3.1. Meganucleases

Meganucleases improved genome editing efficiency by recognising lengthy regions of 14–40 bp DNA, but they were limited by two significant issues (Langner et al., 2018). Firstly, it is unlikely to find a meganuclease that targets a specific location because each has a unique recognition sequence. Secondly, when double-strand breaks occur, most of them are repaired using the error-prone NHEJ DNA repair mechanism, which makes it challenging to incorporate the preferred DNA template at the break sites due to the possibility of random insertions or deletions of DNA segments (Adli, 2018).

3.2. Zinc finger nucleases (ZFNs)

Identifying eukaryotic zinc finger proteins directed in a new phase of genome editing research. Zinc fingers are sequence-specific tiny protein motifs regulated by zinc ions, able to bind to DNA. ZFNs have a reduced specificity since each zinc finger module can target any 18–21 bp genomic region because it can recognise a 3bp DNA sequence (Swarthout et al., 2011). Specific changes were made to zinc finger proteins, enabling the creation of programmable nucleases with the DNA cleavage domain of the FokI endonuclease. FokI can form a homodimer using two distinct zinc finger modules targeting two nearby sites. This results in DNA strand breaks at the target sites (Adli, 2018; Chen et al., 2014).

3.3. Transcription activator-like effector (TALE)

Xanthomonas produce transcription activator-like effector (TALE) proteins that can distinguish between one and three bases (Adli, 2018). Like zinc fingers, TALENs, which are chimeric fusions of the FokI DNA cleavage domain to a set of TALE modules, act as efficient programmable nucleases. While ZFNs and TALENs are valuable engineered nucleases that can improve genome editing efficacy, the challenges associated with their cloning and protein engineering have limited their widespread adoption by researchers (Adli, 2018; Chen et al., 2014).

3.4. CRISPR systems: the CRISPR/Cas9 tool

CRISPR, which stands for clustered regularly interspaced short palindromic repeats, was initially recognised as a unique prokaryotic DNA repeat element. It was later identified as the bacterial immune system, and subsequently utilised as a potent reprogrammable gene-targeting tool (Knott & Doudna, 2018). CRISPR/Cas9 is a powerful and exact genome-editing technique that facilitates gene function analysis and allows the production of targeted mutants with specific traits (Alamillo et al., 2023). The growing number of recent papers on plant genome editing utilising the CRISPR/Cas9 system suggests that this method is practicable due to its increased success rate and ease of usage.

The CRISPR gene editing method uses an RNA-guided endonuclease protein to create specific double-strand breaks DSBs (Knott & Doudna, 2018). Various genome editing tools, such as MNs, ZFNs, TALENs, and CRISPR/Cas9, can cause DSBs, allowing the disruption of desired genes via NHEJ or the insertion of desired genes via HDR. However, the CRISPR/Cas9 technology is the preferred method for researchers in various fields due to its comfort of use and high editing efficiency for genome targeting purposes (Adli, 2018).

RNA hybrid made up of a trans-activating crRNA (tracrRNA) and a CRISPR RNA (crRNA) directs the binding and cleavage activities of Cas9. Combining tracrRNA with crRNA creates a sgRNA (Jinek et al., 2012). A brief (20–23 bp) sgRNA segment is changed to target a new sequence once complementarity identifies the target nucleic acid. The Cas9 HNH and RuvC domains cleave the target DNA’s complementary and non-complementary strands, resulting primarily in blunt-ended DSBs. Subsequently, the host cellular component repairs the DSB (Makarova et al., 2011; Shao et al., 2016).

Despite discovering other Cas9 orthologs (Molla et al., 2020a), Streptococcus pyogenes (SpCas9) Cas9 is the most often employed nuclease for various genome editing research because of its high effectiveness and low requirements for the NGG PAM sequence. A protospacer adjacent motif, or PAM for short, is a conserved sequence of 2–5 base pairs that is needed by Cas9 to identify, bind, and cut target genomic locations (Jiang & Doudna, 2017). Different species’ Cas9 might require different PAMs (Molla et al., 2020a). For Cas9, PAM must be situated directly downstream of the target sequence. While most researchers believe that Cas9 cuts DNA three base pairs upstream of the PAM on both strands (Jiang & Doudna, 2017), a rising body of recent data suggests that Cas9 frequently mediates cleavage at 4-, 5-, and 6-bp upstream in the non-complementary strand (Molla & Yang, 2020b) (Figure 3). Cas12 cleaves at the PAM distal location, resulting in a staggered DNA double-stranded break (Zetsche et al., 2015). Cas13 cleaves single-stranded RNA (Abudayyeh et al., 2016).

