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Genome editing in future crop protection: utilizing CRISPR/Cas9 to improve

crop resistance against diseases, pests, and weeds

Article in Discover Agriculture · November 2024

DOI: 10.1007/s44279-024-00124-0

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Discover Agriculture

Review

Genome editing in future crop protection: utilizing CRISPR/Cas9

to improve crop resistance against diseases, pests, and weeds
Ahmad Faizal1 · Syarul Nugroho2 · Anca Awal Sembada3,4 · Yohanes Theda5 · Tinta Komariyah6 ·
Rizkita Rachmi Esyanti1

Received: 2 August 2024 / Accepted: 13 November 2024

© The Author(s) 2024  OPEN

Abstract
Increasing population and climate change pose significant threats to global food security by imposing stresses on plants,
making them more susceptible to diseases and productivity losses caused by pathogens, pests, and weeds. Traditional
breeding strategies are insufficient for rapid development of new plant traits that can outpace this productivity down-
trend. Modern advances in genome editing technologies, particularly CRISPR/Cas9, have revolutionised crop protection
through precise and targeted genome modifications. This allows for the development of resilient crops with enhanced
resistance against pathogens, pests, and weeds. This review explores various approaches with which CRISPR/Cas9 is
applied for crop protection: knocking out of susceptibility genes, introduction of resistance genes, and modulation of
defence genes. Potential applications of CRISPR/Cas9 in crop protection include the introduction of genes conferring
resistance to pathogens, disruption of insect genes responsible for survival and reproduction and engineering of herbi-
cide-resistant crops. In conclusion, CRISPR/Cas9 holds great promises in advancing crop protection and thus ensuring
food security amidst environmental and population pressures. This review highlights the transformative potential of
genome editing in crop protection and calls for continued research and development in this field.

Keywords CRISPR/Cas9 · Crop protection · Disease resistance · Sustainable agriculture · Genome editing

1 Introduction

The global population is forecast to reach 8.2 billion by 2030 and 9.3 billion by 2050 [1, 2]. This anticipated growth under-
scores the urgent need for a substantial escalation in food production. By 2050, global food demand is expected to rise
by 56%, heightening the risk of hunger [3, 4]. Additionally, climate change poses a significant threat to food security,
as shifts in temperature and precipitation patterns affect land suitability and crop yields [5]. Higher temperatures can
directly influence pest reproduction, survival, spread, and population dynamics, while climate-stressed plants are more
prone to disease [6, 7]. Ensuring plant health is crucial for economic stability, given agriculture’s vital role in the economy
[8, 9]. Plant diseases and pests have been shown to cause substantial economic losses, with direct yield reductions due
to pathogens, pests, and weeds accounting for 20% to 40% of global agricultural productivity losses [10, 11]. Specifically,

* Ahmad Faizal, [email protected] | 1Plant Science and Biotechnology Research Group, School of Life Sciences and Technology,
Bandung Institute of Technology, Bandung 40132, Indonesia. 2Plant Breeding Research Group, Indonesian Oil Palm Research Institute,
Medan 20158, Indonesia. 3Forestry Technology Research Group, School of Life Sciences and Technology, Bandung Institute of Technology,
Bandung 40132, Indonesia. 4Research Center for New and Renewable Energy, Bandung Institute of Technology, Bandung 40132,
Indonesia. 5Department of Biochemical Engineering, University College London, London WC1H 9BT, UK. 6Research Center for Vaccine
and Drug, National Research and Innovation Agency, Bogor 16915, Indonesia.

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pathogens and pests can reduce yields of major crops like maize, potato, rice, soybean, and wheat by up to 40% [12].
Effective crop protection against plant diseases is essential to meet the increasing demand for both food quality and
quantity [13, 14].
One of the aims in modern plant breeding is to produce disease-resistant crops, thereby enhancing yield stability and
crop protection [15, 16]. In contrast to traditional breeding methods, plant genome editing offers the possibility of the
development of novel plant traits [17]. Plant genome editing allows for precise chromosome restructuring, encompassing
small insertions, deletions, substitutions, and complex rearrangements like inversions, duplications, and translocations
[18]. Furthermore, plant genome editing allows for faster and more predictable plant breeding that is widely applicable
for various species and thus plays a critical role in advancing food security and supporting the bioeconomy [19]. As
one of the most significant advancements in modern genome editing, the Clustered Regularly Interspaced Short Pal-
indromic Repeats and its associated proteins Cas (CRISPR/Cas9) technique heralds a new era in plant breeding [20, 21].
CRISPR/Cas9 demonstrates superior applicability compared to other endonuclease-based gene editing techniques like
zinc-finger nuclease (ZFN) or transcription activator-like effector nuclease (TALEN) [22]. By inducing DNA double-strand
breaks (DSBs), CRISPR/Cas9 with its site-specific nuclease activity triggers DNA repair mechanisms within cells, offering
a wide range of genetic alterations depending on repair pathways and the availability of repair templates [23]. Another
avenue through which crop protection can be achieved is by genetically engineering the culprits of crop damages, such
as insects. More specifically, modern genetic engineering approaches on insects could disturb their biological functions
and thus save the otherwise-damaged crops. As will be further elaborated in this article, this could be achieved by pre-
venting mating attempts and introducing bodily defects in insects, among others [24, 25].
The advent of the CRISPR/Cas9 technology opened new possibilities for efficient and effective plant genome editing.
Since the first published application of the CRISPR/Cas9 in plant genome editing in 2013 [26–28], a large body of scientific
publications have shown its versatility for multiple purposes, including conferring resistance against pathogens, insects,
and weeds. In this review, we explore the utilization of the CRISPR/Cas9 system as a genome editing strategy to safeguard
crops against pathogens, insects, and weeds, addressing the challenges of food security. Additionally, we investigate
alternative CRISPR/Cas9-related technologies employed to develop future crop protection measures.

