11A1, prevented peanut-induced allergic diarrhea. Inhibitor treatment decreased the production of cytokines from Th2 and Th17 cells, cell types implicated in the development of asthma and food allergy without impacting CYP11A1 mRNA or protein levels (33–35). Silencing of Cyp11a1 in polarized mouse Th2 CD4⁺ T cells (28) supported the notion (36–39) that CYP11A1 may act downstream of GATA3. Taken together, utilizing an inhibitor and gene silencing approach confirmed CYP11A1 as a central regulator of IL-13 production and as an essential molecule in the development of peanut-induced intestinal anaphylaxis (28).

In humans, peanut allergy is highly heritable (40, 41) and risk factors in innate and adaptive immune pathways have been identified (42, 43). In a study of activated peripheral blood CD4⁺ T cells from peanut-allergic (PA) children, CYP11A1 gene expression was augmented approximately 50-fold and the percentage of CD4⁺CYP11A1⁺ T cells was increased 20-fold compared to non-allergic controls (29). In parallel, IL-4 and IL-13 cytokine production were significantly increased. This was comparable to a previous study where Th2 cytokines (IL-4, IL-13) were the major cytokines produced in peanut-specific T-cell lines generated from PA individuals (44). Levels of CYP11A1 mRNA significantly correlated with elevated IL-13 cytokine production, increased specific IgE to the peanut allergen Ara h 2, and outcomes of double-blind, placebo-controlled oral food challenges to peanut in PA children (29). Recognizing potential limitations in using inhibitors and gene silencing to confirm the importance of CYP11A1 on Th2 cytokine production (28, 29), we directly modified CYP11A1 using CRISPR-Cas9 technology in the in the human CD4⁺ T cell line SUP-T1 (29). Targeting CYP11A1 in this way, reduced CYP11A1 gene expression by more than 50% and IL-13 production was decreased significantly (29). These data suggest that the CYP11A1-CD4⁺ T cell-IL-13 axis may be associated with development of PA in children (Figure 2). Thus, CYP11A1 may represent a novel target for therapeutic intervention in PA children.

Figure 2.

Schematic of the CYP11A1⁺/CD4⁺/IL-13⁺ axis in food allergy (peanut) pathogenesis. Allergen crosslinking of IgE-bound to FcεRI receptors on the surface of mast cells leads to their activation releasing histamine and other inflammatory mediators that cause an immediate allergic reaction including diarrhea, hypothermia, and small intestinal inflammation. In severe cases, anaphylaxis can occur. Dendritic cells (DC) capture allergens which are processed and presented to CD4⁺ T cells. Activated CD4⁺ T cells produce cytokines such as IL-13 which affects many types of cell types involved in the pathogenesis of food allergy. Activated CD4⁺ T cells support B cell development and produce antigen-specific IgE and IgG. FcεRI: Fc epsilon receptor 1, IL-13: interleukin-13, DC: dendritic cell, IgE: immunoglobulin E, IgG: immunoglobulin G. Created with BioRender.com.