The two main mechanisms for repairing genomic double-strand breaks (DSBs) produced by site-specific nucleases are HDR and NHEJ (Figure 4). Higher plants use primarily NHEJ, which is error-prone and results in tiny insertions or deletions (indels). Indel-induced frameshift mutations offer efficient gene knockout. With the development of CRISPR/Cas9 tools, it is now easier to knock off a gene or produce targeted genetic variation (Jinek et al., Jinek et al., 2012). However, homology-directed repair (HDR) in higher plants is not as prevalent as in other organisms. It is utilised for precise adjustments, often requiring an extra source of donor templates containing the desired modification.

Figure 3

Molecular mechanism underlying CRISPR/Cas9 genome editing.

Figure 4

Schematic of CRISPR/Cas9 genome editing system.

4.1. Gene disruption via indels in coding sequences

Numerous prevalent applications of the CRISPR/Cas9 system involve leveraging the error-prone nature of the NHEJ DNA-repair machinery within cells. This process results in insertion or deletion of one or more nucleotides (indel) at the specific location guided by sgRNA. Consequently, a frameshift mutation occurs, disrupting gene transcription. This approach has effectively introduced favourable characteristics into a broad spectrum of crops, including key cereals like wheat and rice (Chen et al., 2014). By manipulating plant’s susceptibility (S) genes, which alters plant-pathogen interaction and reduces the pathogen’s ability to thrive on the host plant, this method has been employed to develop resistance within the disease management framework (Zaidi et al., 2018). The use of CRISPR/Cas9 to introduce mutations in the eukaryotic translation initiation factor 4E proteins (eIF4Es) has been particularly notable. This approach has been successfully employed in Arabidopsis and cucumber (Cucumis sativus) to confer resistance against multiple RNA viruses (Chandrasekaran et al., 2016; Pyott et al., 2016).

Susceptibility (S) genes in host plants promote pathogen invasion and colonisation (Li et al., 2014; Schie & Takken, 2014). Opposed to R genes, manipulating such S genes imposes long-term resistance in the field (Langner et al., 2018). The genes labelled as S genes can be divided into three groups: the first encompasses genes that assist in host recognition by pathogens and their penetration, such as IRG1 in Medicago and MLO in barley and other plants. The second includes genes that facilitate pathogen nutrition and promote the establishment of pathogens, such as SWEET genes in rice and eIF4 genes for viral infection in many plants. The third gene group operates as a negative regulator for host defence responses, with examples like S3H in Arabidopsis and PM4 in tomato (Schie & Takken, 2014). According to recent research, the CRISPR/Cas technique targeting a host gene is becoming increasingly popular for treating disease and pest resistance (Table 1).

S gene editing can offer a broader and more resilient resistance range since changing S genes disrupts compatibility. The most researched S gene is mildew locus O (MLO), found in numerous plant species. It encodes a protein tethered to the membrane and facilitates the entry of powdery mildew fungus into epidermal cells (Büschges et al., 1997). Several plant species lost their vulnerability to MLO when it was edited using CRISPR/Cas (Nekrasov et al., 2017; Wang et al., 2014). Editing effector binding elements (EBEs) and sequences in the promoters of rice SWEET genes have been linked to the development of Xanthomonas oryzae pv. oryzae (Xoo) resistance, according to several studies (Duy et al., 2021; Kim et al., 2019; Zeng et al., 2020). The translation of viral RNA requires the presence of plant translation initiation factors (eIF) 4E and 4G (Wang & Krishnaswamy, 2012). Recent research has shown that CRISPR mutants of the eIF4E and eIF4G genes demonstrated resistance (Macovei et al., 2018; Mushtaq et al., 2020). S genes are a specific group of genes that produce proteins responsible for negatively regulating the plant’s defence mechanisms. These proteins help to modulate and balance the plant’s immune responses, preventing overreactions that could be harmful to the plant itself. For instance, the cotton gene Gh14-3-3d engages in BR signalling and interferes with Verticillium dahlie resistance (Gao et al., 2013).