2 The CRISPR/Cas9 system in modern genome editing

CRISPR/Cas9 stands as a pivotal technology in contemporary genome editing techniques and has drastically revolution-
ized the field of genetic manipulation [19, 29]. Approximately 84% of archaea and 45% bacteria are equipped with CRISPR/
Cas systems, showcasing their natural prevalence [30]. The CRISPR array consists of small repetitive sequences that are
interspersed with unique spacers which originate from viral nucleic acid fragments, serving as molecular memory to
combat future infections [31]. Paired with Cas9, an endonuclease from Streptococcus pyrogenes, the CRISPR system acts
as a precise genetic engineering tool [32].
First discovered in the DNA of Eschericia coli by Ishino et al., the function of the CRISPR system was not immedi-
ately clear [33, 34]. It was only later that its function became clearer, with studies such as that of Mojica et al. that
hypothesised its function in the bacterial immune system [35]. Twenty years from its discovery, the first experimen-
tal demonstration of the immunological role of CRISPR was conducted by Barrangou et al. through their work with
Streptococcus thermophilus [29]. Along with the development of CRISPR, the discovery and development of CRISPR-
associated (Cas) enzymes was also taking place. The most studied Cas enzymes are those who possess the capability
to cut nucleic acid, of which Cas9 is a prominent exemplar. The first association between Cas9 and CRISPR was made
by Bolotin et al. in a study on S. thermophilus [36]. Copious amount of research has been then concerted towards
studying and exploiting CRISPR and Cas9, which eventually culminated in the works that led Jennifer Doudna and
Emmanuelle Charpentier to win the 2020’s Nobel Prize in Chemistry. Within the CRISPR/Cas9 system, Cas9 employs
a specific guide RNA (gRNA) duplex consisting of CRISPR RNA (crRNA) and trans-activating CRISPR RNA (tracrRNA),
as illustrated by Fig. 1A. crRNA is 18–20 nt in length and plays a crucial role in targeting a certain DNA sequence by
pairing with it. tracrRNA, on the other hand, is a longer piece of RNA, 50–150 nt in size and is an integral part of the
Cas9 DNA cutting mechanism [37, 38]. For modern genetic engineering purposes, the duplex is combined into a
single molecule termed the single guide RNA (sgRNA) [39], as shown by Fig. 1B. More specifically, sgRNA is created
by attaching a linker to the 3’ end of crRNA and 5’ end of tracrRNA. sgRNA retains two crucial features: a 20-nucleo-
tide sequence at the 5′ end that specifies the DNA target, and a double-stranded structure at the 3′ end that binds
to Cas9 [39].

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Fig. 1  The basics of the CRISPR/Cas9 technology in genome editing. This technology exploits the naturally occurring genomic properties
found in archaea and bacteria to precisely edit the genome of an organism. In the native system A the Cas9 enzyme is guided by a guide
RNA (gRNA) duplex consisting of trans-activating CRISPR RNA (tracrRNA) and CRISPR RNA (crRNA). In the modern CRISPR/Cas technology B
a synthetic guide RNA used in genome editing combines tracrRNA and crRNA into single guide RNA (sgRNA). In both cases, Cas9 introduces
double strand breaks (DSBs), which can be repaired through one of two mechanisms (C): non-homologous end joining (NHEJ) and homol-
ogy-directed repair (HDR)

In both the native and synthetic CRISPR/Cas9 systems, a protospacer adjacent motif (PAM) acts as a marker for
the target sites. PAMs are a prerequisite for precise nucleic acid cleavage, since they “instruct” Cas9 to cleave a DNA
strand at a precise location [40]. The length of a PAM can vary between two to six nucleotides, although prior research
has shown that most well-conserved PAMs have three or for nucleotides, such as NGG, NAG, CTT, and TTTV (V is A,
C, or G) [41]. The seed region is a short DNA sequence consisting of 10–12 base pairs adjacent to the PAM. It has a
crucial role in determining Cas9 specificity, and it makes this region more essential than other regions in the SgRNA
[39]. The interplay between these components enables Cas9 to introduce double strand breaks (DSBs) in the DNA
precisely [42]. DSBs then triggers their reparations, which can be achieved via one of two main mechanisms (Fig. 1C):
non-homologous end joining (NHEJ) or homology-directed repair (HDR); both of which can be exploited to intro-
duce genetic alterations [42, 43]. NHEJ typically results in random insertions or deletions, often causing mutations
due to frameshifts and effectively knocking out the targeted gene [23]; whereas HDR allows for more precise gene
insertions, known as ‘knock-ins’ [44]. In the absence of a homologous template, DSBs trigger the NHEJ mechanism
[45], which is particularly efficient when overhangs are generated [46]; otherwise, when a homologous template is
present, the HDR mechanism can be triggered. In the HDR system, the Cas9-gRNA complex plays a crucial role in this
process by guiding the delivery of gene-carrying fragments flanked by homologous sequences to the DSB site [47].
Both NHEJ and HDR offer versatility and precision in plant genome editing for crop protection. CRISPR/Cas9-
mediated gene knockouts have demonstrated efficacy in conferring resistance to diseases and insect pests [48–50].
However, harnessing HDR for DNA knock-in remains a challenging endeavour due to the limitations in supplying
sufficient repair templates, such as donor DNA [51]. Furthermore, in plants, several delivery methods exist, including
Agrobacterium-mediated delivery, bombardment-mediated delivery, and PEG-mediated delivery. Of these methods,
Agrobacterium-mediated delivery is the most established one [52]. Several future potential delivery methods include
pollen magnetofection-mediated delivery and nanoparticle-mediated delivery, which do not need tissue culture
[53]. Despite the challenges, as will be discussed in this article, the genome editing potential of CRISPR/Cas9 enables
unprecedented precision and versatility in plant genome editing, which can be materialised into the conferment of
beneficial traits into plants, such as disease resistance and pest resistance.