TARGETING CYP11A1 ALTERS THE ASTHMA PHENOTYPE

Allergic asthma is characterized by chronic airway inflammation and reversible airway hyperresponsiveness. The roles of CD8⁺ T cells and the production of Th2-related cytokines in asthma have been defined (45). Moreover, numbers of CD8⁺IL-13⁺ T cells are increased in asthmatics and experimental asthma in mice (46–48). The conversion of CD8⁺ effector T cells from IFN-γ to pathogenic IL-13-producing effector cells contributes to an asthma phenotype (49–51), Upregulation of CYP11A1 was demonstrated following differentiation of CD8⁺ T lymphocytes to a Tc2 (IL-13-producing) phenotype (51). In mice, Cyp11a1 gene expression was 30-fold higher in Tc2 cells compared to Tc1 (IFNγ-producing) cells (52). CYP11A1 protein and pregnenolone levels were also increased in cells differentiated in the presence of IL-4 (38, 51). Unlike transfer of untreated CD8⁺ T cells, adoptive transfer of AMG-treated CD8⁺ T cells into sensitized and challenged CD8-deficient mice failed to restore airway hyperresponsiveness and airway inflammation (51). Inhibition of CYP11A1 enzymatic activity with AMG or silencing of Cyp11a1 using shRNA blocked the functional conversion of CD8⁺ T cells from IFNγ- to IL-13-producing T cells without affecting expression of the lineage-specific transcription factors T-bet or Gata3 (51). Association studies showed a link between genetic variants in CYP11A1, vitamin D receptor (VDR), and the susceptibility to childhood asthma (38). In mice, the Cyp11a1 promoter region contains several binding sites for the key transcription factor VDR in the vitamin D pathway. The active form of vitamin D3, calcitriol, prevented CD8⁺ Tc2 skewing and asthma development through changes in VDR binding to the Cyp11a1 promoter, thus regulating Cyp11a1 expression. As in peanut allergy, these data suggest that CYP11A1 serves as a key regulator in the development of asthma (38, 51).

The application of CRISPR-Cas9 tools in a number of chronic respiratory diseases such as cystic fibrosis, lung cancer, and acute respiratory distress syndrome have now been described (53). Shaikh et al. stressed the urgent need to extend CRISPR-Cas9 gene-editing tools additionally to other lung diseases such as chronic obstructive pulmonary disease (COPD) and idiopathic pulmonary fibrosis (IPF) as well as asthma (53) but have to the best of our knowledge not successfully been applied to date.

GENE EDITING MODIFIES DENDRITIC CELL FUNCTION IN ALLERGIC DISEASE

Dendritic cells (DCs) serve as important antigen-presenting cells and act as messengers linking the innate and adaptive immune system. DCs capture allergens which are then processed and presented to T lymphocytes, inducing T-cell differentiation and cytokine production (54). In a mouse model of peanut allergy, adoptive transfer of peanut-pulsed bone marrow derived DCs into non-sensitized mice prior to oral peanut challenge, triggered the full spectrum of allergic responses including diarrhea, inflammatory cell accumulation in the small intestine, and increased antigen-specific IgE, IgG1, and IgG2a (55). In addition to peanut-induced intestinal allergy (55, 56), DCs are critical in the effector phase of allergic disease through the release of numerous cytokines and chemokines recruiting effector Th2 cells to local immune sites, thus enhancing inflammation (57, 58). In milk allergic patients, DC antigen presenting cell function was augmented (59). DCs from these food allergic patients co-cultured with autologous CD4⁺ T cells, produced higher levels of Th2 cytokines (IL-5, IL-13). Recently, Kim et al. used CRISPR-Cas9-engineered DCs in the treatment of allergic rhinitis (AR). This approach led to an upregulation of vacuolar protein sorting-associated protein 37B (VPS37B) in DCs from these AR patients (60). VPS37B is part of a complex sorting ubiquitinated protein cargoes into multivesicular bodies (61). After gene editing to knockout the vacuolar protein sorting-associated protein 37A (VPS37A) and VPS37B genes in human peripheral blood DCs, co-culturing with CD4⁺ T cells resulted in markedly reduced Th2 cytokine production (60). The relevance of this approach was further substantiated in mice, as the administration of bone marrow-derived DCs depleted of Vps37a and Vps37b led to reduced airway responsiveness in a mouse model of AR. Together, these data suggest that CRISPR-Cas9 editing of this gene family in DCs may provide a potential new target for treatment and prevention of airway disease through the manipulation of DC and T lymphocyte immune functions. One may speculate that CRISPR-engineered DCs may also be used in other allergic diseases.