According to Schie and Takken (2014), the majority of S genes that have been identified are monogenic; numerous genes can encode susceptibility factors, and CRISPR/Cas multiplexing offers an alluring way to target them all at once. Comparison of transcriptomes or proteomes after host-pathogen interaction may help identify more unique S gene targets in the plant system. Numerous S genes are crucial for the growth and development of crops. Crop productivity and growth would suffer if these S genes were functionally knocked out. For instance, while conferring disease resistance, the null mutants of OsSWEET14/Os11N3 (Antony et al., 2010) and mlo (Li et al., 2022) also showed growth penalties and yield losses.

4.2. Gene disruption via indels in promoter regions

There are two strategies for utilising CRISPR-mediated promoter editing in genetic modification. The first involves disrupting the promoter sequence to halt gene production entirely. The second approach focuses on disrupting an effector-binding site to prevent a pathogen effector from binding to the promoter, thereby reducing the plant’s susceptibility to pathogens. By modifying the rice sugar transporter OsSWEET14 gene’s promoter, the latter method has been successfully employed to confer blight resistance by severing the gene’s interaction with an effector from a bacterial blight pathogen (Blanvillain-Baufumé et al., 2017; Xu et al., 2019). CRISPR, a revolutionary gene-editing technology, has demonstrated its capability to not only edit promoters but also modify gene regulation by targeting regions upstream of open reading frames (ORFs) and editing cis-regulatory elements (Si et al., 2020).

4.3. Gene deletion via multiplex sgRNAs

Multiple sgRNAs can be utilised in CRISPR systems to introduce numerous DSBs at specific target genomic regions. For example, two sgRNAs binding to the gene of interest before its start codon and after its finish codon will result in DSBs at the corresponding places. Before the NHEJ mechanism can fix the breaks, the DSBs may lead to the loss of the DNA fragment that encodes the desired protein. This method can be employed to delete individual genes and substantial chromosomal segments (Cai et al., 2018; Zhou et al., 2014). Developing a logically constructed Cas9, known as SpCas9-NG, has enabled the identification of NG PAMs with a relaxed stringency compared to the usual NGG PAMs. This advancement further facilitates the process (Nishimasu et al., 2018). In S gene clusters, when many S genes are located on contiguous chromosomes (Ashfield, 2012), removing the chromosomal region will likely result in long-term resistance to the infectious agents.

4.4. Gene insertion via homology-directed repair

Altering the significant and multifunctional proteins that are synthesised by S genes may lead to unforeseen consequences for both plant health and agricultural productivity. When gene disruption is not the preferred method, other options, like editing the cis-regulatory elements and promoters, can be utilised to modify gene expression. However, in cases where the plant-pathogen interaction is not comprehensively captured, and the S genes have not been thoroughly studied, resistance (R) genes against pathogens are often necessary. In such situations, R genes can be inserted using the CRISPR toolbox. The Cas9 protein and the sgRNAs are combined with a delivery fragment that includes an R gene, flanked by a sequence matching the ends of a DSB. The R gene is placed between the two DSB sites with the help of this cassette using HDR. This method has been used to insert one or more genes at precise positions in the genome (Van Vu et al., 2019).

Still, a significant obstacle to HDR’s frequent deployment is its low frequency in higher plants (Molla et al., 2020a). A variety of strategies have been investigated with varying degrees of success to improve HDR efficiency in plants, including viral replicons (Čermák et al., 2015), bacterial retrons (Sharon et al., 2018), transcript-templated HDR (Li et al., 2019a), tandem repeat HDR (Lu et al., 2020b), and chimeric guide RNA (Butt et al., 2017). Ali et al. (2020) developed a Cas9 and VirD2 relaxase fusion to create targeted DNA double-strand breaks. This allows the repair template to be positioned close to the DSBs, aiding in HDR.

4.5. Biomimicking via promoter, allele, or gene replacement

When specific changes are made to the sequence of an intended gene using the CRISPR technology, resulting in a gene matching that of a disease-resistant type, this process is known as “biomimicking.” Suppose the nucleotide variances between the gene of interest in domesticated and wild varieties do not significantly impact the plant’s ability to survive and produce. In that case, the researcher will only introduce the particular mutations linked to the disease resistance trait instead of swapping out the entire gene. Fusing a nuclease-dead Cas9 or nickase with cytidine deaminase enables high-precision point mutagenesis. This innovative method has demonstrated practical application in gene modification across various species (Komor et al., 2016).