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3 CRISPR/Cas9 for crop protection to control pathogens

Plant diseases pose significant challenges to agriculture, impacting various stages from cultivation to post-harvest,
with the principal pathogen categories encompassing viruses, bacteria, fungi, nematodes, and parasitic plants. These
biotic constraints pose a threat to food security [13]. The extent of yield loss induced by viral infections varies widely,
ranging from 0 to 100%, influenced by factors such as the virus source, vector, and environmental conditions [54]. Fun-
gal diseases represent a crucial yield-limiting factor, with potential yield reductions ranging from 15 to 20% in cereals
and, in severe cases, escalating up to 60% [55]. In parallel to fungal- and viral-induced losses, damages from bacterial
infection can lead to significant quality decreases and yield losses, as exemplified by the documented 20.27% losses
of maize yields due to bacterial stalk rot and 50% yield loss in rice yields from bacterial leaf streak disease [56, 57].
The CRISPR/Cas9 technology is able to introduce artificial mutations that alter the sequence of the target gene
to resemble a disease-resistant type, provided the nucleotide variations do not adversely affect plant viability or
productivity [58]. Not only does this approach have the potential to mitigate yield losses but it also contributes to
the assurance of food security by enhancing the productivity and sustainability of agricultural systems. As such, lev-
eraging the CRISPR/Cas9 technique for crop protection represents a pivotal step towards addressing the multifaceted
challenges posed by plant diseases in agriculture.

3.1 Harnessing CRISPR/Cas9 for precision virus resistance in crops

The CRISPR/Cas9 technology can also be employed to introduce mutations in alleles associated with virus resist-
ance, such as eukaryotic initiation factor 4E (eIF4E). RNA viruses rely on eIF genes like eIF4E to complete their life cycle
[59]. The eIF protein plays a crucial role in initiating protein translation by recognizing and interacting with mRNA
cap structures and ribosomes. Mutations in these host factors can impede viral proliferation and host infection [60].
Notably, the eIF4E mutation has been demonstrated to confer virus resistance in various plants, including tomato,
Arabidopsis, cucumber, and Chinese cabbage [61–64].
The CRISPR/Cas9 technology can also be employed for direct targeting of the viral genome, offering a promising
approach to generate virus resistance [58]. For instance, in tomato plants, CRISPR/Cas9 has been utilized to target
the genome of the tomato yellow leaf curl virus (TYLCV), resulting in interference with viral replication. Notably,
plants expressing sgRNA sequences targeting TYLCV’s coat protein sequence exhibited enhanced resistance to the
virus [65]. The viral coat protein plays a critical role in encapsulating the viral genome, facilitating virus movement,
and recognizing vectors [66]. Similarly, tobacco plants employ a similar mechanism to resist infection by the cotton
leaf curl mutant virus (CLCuMuV) [67]. By precisely targeting viral genomic sequences essential for infection and
replication, CRISPR/Cas9 technology offers a potent strategy to confer durable resistance against viral pathogens in
crops. In addition, the CRISPR/Cas9 technology has been utilised to edit the AC2 and AC3 genes of the African cassava
mosaic virus (ACMV) genome. The AC2 gene encodes the multifunctional TrAP protein, which plays crucial roles in
gene activation, viral pathogenicity, and suppression of gene silencing, while the AC3 gene encodes the REn protein,
involved in increased viral replication [68]. The engineered plant possessed an ACMV-resistant trait [69]. Jogam et al.
observed the induction of CRISPR/Cas9 mutations of the Tobamovirus multiplication 1 (TOM1) gene successfully
confers tobacco resistance to the Tobacco mosaic virus (TMV) [70].
Furthermore, the CRISPR/Cas9 technology has been shown to have conferred resistance to banana streak virus
(BSV) in bananas by targeting the endogenous virus sequences (ORF1, ORF2, and ORF3), thereby disrupting proper
transcription and translation of functional viral proteins [71]. By exploiting the inactive Cas9 nuclease domain (dCas9),
the CRISPR/Cas9 technology offers a promising strategy for combating viral infections in plants. In the case of cotton
leaf curl virus (CLCuV) in tobacco, researchers have demonstrated the effectiveness of utilising dCas9 to suppress virus
replication [72]. This approach involves targeting the promoter region of the AC1 gene, crucial for virus replication,
with site-specific DNA-binding proteins to inhibit the function of the Rep protein, essential for viral replication [73].
CRISPR/dCas9 can serve as a DNA-binding protein to effectively block the promoter region of target genes, thereby
impeding virus replication and reducing viral infection rates [72]. By harnessing dCas9/sgRNA complexes, along
with transcription effector complexes, researchers induced transcriptional interference at the promoter regions of
downstream target genes. This interference disrupts RNA polymerase binding or elongation, further inhibiting virus
replication and minimizing the spread of viral infection within plant tissues [74]. Overall, these findings highlight

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the potential of CRISPR/Cas9-mediated transcriptional interference as a powerful tool for developing viral-resistant
crops and enhancing plant defence mechanisms against viral pathogens.

3.2 CRISPR/Cas9 targeted gene editing for fungal disease resistance

CRISPR/Cas9 technology offers a promising approach to developing fungal-resistant plants by targeting genes associated
with susceptibility to fungal diseases. Several past studies have showcased the potential of CRISPR/Cas9 in engineering
fungal-resistant crops by targeting key genes involved in plant-fungal interactions. For instance, knockout of the mildew-
resistance locus (MLO) gene using CRISPR/Cas9 has led to resistance against powdery mildew in various crops such as
grapevine, tomato, bread wheat, cucumber, and soybean [49, 75–78]. The MLO locus encodes proteins that render plants
susceptible to powdery mildew fungi, and loss-of-function mutations result in enhanced resistance to this disease [79].
Similarly, resistance to downy mildew disease can be achieved by knocking out the homoserine kinase (HSK) gene,
which plays a crucial role in conditioning susceptibility to downy mildew in sweet basil [80]. Additionally, knocking out
pathogenesis-related proteins (PR) has been shown to increase susceptibility to downy mildew in grapevine, accompa-
nied by reduced accumulation of reactive oxygen species around the stomata [81]. These findings underscore the poten-
tial of CRISPR/Cas9 in engineering fungal-resistant crops by targeting key genes involved in plant-fungal interactions.
The CRISPR/Cas9 technology has also demonstrated efficacy in conferring resistance to rice blast disease by targeting
specific genes involved in the plant’s defence mechanisms. For instance, mutations induced in the ethylene responsive
factors (ERF) gene using CRISPR have been shown to enhance rice blast resistance [82]. ERF serves as a negative regula-
tor of blast resistance in rice, and the induced frameshift mutations in this gene contribute to increased resistance to
rice blast pathogens.
Knocking out susceptible genes in plants has also been proven in the cacao plant (Theobroma cacao) by repressing
the expression of the TcNPR3 which had been previously shown to repress pathogen defence responses [83]. Fister et al.
has successfully express CRISPR/Cas9 machinery targeting TcNPR3 in cacao plants and enhance their resistance against
Phytophthora tropicalis, a bane in cacao farming [84]. Furthermore, disruption of the subunit of the exocyst complex (SEC3A)
using CRISPR/Cas9 has also been linked to enhanced resistance against rice blast disease [85]. This disruption leads to
an increase in salicylic acid synthesis, a key signalling molecule involved in plant defence responses against pathogens.
The elevated levels of salicylic acid contribute to bolstering the plant’s immune response, thereby improving its ability
to withstand rice blast infections.