TARGETING OF JANUS KINASE 3 THROUGH GENE EDITING

Janus kinase 3 (JAK3) is a tyrosine kinase that functions in signal transduction and interacts with members of the signal transducer and activator of transcription (STAT) family leading to Th2 cytokine production. The role of JAK3 in regulating the pathogenesis of allergic asthma is well-described (62). JAK3 is highly expressed in immune cells and deficiency of JAK3 leads to reduced numbers of circulating natural killer (NK) cells, T cells, and normal numbers of B cells but with impaired B cell function (63). CRISPR-Cas9 mediated correction of the JAK3 gene deficiency in induced pluripotent stem cells from patients with severe combined immunodeficiency restored normal human T-cell development and normal numbers of NK and T cells. With this in mind, the CRISPR-Cas9 systems may be used to reprogram JAK3 in Th2 cells, providing a means to prevent development of allergic disorders. As a cautionary note, introducing mutations of genes such as JAK3 that have pleiotropic functions may also trigger development of other diseases (64) and will require precision and fine-tuning to achieve desired outcomes.

THE ROLE OF MODIFIED MUCINE 18 IN ALLERGIC DISEASE

Mucine 18 (MUC18), also known as CD146 or melanoma cell adhesion molecule, is a transmembrane glycoprotein and a member of the immunoglobulin superfamily (65, 66). It served originally as a marker of tumor progression in human melanoma. MUC18 was upregulated in airway epithelial cells from asthmatics and patients with COPD and was expressed in basal and ciliated airway epithelial cells (67). CRISPR-Cas9 knockout of the MUC18 gene in human primary nasal airway epithelial cells resulted in a marked reduction of IL-8 levels, a pro-inflammatory chemokine following Toll-like receptor (TLR2, TLR3, and TLR4) agonist stimulation (68). These data suggest that MUC18 has a pro-inflammatory function in airway epithelial cells which contributes to airway inflammation in response to bacterial and/or viral stimulation, and potentially in asthma.

GENE EDITING OF ALLERGENS

As implementation of immunotherapy for allergic diseases increases, much attention has focused on manipulation of causative allergens through gene editing (69). The overarching goal is to delete allergenic genes decreasing allergies or to minimize allergenicity.

Modifying Allergenicity of Cat Allergen

Cat allergy affects about10–30% of the population in Western countries with an increasing incidence rate (70, 71). In the 2000s, a much-hyped start-up claiming to have bred hypoallergenic cats collapsed leaving some allergic customers with pets that still made them wheeze. Fel d 1, the major cat allergen, causes IgE-mediated sensitization in more than 90% of cat allergic individuals (72). Clinical trials of “tolerogenic, non-allergenic” peptides were initiated but later discontinued due to the lack of success (73). CRISPR-Cas9-mediated deletion of Fel d1 has been reported. Brackett et al. targeted Fel d 1 in feline kidney epithelial cells in vitro through Fel d 1-specific sgRNAs (72). Each of these sgRNAs was generated to lead to frameshift mutations with varying editing efficiency. Comparable to other allergen gene-editing approaches (73), in vitro Fel d 1 deletion may provide a means to overcome the failed generation of hypoallergenic cats, or the generation of Fel d 1-allergen free cats benefiting cat-sensitive individuals.

Approaches to Alter the Allergenicity of Food Allergens

Peanuts, tree nuts, eggs, milk, fish, crustacean shellfish, wheat, and soybeans are responsible for 90% of food allergies and serious allergic reactions in the United States (74). Editing of allergen genes has been described and genome engineering, CRISPR editing in particular, provides potential to effectively knockout culprit allergen genes in crop seeds or plants.