Wang et al. (2017) demonstrated the use of the CRISPR/Cas9 technology for biomi­micking via promoter replacement to enhance disease resistance in rice. The researchers achieved targeted gene expression alterations by substituting the native promoter of particular genes associated with disease susceptibility, increasing resistance to bacterial and viral pathogens. This example shows how promoter replacement using CRISPR may mimic natural plant defence systems and confer disease resistance in crop plants.

Zhang et al. (2017) used the CRISPR/Cas9 technology to create disease-resistant wheat plants for allele replacement. The researchers edited three homoeologs of the TaEDR1 gene, which is linked to powdery mildew susceptibility, to create precise mutations that mimic natural genetic variability and improve disease resistance. The resultant wheat plants demonstrated greater tolerance to powdery mildew infection.

By targeting a susceptibility gene involved in geminivirus infection, Ali et al. (2015) replaced the susceptible allele with a resistant allele using CRISPR/Cas9. The plants that were produced showed improved resistance to geminivirus infections.

4.6. Need for synthetic R gene-mediated resistance

The largest group of R genes in plants consists of genes that encode nucleotide-binding site (NBS)–leucine-rich repeat (LRR) proteins. If altered, the LRR domain may be able to identify elicitors from a wide range of pathogens in addition to pathogen-derived elicitors that confer resistance. Resistance to certain strains of potato virus X (PVX) is granted by the potato R protein (Rx) LRR domain. Wheat R protein, Pm3F, with two amino acid substitutions, increases its potency against other pathogens (Stirnweis et al., 2014). For identifying isolate-specific Avr effectors (AVRa7, AVRa9, AVRa10, and AVRa22) of Blumeria graminis f. sp. hordei, the barley Mla locus contains many allelic variants (MLA7, MLA9, MLA10, and MLA22) (Saur et al., 2019). Therefore, recognition specificity can be changed via knowledge-based modification of the molecular structure of R genes. Using CRISPR/Cas tools, particularly prime and base editors, can be beneficial in modifying R genes for broad-spectrum resistance in vivo.

To create resistance against TuMV, Kim et al. (2016) modified the AvrPphB cleavage site in PBS1 to incorporate the cleavage site of the NIa protease from turnip mosaic virus (TuMV). This strategy has recently been expanded to soybeans to establish resistance against the soybean mosaic virus (SMV) (Pottinger et al., 2020). Utilising advanced genome editing techniques such as prime editing and HDR, we can precisely modify the endogenous PBS1 protein to confer resistance to diseases for which robust hereditary resistance is currently lacking. Integrated domains (IDs), present in specific NBS-LRR proteins (for example, Rice Pik-1), are believed to be crucial in detecting pathogen effectors. As a result, targeting IDs for receptor engineering holds significant promise in enhancing disease resistance. Kourelis et al. (2023) conducted a groundbreaking study that showed how resistance against a virus can be gained by replacing the integrated HMA domain of Pik-1 with an effector-binding VHH-nanobody. To create “Pikobodies,” which provide novel resistance to any pest or pathogen that delivers effectors inside plant cells, nanobodies capable of binding the desired effectors can be introduced into Pik-1 to replace the HMA domain.

It should be remembered that altering an already described R gene to produce novel resistance could cause it to lose its original function and become susceptible to a different infection. Saturated mutagenesis via prime editors (Xu et al., 2021) and dual base editors (Li et al., 2020b) will enable knowledge-guided directed evolution of R genes to broaden their resistance range. The HDR-mediated targeted insertion of multiple R genes into a specific genomic locus provides an attractive method for stacking R genes in a single background. Furthermore, it is essential to address the insert size restrictions and low efficiency of this approach (Dong et al., 2020). Novel R gene stacks created with HDR would be considered genetically modified organisms (GMOs) and might be subject to strict restrictions. Gain-of-function mutagenesis techniques such as CRISPR base and prime editors are employed to create disease resistance.

4.7. Developing CRISPR-like artificial immunity

To defend against the pathogen of interest, host plants that express the Cas9-sgRNA complex targeted to virus genomes may build a bacterial immune system akin to CRISPR. Past research has employed this strategy to build immunity against viruses (Ali & Mahfouz, 2021; Mushtaq et al., 2020). Assume a host cell possesses a homologous sgRNA protospacer and an active Cas effector, both essential viral genome components. The viral life cycle can then be stopped by the Cas protein, causing damage to the viral genome. While Cas13 is ideal for targeting viral RNA genomes, Cas9 and Cas12 are suitable for targeting viral DNA genomes.