3.3 Enhancing plant resistance to bacterial infections using CRISPR/Cas9

Development of plants with resistance against bacterial infections has also been done using the CRISPR/Cas9 technol-
ogy. For instance, modifying the promoter of the lateral organ border (LOB) gene can increase resistance to citrus canker
in grapefruit and oranges [86, 87]. The primary transcription activator-like (TAL) effector of Xanthomonas citri subsp. citri
(Xcc), known as PthA4, binds specifically to the effector binding element, EBEPthA4, in the LOB promoter. This binding
activates gene expression, accelerating the growth of citrus canker. Mutations in EBEPthA4 can reduce or even eliminate
the PthA4-inducible activity of the LOB promoter, thereby enhancing resistance [88].
Similarly, the TAL effector of Xanthomonas oryzae pv. oryzae, known as PthXo1, binds to the effector binding ele-
ment, EBEPthXo1, in the promoter region of Os8N3, a member of the SWEET family of sugar transporters. This interaction
promotes bacterial blight disease in tomatoes. By using CRISPR/Cas9 to disrupt the SWEET gene, plants can achieve
increased tolerance to bacterial blight [89]. Another example of knockouts of susceptible genes has been demonstrated
in a study by Pompili et al., in which the authors knocked out the gene encoding the MdDIPM4 protein in apple plants
(Malus domesticus) [90]. The MdDIPM4 protein plays a crucial role in the infection mechanism of Erwinia amylovora which
causes fire blight infection in apple plants. This technique highlights the potential of CRISPR/Cas9 to target specific
genes and regulatory elements, thereby providing an effective strategy to enhance plant resistance against bacterial
pathogens. The ability to precisely edit genes associated with susceptibility allows for the development of crops that are
better equipped to withstand bacterial infections, contributing to improved agricultural productivity and sustainability.
Furthermore, CRISPR/Cas9 can enhance resistance to bacterial speck in tomatoes by editing the jasmonate zim domain
(JAZ) repressor proteins [91]. The JAZ protein is essential for coronatine to stimulate stomatal opening, which facilitates
bacterial leaf colonization. Editing the JAZ gene using CRISPR/Cas9 prevents stomatal reopening, thereby hindering
bacterial colonization [92]. Furthermore, CRISPR/Cas9 can target the downy mildew resistance (DMR) gene to control
banana xanthomonas wilt disease (BXW). The DMR gene is a susceptibility gene that is upregulated during pathogen

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infection, and its modification can provide broad-spectrum resistance to bacterial pathogens [93]. Kim et al. reported
the mutation of the U-box type E3 ubiquitin ligase (PUB) gene showed resistance to bacterial leaf blight in rice [94]. The
Pi21 gene and the effector-binding element of the OsSULTR3;6 gene mutagenesis exhibited resistance to bacterial leaf
streak in rice [95]. These approaches highlight the versatility of CRISPR/Cas9 in developing disease-resistant crops by
targeting genes involved in susceptibility and enhancing the plant’s natural defense mechanisms. By precisely editing
specific genes, CRISPR/Cas9 can significantly improve plant resilience against various bacterial infections, contributing
to sustainable agricultural practices and increased crop yields.

3.4 CRISPR/Cas9 for crop protection against insect pests

Two-thirds of insects are phytophagous, meaning they feed on living plant components, making them a significant threat
to agriculture. Insects represent the most diverse group of arthropods, highlighting the breadth of potential pests [96].
Their impact is profound, causing annual yield losses of 18–20% of the world’s crop production, amounting to over $470
billion in estimated losses [97]. Moreover, insects play a crucial role as vectors for transmitting various plant diseases,
including viruses, phytoplasmas, and bacteria, further exacerbating agricultural losses [7]. With the exacerbating effects
of climate change, there is a looming threat of increased insect pest outbreaks and their global spread. In new habitats,
invasive pest species often exhibit rapid proliferation, leading to heightened concerns about insect-borne plant diseases
[98]. Consequently, there’s a pressing need for genome editing to counteract insect pests. Insect genome editing presents
two viable approaches for genetic modification to enhance resistance to insect pests [99].
Genome editing techniques offer promising avenues for controlling insect pests by targeting crucial developmental
genes (Fig. 2). CRISPR/Cas9-mediated knockout of vitellogenin, a precursor for egg yolk essential for insect egg matura-
tion and embryonic development, has resulted in incomplete embryo development in Plutella xylostella [100]. Similarly,
knockout of the abdominal-A homeotic gene induces severe abdominal morphological defects in Plutella xylostella and
Spodoptera frugiperda [101, 102]. The abdominal-A (abd-A) gene, a member of the homeotic (Hox) gene family, plays a
pivotal role in segmental identity during embryogenesis by encoding transcription factors that modulate segment devel-
opment through interactions with downstream target genes [103]. Mutations in abdominal-A lead to various deleterious
phenotypes, including deformed segments, abnormal prolegs, abnormal gonads, and even embryonic lethality, with
abnormal gonads potentially contributing to male and female infertility [101].
Additionally, knockout of pheromone binding proteins (PBPs) in Spodoptera litura disrupts female sex pheromone
perception, thereby preventing mating attempts [24]. Similarly, knockout of the intersex (ix) gene in Bombyx mori results
in infertility and irregular external genitalia, accompanied by developmental defects in imaginal discs, affecting wings,
antennae, and legs [25]. These findings underscore the potential of CRISPR/Cas9 technology to precisely target key genes
involved in insect development, offering a promising strategy for controlling insect pests in agriculture.
On the side of the plants, insect-resistant plants can be achieved by amplifying the generation of salicylic acid to repel
insects [104]. In rice, the cytochrome P450 gene CYP71A1 encodes tryptamine 5-hydroxylase, which catalyses the conver-
sion of tryptamine to serotonin [105]. By using CRISPR/Cas9 to inactivate the CYP71A1 gene, serotonin production can
be halted, resulting in higher levels of salicylic acid. This genetic modification makes the engineered plant more resistant
against insect pests such as planthoppers and stem borers [50]. In addition, salicylic acid also acts as an immune signal,