Peanut is one of the major causes of food allergy (74). Out of the eighteen peanut allergens. Ara h 2 is recognized by IgE antibodies in more than 90% of peanut allergic individuals (76, 77). Dodo et al. developed allergen-reduced peanut using RNA interference (RNAi) silencing of the Ara h 2 gene (78). Ara h 2 was eliminated from modified peanuts by infecting peanut explants with Agrobacterium-mediated transformation. Stable integration of the transgene was obtained in 44% of the plants (78). Levels of Ara h 2 were significantly reduced or even undetectable in several genetically altered peanut seeds, and IgE binding of peanut-allergic patient serum was decreased compared to wild-type seeds. Studies depleting Ara h 2 and other major peanut allergens (Ara h 1, Ara h 3, and Ara h 6) using the CRISPR-Cas9 have been initiated to develop non-allergenic peanuts to protect peanut-sensitive individuals (79). Recently, using multiplex CRISPR-Cas9 genome editing, the disruption of the Ara h 2 gene in peanut protoplasts was proven feasible in the presence of an endogenous transfer RNA-processing system (80). These approaches provide a rapid and effective tool towards understanding gene functions and molecular pathways in peanut allergy to potentially reduce peanut allergy.

Egg allergy is the second most common food allergy in children and may persist into adulthood (74, 81). Most of the egg allergens are in egg white and include ovomucoid (Gal d 1), ovalbumin (Gal d 2), conalbumin (Gal d 3), and lysozyme (Gal d 4) (82). Gal d 1 is responsible for most allergic reactions, and unlike other egg allergens, retains allergenicity even after extensive heating. Heterozygous mutants in the ovalbumin (OVA) gene were generated by editing primordial germ cells through the TALEN technology (83), an earlier approach to genome editing. TALEN-mediated knockout of the OVA gene in chicken primordial germ cells (PGCs) was associated with loss of OVA gene function. Although OVA is the most abundant allergenic protein, ovomucoid (OVM) is the more dominant allergen related to egg allergy (82). Oishi et al. reported CRISPR-Cas9-mediated knockout of both egg white genes, OVA and OVM, in cultured chicken PGCs with an efficiency of more than 90% (84). In addition, transplantation of OVM-disrupted PGCs into recipient chicken embryos generated homozygous OVM knockout chickens by the second generation. An independent study showed that the OVM knockout by CRISPR-Cas9 almost completely eliminated OVM from eggs without abolishing fertility (85). In egg-allergic individuals, OVM-depleted eggs have the potential to be a hypoallergenic food source and reduce or eliminate allergic reactions to egg.

Cow’s milk allergy is the most common food allergy in children affecting 2–3% of infants in developed countries (86). Cow’s milk contains both casein proteins (α-s1-, α-s2-, β-, and κ-casein) and whey proteins consisting of α-lactalbumin (ALA) and β-lactoglobulin (BLG) (87). BLG allergen accounts for about 50% of whey proteins and about 10% of total milk protein. Most cow’s milk allergic individuals are sensitive to both casein and whey protein (88). Sun et al. generated DNA-free BLG knockout cows using zinc finger nuclease mRNA, which produced hypoallergenic milk (89). Unlike milk produced from wild-type cows, levels of BLG were not detectable in the milk produced from BLG knockout cows. The lower allergenicity of BLG-free milk was assessed in a mouse model of milk allergy. BLG-free milk triggered lower allergic reactions including rectal temperature drop and reduced allergen-specific IgE production.

Goat’s milk contains various allergens, predominantly BLG. Editing of BLG was achieved in goat fibroblasts using CRISPR-Cas9 (90, 91). Zhou et al. targeted the BLG locus to generate BLG knockout goats by injecting different sgRNAs into goat embryos (91). Among the offspring, the genome-targeting efficiency of injecting one specific sgRNA was 12.5% while the efficiencies increased up to 28.6% using two sgRNAs. BLG protein was eliminated in the milk of the BLG knockout goats and BLG expression was significantly decreased in the mammary glands of these goats. A similarly successful approach was taken by Ni and co-workers in generating BLG knockout goats (90). These studies provide a basis for removal of allergenic proteins in milk to produce hypoallergenic or non-allergenic milk to benefit milk-allergic individuals in the future.

Targeting Genes in Soybean, Wheat, and Mustard

In the United States and Europe, 5–8% of babies and 2% of adults are allergic to soybean (92). Glycoprotein Gly m 8 and Gly m 5 are the major allergenic proteins in soybean seeds (93, 94). Sugano et al. used two sgRNAs in soybean plants to simultaneous knockout both of these critical genes (95). The authors proposed the applicability of CRISPR-Cas9 for the production of hypoallergenic soybean plants.