One drawback of this strategy is that a transgene-free immunity system is impossible because the Cas9 and sgRNA expression cassettes must always be present in the plant cell. Furthermore, viruses can develop mutations resistant to Cas cleavage, shielding them from CRISPR-like plant immunity.

5.1. CRISPR/Cas9-based resistance to viruses

Plant viruses are obligate parasites and rely on host machinery for survival. Managing viral diseases is challenging because viruses evolve rapidly with insect vectors. The conventional strategy based on pathogen-derived resistance (PDR) is time-consuming. However, the CRISPR/Cas9 tool has shown significant promise in controlling viral infection and spread (Baltes et al., 2015; Ji et al., 2015; Liu & Zhang, 2020) (Table 2).

5.2. CRISPR/Cas9-based resistance to fungi

The “mildew resistance locus” (Mlo) is a well-known S-gene mutation that provides resistance to powdery mildew in barley. By utilising loss-of-function Mlo alleles, barley variants with powdery mildew resistance have been developed. These resistant plants have demonstrated long-term resilience to highly pathogenic strains of powdery mildew in field cultivation, showcasing the effectiveness of S-gene-based resistance (Jørgensen, 1992). The discovery of the barley Mlo gene through cloning showed that it is present in various plant species and is found as a multi-copy gene family in higher plants. This suggests that the Mlo gene is widely conserved across the plant kingdom and plays a significant role in the genetic makeup of higher plants (Büschges et al., 1997; Devoto et al., 2003). Furthermore, many plant species, including Arabidopsis, tomato, pea, pepper, tobacco, bread wheat, and possibly grapevine and peach, have been linked to powdery mildew susceptibility due to Mlo like genes (Appiano et al., 2015; Bai et al., 2008; Consonni et al., 2006; Feechan et al., 2008; Humphry et al., 2011; Wang et al., 2014; Zheng et al., 2013). Grapevine powdery mildew resistance was increased by the CRISPR/Cas9 deletion of VvMLO7 and VvMLO3 (Malnoy et al., 2016; Wan et al., 2020). The RNP-based technique has been effectively applied to alter three (DIPM-1, DIPM-2, and DIPM-4) genes in apple (Malus domestica) protoplasts to produce resistance against Erwinia amylovora, the fire blight (Malnoy et al., 2016). Four guide RNAs were employed in the CRISPR/Cas9 strategy to target the VvWRKY52 transcription factor. In contrast to monoallelic mutant plants, the results revealed 21% of B. cinerea (Wang et al., 2018). The CRISPR/Cas9 system temporarily targeted the immune system suppressor gene, Pathogenesis-Related 3 (NPR3), in cocoa leaves, increasing resistance to Phytophthora tropicalis. The application of the CRISPR/Cas9 technology has led to the identification of a novel selection marker for Phytophthora species, a group of oomycetes known for causing devastating plant diseases. This significant breakthrough emerged from genetically modifying a fungicide-resistant gene known as PcMuORP1 (Wang et al., 2019). Resistance against rice blast disease is conferred by OsSEC3A and OsERF922 gene disruption mediated by CRISPR/Cas9 (Ma et al., 2018; Wang et al., 2016). Furthermore, rice with the pi21 gene exhibited long-lasting resistance against rice blast (Zhou et al., 2022). Additionally, the production of OsSEC3A mutants improved rice resistance to disease caused by Magnaporthe oryzae and resulted in a pleiotropic phenotype with a rise in salicylic acid (SA) concentration. Additionally, many genes linked to pathogenesis and SA were upregulated (Ma et al., 2018). By leveraging the CRISPR/Cas9 technology, researchers have successfully enhanced rice’s resistance to the devastating rice blast disease caused by the fungus M. oryzae. This advancement was achieved by targeting the negative regulators associated with the plant’s defence mechanism. Moreover, a groundbreaking CRISPR/Cas9 chromosomal rearrangement technique for soybeans has also been revealed (Nagy et al., 2021). Through the artificial creation of chimeric paralogs, this work generated two NLR gene clusters, named Rpp1-like and Rps1, which are associated with resistance to Phakopsora pachyrhizi and P. sojae, respectively. It also demonstrated a potential cutting-edge method for managing plant disease. In summary, the advantages, effectiveness, and potential of the CRISPR/Cas-based editing technology to improve crop plant resistance were ascertained by all of these successful fungal disease resistance outcomes (Table 3).