Fig. 2  The CRISPR/Cas9 sys-

tems approach for develop-
ing resistant crops to control
diseases, insect pests, and
weeds

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and its increase often coincides with elevated expression of antimicrobial pathogenesis-related (PR) genes, leading to
enhanced disease resistance [106]. By targeting specific genes and pathways, scientists can develop crops with improved
resistance to pests, contributing to sustainable agricultural practices and reducing the reliance on chemical pesticides.
This strategy not only helps in protecting crops from damage but also supports environmental conservation efforts by
minimizing pesticide use.
Gene drives are utilized by introducing an effector gene that causes mortality or sex bias that leads to a suppressed
population in each generation. Besides, this strategy can also be used to modify the population by spreading a genetic
variant that removes a harmful trait but sustains the population of the target [107–109]. Several selfish genetic elements
have been adopted for gene drive systems, including meiotic drives such as transposons and maternal effect dominant
embryonic arrest (Medea), which is a region of nuclear DNA found only in some flour beetles [110, 111]. Moreover, syn-
thetic systems such as artificial Medea systems, engineered under dominance or homing endonuclease genes (HEGs)
have been proposed to drive genes into a population [112]. Asad et al. developed a single CRISPR/Cas9-mediated gene-
drive construct for Plutella xylostella, a highly destructive lepidopteran pest of cruciferous crops [113]. The gene drive
constructed contains a Cas9 gene, a gRNA sequence, and a marker gene (EGFP), with their promoters to the target sites.
This study resulted in gene-drive efficiency due to HDR being 6.67–12.59% and resistant-allele formation due to NHEJ
being 80.93–86.77%.

3.5 CRISPR/Cas9 for crop protection to control weeds

Weeds are among the most significant biotic limitations on agricultural output, posing great potential yield losses to
crops alongside viruses and pests [11]. Weeds compete with crops for essential resources such as space, sunlight, water,
and nutrients, significantly reducing crop productivity. Furthermore, they serve as hosts for various insects and viruses
that can harm crop plants, exacerbating the damage. Beyond agricultural impacts, weeds also devastate native habitats,
threatening local flora and fauna and disrupting ecosystems [114].
Addressing these challenges is critical for ensuring food security and maintaining biodiversity. One promising solu-
tion is the creation of herbicide-resistant plants through genome editing. This approach allows engineered plants to
withstand herbicide and thus its application can selectively eliminate unwanted weeds. The CRISPR/Cas9 technology
offers precise and efficient methods for developing such herbicide-resistant crops, contributing to sustainable agricul-
tural practices and better parasite management. In addition, through plant genome editing, it is possible to engineer
crops that not only resist herbicides but also adapt to the dynamic and evolving challenges posed by weed species. The
herbicide glyphosate inhibits the biosynthesis of the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) enzyme in
plants, disrupting the shikimate pathway and leading to plant death upon exposure to glyphosate [115]. Glyphosate
interferes with the production of essential amino acids by binding to the EPSPS enzyme, which is crucial for plant growth
and survival. To combat this, genetic engineering can be employed to create glyphosate-resistant plants.
A more conventional approach is transferring the EPSPS gene from Agrobacterium strain CP4 to crops such as potatoes.
The bacterial EPSPS enzyme from Agrobacterium has a small modification that prevents glyphosate binding, thereby
allowing the plant to continue producing amino acids even in the presence of glyphosate [116]. This modification ensures
that while the plant’s native EPSPS is inhibited by glyphosate, the bacterial EPSPS remains functional, conferring resist-
ance to the herbicide. A more modern approach is to use the CRISPR/Cas9 technology to achieve constitutive expression
of the EPSPS gene and thus heightened glyphosate resistance. Hummel et al. succeeded in replacing the endogenous
EPSPS promoter and the first two exons with a strong constitutive promoter, resulting in glyphosate tolerance in cassava
[117]. Li et al. developed gene replacement and insertion strategies targeting general introns via the NHEJ pathway using
CRISPR/Cas9 [118]. This strategy was successfully employed to introduce the targeting induced local lesions in genomes
(TILLING) amino acid substitution to the rice EPSPS gene, conferring resistance to glyphosate in rice.
The base editing technique mediated by CRISPR/Cas9 in the acetolactate synthase (ALS) gene has been used to gener-
ate herbicide resistance in rice, soybean, and watermelon [119–121]. In plants, ALS is an essential enzyme for the produc-
tion of the branched-chain amino acids valine, leucine, and isoleucine. Single point mutations at multiple conserved
locations of ALS genes can inhibit the interaction between the ALS protein and herbicides, offering a significant level of
herbicide resistance across various plant species [122, 123]. The CRISPR/Cas9 technique has proven viable and successful
in precise gene replacement. For instance, two specific amino acid residues in the ALS gene were precisely replaced to
develop herbicide-resistant rice plants with homozygous resistance [124]. This precise gene editing approach provides a
robust method for developing herbicide-resistant crops, offering an efficient alternative to traditional genetic modifica-
tion techniques. By targeting specific genes and inducing precise mutations, CRISPR/Cas9 facilitates the development of