A research team has successfully used genome editing to precisely and efficiently edit target genes in bread wheat (96). Wheat grain contains gluten proteins including α-gliadins, which are the primary cause of celiac disease and gluten sensitivity. The levels of α-gliadins were markedly reduced in gene-edited wheat lines and immunoreactivity was reduced by 85% (96). This study provides evidence that hypoallergenic wheat products can be generated which may reduce or even alleviate allergic reactions in wheat allergic patients.

Mustard is a hidden allergen in infant diets (97). Brassica juncea 1 (Bra j 1, 2S albumin seed storage protein), the prolamin superfamily major allergen in brown mustard, was found in the 2S albumin fraction and mustard specific IgE antibodies were detected in sensitive individuals (98). Assou et al. deleted the Bra j 1 gene from brown mustard by CRISPR-Cas9 (99) and provided hypoallergenic or non-allergenic protein in mustard-derived products. Creating hypoallergenic mustard plants is now feasible, potentially improving safety in mustard-sensitized individuals.

Conclusion

Allergic diseases such as food allergy, asthma, atopic dermatitis, and allergic rhinitis are complex with a shared common immunopathology, one dependent on the target organ and immune/inflammatory cells. Many cell types such as DCs and CD4⁺ T cells, mediators, and Th2 cytokines play critical roles. Targeting sentinel genes in these cells and proteins individually or targeting multiple genes with gene editing tools such as CRISPR-Cas9 will dramatically improve our understanding of the pathogenic mechanisms underlying allergic disease. The use of this technology is only the beginning on the gene-editing highway. It has already been successfully applied and investigated in human clinical trials in patients with cancer in China and in the United States (100, 101). With researchers developing newer versions of CRISPR technology and increasingly efficient delivery systems, editing additional genomic targets simultaneously and more efficiently without causing harmful side effects, the application of gene editing in allergic diseases and in modifying causal genes and allergens promises future novel strategies for disease intervention.

ABBREVIATIONS

ABE

Adenine base editor

A

Adenine

ALA

α-lactalbumin

AMG

Aminoglutethamide

AR

Allergic rhinitis

ATI

α-amylase/trypsin inhibitor

BLG

β-lactoglobulin

Bra j

Brassica juncea

Cas9

CRISPR-associated protein 9

CBE

Cytosine base editor

COPD

Chronic obstructive pulmonary disease

CRISPR

Clustered regularly interspaced short palindromic repeats

CYP11A1

Cytochrome P450, family 11, subfamily A, polypeptide 1

C

Cytosine

DCs

Dendritic cells

dCas9

Dead Cas9

DSB

Double strand breaks

FcεRI

Fc epsilon receptor 1

HDR

Homology-directed repair

G

Guanine

I

Inosine

IPF

Idiopathic pulmonary fibrosis

IgE

Immunoglobulin E

IgG

Immunoglobulin G

IL

Interleukin

IUIS

International Union of Immunological Societies

JAK3

Janus kinase 3

MUC18

Mucine 18

NHEJ

Non-homologous end joining

nCas-9

Nickase Cas9

NK

Natural killer

OVA

Ovalbumin

OVM

Ovomucoid

PA

Peanut-allergic

PAM

Protospacer-adjacent motif

PGCs

Primordial germ cells

RNAi

RNA interference

shRNA

Short hairpin RNA

sgRNA

Single guide RNA

STAT

Signal transducer and activator of transcription

TALEN

Transcription activator-like effector nuclease

Th2

T helper 2

TLR

Toll-like receptor

T

Tyrosine

U

Uracil

UGI

Uracil glycosylase inhibitor

VPS37A

Vacuolar protein sorting-associated protein 37A

VPS37B

Vacuolar protein sorting-associated protein 37B