According to Frye et al. (2001), edr1 mutant plants in Arabidopsis demonstrated tolerance against bacteria and oomycetes. On the other hand, Taedr1 mutant plants in wheat produced immunity to Bgt after all homologs of EDR1 were simultaneously altered using CRISPR/Cas9 (Zhang et al., 2017). According to Nekrasov et al. (2017), tomato resistance to Oidium neolycopersici was enhanced by SlMlo1 deletion using CRISPR/Cas9. Nevertheless, powdery mildew resistance 4 (PMR4), a gene encoding callose synthase, has also been altered by GET, which has resulted in the establishment of resistance to O. neolycopersici in tomato and Arabidopsis as well. Similar to this, rice plants’ resistance to M. oryzae has increased due to a mutation in the Pi21 gene’s coding area generated by CRISPR/Cas9 (Li et al., 2019b). In addition, Xie and Yang (2013) reported that CRISPR/Cas9 was also applied to rice, specifically targeting the coding area of Oryza sativa mitogen-activated protein kinase 5 (OsMAPK5) to enhance resistance to Magnaporthe grisea. In a study by Wang et al. (2018), it was discovered that grapevines with homozygous mutants of VvWRKY52 showed improved resistance to Botrytis cinerea. The researchers developed four gRNAs targeting the VvWRKY52 transcription factor using the CRISPR/Cas9 system. Additionally, the application of CRISPR/Cas9 has led to heightened resistance against Phytophthora tropicalis and Phytophthora palmivora in papaya (Gumtow et al., 2018) and cacao (Fister et al., 2018) plants, respectively. These instances of CRISPR/Cas9 applications highlight its vital role in genetic engineering technologies, especially in enhancing plant resistance to fungal pathogens.

5.3. CRISPR/Cas9-based resistance to bacteria

Significant risks to agriculture are posed by phytopathogenic bacteria, which can produce rots, mosaics, spots, and other difficult-to-control illnesses that severely reduce productivity. Bacteria become resistant to antibacterial agents and proliferate infections due to their constantly changing environment and horizontal gene transfer. Bacteria typically gain entry into cells through mechanical wounds, stomata, stigma, and other plant openings. Additionally, they can penetrate cells by releasing bioactive chemicals that facilitate their entry (Zeng et al., 2010). Upon identifying these signals, the plant defence mechanism takes the necessary action to eradicate the infection (Figure 1). Nonetheless, numerous host plant genes, including a few “S” genes, aid in the complicated signal cascade that successful pathogens employ to overcome plant protection and start an infection (Das et al., 2018; Dong & Ronald, 2019). CRISPR/Cas9 techniques were utilised to modify tomato plants that were under attack by bacterial plant infections, such as Pseudomonas syringae and Xanthomonas spp. This led to a decrease in tomato yield and significant economic damage (Schwartz et al., 2015). According to Zeilmaker et al. (2015) and Langner et al. (2018), P. syringae pv. tomato infection was found to increase the expression of DMR6 (downy mildew resistance 6), function as a negative regulator of plant immunity, and aid in the spread of infection. By utilising CRISPR/Cas9, scientists were able to remove the tomato counterpart SlDMR6-1, resulting in an improved resistance of tomato plants to multiple diseases, such as P. syringae pv. tomato and Phytophthora capsici. Remarkably, this enhancement in disease resistance was achieved without causing any damage to the overall health of the plants (Thomazella et al., 2021). In another case, OsSWEET13, a sensitive host gene implicated in sucrose transfer during disease, was edited using CRISPR/Cas9 to confer resistance against the bacterial blight pathogen Xanthomonas oryzae pv. oryzae (Zhou et al., 2014). When Zhou et al. (2015) deleted the OsSWEET13 gene from rice, a null mutant with increased resistance to X. oryzae was produced. Similar to this, DspE is the name of the pathogen effector protein produced by Erwinia amylovora. The dspE protein interacts with DspE-interacting proteins of Malus (DIPM 1, 2, 3, 4) genes (Gimenez-Ibanez et al., 2017; Das et al., 2018). This interaction has been found to enhance plant susceptibility to fire blight disease. To build resistance against this disease, researchers utilised CRISPR/Cas9 to truncate these DIPM genes in apple protoplasts (Malnoy et al., 2016). A viable strategy for sustainable agriculture could be transgenic crops with unique alleles and broad disease resistance (Table 4).