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crops that can withstand herbicide application, ensuring higher yields and more sustainable agricultural practices. The
success of CRISPR/Cas9 in editing the ALS gene underscores its potential as a powerful tool in crop improvement and
herbicide resistance management. Here is a comprehensive list (Table 1) detailing the various applications of CRISPR/
Cas9 technology in crop protection, specifically aimed at managing plant diseases, pests, and weeds.
These methods illustrate how CRISPR/Cas9 can be used to create herbicide-resistant crops by precisely modifying
specific genes or regulatory elements. This approach not only offers an alternative to traditional genetic engineering
methods but also provides a more targeted and efficient way to enhance crop resilience against herbicides. Over the
last eight decades, the constant use of herbicides has resulted in the widespread evolution of herbicide resistance in
numerous weed species. Currently, there are 269 weed biotypes (154 dicots and 115 monocots) have been reported
to be resistant to 21 of the 31 known herbicide sites of action to date [125]. Therefore, the CRISPR/Cas9 gene drive has
come as a novel genetic control strategy in agricultural weed management. There are several benefits of the utilization
of gene drive in weed control, such as the specific elimination of invasive species. Its precise manner of gene drive could
prevent harmful effects on non-target organisms and closely related species [126]. As an alternative to herbicides, gene
drive has no safety concerns of exposure to hazardous chemicals that can lead to a lack of disturbances to the soil or
environment [127]. Added advantages of the use of gene drive is reduced cost for invasive plant management and would
require minimal human intervention [126].
Regulations on genome editing crops vary significantly across countries, reflecting different public perception and
agricultural policies. In the U.S., the regulation of genome-edited crops is primarily overseen by three agencies: the U.S.
Department of Agriculture (USDA), the Environmental Protection Agency (EPA), and the Food and Drug Administration
(FDA) [133]. The USDA’s 2020 SECURE (Sustainable, Ecological, Consistent, Uniform, Responsible, Efficient) rule clarified
that certain genome-edited crops, such as those with small deletions or edits that could be achieved by conventional
breeding, are not subject to the same regulations as traditional GMOs [134]. The FDA typically requires a safety assess-
ment if the genome-edited crop introduces a new protein or has the potential to change the crop’s nutritional content
[135]. The European Union (EU) has a more stringent approach to regulating genome-edited crops [136]. Genome-edited
organisms are generally regulated under the same framework as genetically modified organisms (GMOs), following the
precautionary principle. Regulation is mainly governed by Directive 2001/18/EC, which requires a thorough risk assess-
ment, labelling, and traceability for GMOs [137]. China regulates genome-edited crops through the Ministry of Agriculture
and Rural Affairs (MARA) [138]. The regulatory system is evolving as China invests heavily in biotechnology research.
Canada regulates genome-edited crops based on the "novelty" of the trait rather than the technique used to develop
it [139]. The Canadian Food Inspection Agency (CFIA) and Health Canada are the primary regulatory bodies [140]. CFIA
oversees environmental safety, while Health Canada evaluates food safety. Japan’s regulatory approach is somewhat
similar to that of Canada, focusing on the novelty of the product rather than the technique. The Ministry of Agriculture,
Forestry and Fisheries (MAFF) and the Ministry of Health, Labour and Welfare (MHLW) are the primary bodies overseeing
genome-edited crops [141].

4 Conclusion and future perspectives

As we look to the future, the potential of CRISPR/Cas9 technology in crop protection is both vast and promising. The
increasing global population and the impacts of climate change will continue to exert pressure on agricultural systems,
necessitating innovative approaches to enhance crop resilience and productivity. CRISPR/Cas9 stands at the forefront of
these innovations, offering unprecedented precision and efficiency in genome editing. One of the key future perspectives
is the integration of CRISPR/Cas9 with other emerging technologies, such as synthetic biology and bioinformatics, to
create multi-faceted solutions for crop protection. By combining CRISPR/Cas9 with advanced data analysis and model-
ling techniques, researchers can better predict the outcomes of genetic modifications and optimize editing strategies
for maximum effectiveness. This integrative approach will enable the development of crops that are not only resistant to
diseases, pests, and weeds but also tailored to thrive in specific environmental conditions. Additionally, there is significant
potential for CRISPR/Cas9 to be used in developing crops that can adapt to abiotic stresses such as drought, salinity, and
extreme temperatures. Shi et al. utilized CRISPR/Cas9-enabled advanced breeding technology to create new variants
of ARGOS8 [142]. In their earlier research, they demonstrated that transgenic plants with constitutive overexpression of
ARGOS8 exhibit reduced sensitivity to ethylene and enhanced grain yield under drought stress conditions. Zhang et al.
reported enhancing rice salinity tolerance by engineering a vector expressing Cas9 and OsRR22-gRNA, which targets the

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 Table 1  A comprehensive list of CRISPR/Cas9 applications in the field of crop protection to address plant diseases, pests, and weeds
Species Transformation method Applications of CRISPR/Cas9 Target New traits Detection method Refs.

Resistance against viruses

Arabidopsis (A. thaliana) A. tumefaciens Knock out gene Eukaryotic translation initia-
Resistant to clover yellow GFP, PCR, sequencing [62]
tion factor 4E (eIF4E) vein virus (ClYVV) in
Arabidopsis
Discover Agriculture

Banana (Musa spp.) A. tumefaciens Knock out gene Endogenous banana streak Resistant to banana streak PCR, sequencing [71]
virus (eBSV) virus (BSV)
Barley (Hordeum sponta- A. tumefaciens Knock out gene Protein disulfide isomerase Resistance to barley mild PCR, sequencing [128]
neum) like 5–1 (PDIL5-1) mosaic virus (BaMMV)
Cassava (Manihot escu- A. tumefaciens Knock out gene AC2 and AC3 genes of ACM Resistant to African cassava PCR, sequencing [69]
lenta) genome mosaic virus (ACMV)
(2024) 2:104

Chinese cabbage (Brassica A. tumefaciens Insertion and deletion gene Eukaryotic translation initia- Resistance to Turnip Mosaic PCR, sequencing [64]
rapa) tion factor 4E (eIF4E) Virus (TuMV)
Cucumber (Cucumis A. tumefaciens Knock-out gene Eukaryotic translation initia- Resistant to cucumber vein PCR, sequencing [63]
sativus) tion factor 4E (eIF4E) yellowing virus (CVYV)
Red Chili (Capsicum A. tumefaciens, in planta Knock-out gene Proliferating cell nuclear Resistant to pepper yellow PCR, sequencing [129]
annuum) antigen (PCNA) leaf curl virus (PepYLCV)
Rice (Oryza sativa) A. tumefaciens Knock-out gene Translation initiation factor 4 Resistant to rice tungro PCR, sequencing [48]
gamma gene (eIF4G) spherical virus (RTSV)
Tobacco (Nicotiana A. tumefaciens Optimising gene regulation Promoter region of the AC1 Resistant to cotton leaf curl PCR, sequencing [72]
benthamiana) (dCas9) gene virus (CLCuV)
Tobacco (N. benthamiana) A. tumefaciens Knock out gene Tobamovirus multiplication Resistance to Tobacco PCR, sequencing [70]
1 (TOM1) mosaic virus (TMV)
Tobacco (N. benthamiana) A. tumefaciens Knock out gene Coat protein of CLCuMuV Resistant to leaf curl multan PCR, sequencing [67]
genome virus (CLCuMuV)
Tomato (Solanum lycoper- A. tumefaciens Knock out gene Coat protein of TYLCV Resistant to yellow leaf curl PCR, sequencing, T7EI [65]
sicum) genome virus (TYLCV)
Tomato (S.lycopersicum) A. tumefaciens Knock-out gene Eukaryotic translation initia- Resistant to pepper mottle PCR, sequencing [61]
tion factor 4E (eIF4E) virus (PepMoV)
Resistance against fungi
Bread wheat (Triticum Protoplast transformation, Knock out gene Mildew-resistance locus Resistant to powdery GFP, PCR, sequencing [49]
| https://doi.org/10.1007/s44279-024-00124-0

aestivum) particle bombardment (MLO) mildew Blumeria graminis

f. sp. tritici
Cucumber (Cucumis A. tumefaciens Insertion and deletion gene Mildew locus O 8 (MLO8) Enhanced resistance to PCR [77]
sativus) powdery mildew resist-
ance
Grapevine (Vitis vinifera) A. tumefaciens Knock out gene Pathogenesis-related pro- Resistant to powdery mil- PCR, sequencing [81]
teins (PR) dew Plasmopara viticola
Grapevine (V. vinifera) A. tumefaciens Knock out gene Mildew-resistance locus Resistant to powdery mil- PCR, sequencing [75]
(MLO) dew Erysiphe necator

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 Table 1  (continued)
Species Transformation method Applications of CRISPR/Cas9 Target New traits Detection method Refs.
Review

Maize (Zea mays) A. tumefaciens Knock out gene ZmJAZ15 Resistance to Gibberella PCR [130]
stalk rot (Fusarium

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graminearum)
Maize (Z. mays) A. tumefaciens Knock out gene ZmCOla Immunity to Gibberella stalk PCR [130]
rot (Fusarium gramine-
arum)
Oil palm (Elaeis guineensis) A. tumefaciens Knock out gene Early methionine-labeled Resistant to Ganoderma PCR, sequencing [131]
polypeptide (EMLP) boninense
Rice (Oryza sativa) A. tumefaciens, protoplast Knock out gene Ethylene responsive factors Resistant to rice blast Mag- PCR, sequencing [82]
transformation (ERF) naporthe oryzae
Rice (O. sativa) Protoplast transformation Knock out gene Subunit of the exocyst com- Resistant to rice blast Mag- GFP, PCR, Lipid bind- [85]
plex (SEC3A) naporthe oryzae ing assay
Soybean (Glycine max (L.) A. tumefaciens Knock out gene Mildew locus O (MLO) Enhanced resistance to PCR, sequencing [78]
Merrill) powdery mildew resist-
ance
Discover Agriculture

Sweet basil (Ocimum Biolistic bombardment Knock out gene Homoserine kinase (HSK) Resistant to rice blast Per- PCR, sequencing [80]
basilicum) onospora belbahrii
Tomato (S. lycopersicum) A. tumefaciens Knock out gene Mildew-resistance locus Resistant to powdery mil- PCR, sequencing [76]
(MLO) dew Oidium neolycopersici
Resistance against bacteria
Apple (Malus domestica) A. tumefaciens Knock out gene MdDIPM4 Resistance to fire blight PCR [90]
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disease
Apple (M. domestica) Protoplast transformation Knock out gene DIPM-1, DIPM-2, DIPM-4 Resistance against fire PCR, sequencing [132]
blight disease
Banana (Musa spp.) A. tumefaciens Knock out gene Downy mildew resistance Resistant to banana PCR, sequencing [93]
(DMR) xanthomonas wilt
disease (BXW) Xan-
thomonas campestris pv.
musacearum (Xcm)
Grapefruit (Citrus paradisi A. tumefaciens Knock out gene Lateral organ boundaries Resistant to citrus bacte- PCR, sequencing [86]
Macf.) (LOB) rial canker (CBC) Xan-
thomonas citri subsp. Citri
(Xcc)
Orange (Citrus sinensis A. tumefaciens Knock out gene Lateral organ boundaries Resistant to citrus bacte- PCR, sequencing [87]
Osbeck) (LOB) rial canker (CBC) Xan-
thomonas citri subsp. Citri
(Xcc)
Rice (O. sativa L) A. tumefaciens Knock out gene U-box type E3 ubiquitin ligase Resistance to bacterial leaf Sequencing [94]
(PUB) blight
Rice (O. sativa L) A. tumefaciens Knock out gene Pi21 and SULTR3;6 EBE Resistance to rice blast PCR [95]
(effector-binding element)
| https://doi.org/10.1007/s44279-024-00124-0
 Table 1  (continued)
Species Transformation method Applications of CRISPR/Cas9 Target New traits Detection method Refs.

Rice (O. sativa L) A. tumefaciens Knock out gene Pi21 and SULTR3;6 EBE Resistance to bacterial leaf PCR [95]
(effector-binding element) streak
Tomato (S. lycopersicum) A. tumefaciens Knock out gene Jasmonate zim domain (JAZ) Resistant to tomato bacte- PCR, sequencing [91]
repressor proteins rial speck disease Pseu-
Discover Agriculture

domonas syringae
Tomato (S. lycopersicum) A. tumefaciens Knock out gene Sugar will eventually be Resistant to bacterial blight PCR, sequencing [89]
exported transporters disease Xanthomonas
(SWEET) oryzae pv. oryzae (Xoo)
Resistance against insects
Rice (O. sativa) A. tumefaciens Knock out gene CYP71A1 encodes a Resistant to rice brown PCR, sequencing [50]
cytochrome P450 planthopper (BPH; Nilapa-
(2024) 2:104

monooxygenase rvata lugens Stål) and

striped stem borer (SSB,
Chilo suppressalis)
Common cutworm (Spo- Microinjection Knock out gene Binding proteins (PBPs) Female sex pheromone PCR, sequencing [24]
doptera litura) perception
Diamondback moth (Plu- Microinjection Knock out gene Vitellogenin (Vg) Incomplete embryonic PCR, sequencing [100]
tella xylostella) development
Diamondback moth (P. Microinjection Knock out gene Abdominal-A homeotic Disruption of gonad devel- PCR, sequencing [102]
xylostella) (abd-A) opment
Fall armyworm moth (Spo- Microinjection Knock out gene Abdominal-A homeotic Disruption of gonad devel- PCR, sequencing [101]
doptera frugiperda) (abd-A) opment
Silkworm (Bombyx mori) Microinjection Knock out gene Intersex (ix) Sterile strain or a flightless PCR, sequencing [25]
moth
Resistance against weeds
Cassava (M. esculenta) A. tumefaciens, protoplast Optimising gene regulation 5-enolpyruvylshikimate- Tolerance to herbicide PCR, sequencing [117]
transformation 3-phosphate synthase glyphosate
(EPSPS) promoters
Rice (O. sativa) Particle bombardment Knock in gene Acetolactate synthase (ALS) Tolerance to herbicide PCR, sequencing [124]
bispyribac sodium
| https://doi.org/10.1007/s44279-024-00124-0

Rice (O. sativa) Protoplast transformation Knock in gene 5-enolpyruvylshikimate- Tolerance to herbicide PCR, sequencing [118]
3-phosphate synthase glyphosate
(EPSPS) promoters
Rice (O. sativa) A. tumefaciens Knock out gene Acetolactate synthase (ALS) Tolerance to herbicide PCR, sequencing [119]
imazethapyr
Soybean (G. max) A. tumefaciens, in planta Knock out gene Acetolactate synthase (ALS) Tolerance to herbicide PCR, sequencing [120]
chlorsulfuron
Watermelon (Citrullus A. tumefaciens Knock out gene Acetolactate synthase (ALS) Tolerance to herbicide PCR, sequencing [121]
lanatus) tribenuron

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OsRR22 gene in rice [52]. Malzahn et al. compared the editing efficiencies and NHEJ repair profiles of AsCas12a, FnCas12a,
and LbCas12a across four different temperatures in rice [143].
Another promising area is the use of CRISPR/Cas9 for the enhancement of plant nutritional profiles and the reduc-
tion of allergens and antinutritional factors. By precisely editing genes involved in metabolic pathways, crops can be
engineered to produce higher levels of essential nutrients, vitamins, and antioxidants, contributing to improved human
health and nutrition. Some prominent examples include the study by Sun et al. in which CRISPR/Cas9 was used to regulate
the expression of MdMKK9 to increase anthocyanins in apple (Malus sp.) and one by Endo et al. where CRISPR/Cas9 was
used to modify the OsOr gene in rice which resulted in β-carotene hyper accumulation [144, 145]. These examples are still
in their nascent periods and are promising for further development. Editing genes associated with stress tolerance can
create crops that maintain high yields even under adverse conditions, thus contributing to global food security. Future
research should focus on identifying and validating target genes involved in abiotic stress responses and developing
robust editing protocols to enhance crop resilience.
Another promising area is the use of CRISPR/Cas9 for the enhancement of plant nutritional profiles and the reduc-
tion of allergens and antinutritional factors. By precisely editing genes involved in metabolic pathways, crops can be
engineered to produce higher levels of essential nutrients, vitamins, and antioxidants, contributing to improved human
health and nutrition. Furthermore, as regulatory frameworks evolve, the commercialization of CRISPR/Cas9-edited crops is
expected to accelerate. It is crucial for policymakers, scientists, and stakeholders to work together to establish guidelines
that ensure the safety and efficacy of genome-edited crops while addressing public concerns and ethical considera-
tions. In conclusion, the future of crop protection lies in the continued advancement and application of CRISPR/Cas9
technology. By leveraging its potential, we can develop crops that are better equipped to withstand biotic and abiotic
stresses, thereby ensuring sustainable agriculture and food security for future generations. The transformative impact
of genome editing in agriculture underscores the importance of sustained research, collaboration, and innovation in
this rapidly evolving field.

Author contributions Conceptualization and supervision were performed by A.F. and R.R.E. References collection was performed by S.N.,
A.A.S., Y.T. and T.K. Visualization was performed by S.N. and A.A.S. The first draft of the manuscript was written by A.F. and S.N., and all authors
commented on previous versions of the manuscript. All authors read and approved the final manuscript.

Funding No funding was received to assist with the preparation of this manuscript.

Data availability Not applicable.

Code availability Not applicable.

Declarations
Ethics approval and consent to participate Not applicable.

Informed Consent Not applicable.

Competing interests The authors have no relevant financial or non-financial interests to disclose.

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adapta-
tion, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source,
provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article
are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in
the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will
need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

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