Muhammad Kashif Zahoor et al. Science Reviews - Biology, 2024, 3(3), 22-40
22
Genome Editing in Insects: CRISPR Technology and
its Prospects
Muhammad Kashif Zahoor*, PhD, Aftab Ahmad
1
, PhD and Muhammad Zulhuss-
nain
2
, PhD
*
Dr. Muhammad Kashif Zahoor (Corresponding Author)
Department of Zoology, Government College University Faisalabad, Pakistan; kashif.zahoor@gcuf.edu.pk
https://orcid.org/0000-0003-0309-9758
1
Dr. Aftab Ahmad
Department of Biochemistry/US-Pakistan Center for Advance Studies in Agriculture and Food Security (USPCAS-AFS), University
of Agriculture Faisalabad, Pakistan
https://orcid.org/0000-0002-2792-9771
2
Dr. Muhammad Zulhussnain
Department of Zoology, Government College University Faisalabad, Pakistan
https://orcid.org/0009-0009-3107-118X
https://doi.org/10.57098/SciRevs.Biology.3.3.3
Received September 23, 2024. Revised October 06, 2024. Accepted October 07, 2024
Abstract: Insects, as one of the largest animal groups, play a crucial role due to their vast diversity, economic
significance in agriculture and cottage industries, and their ecological functions as pollinators and vectors of
various diseases. Significant advancements in genetics have provided extensive information on gene identity
and sequences for many insect species. These genetic resources have facilitated genome editing studies aimed
at developing improved genetic traits. One such strategy is the Sterile Insect Technique (SIT), which has been
effectively employed against the screwworm in North America and continues to be used for managing insect
pests. Gene silencing via RNA interference (RNAi), a fundamental genomic tool in model insect research, has
also been applied in various biological studies. However, its variable efficiency among insect pests has limited
its widespread use. Other gene-editing approaches include the induction of Double-Strand Breaks (DSBs) in
DNA using Zinc-Finger Nucleases (ZFNs) and Transcription Activator-Like Effector Nucleases (TALENs),
which stimulate non-homologous end joining or homology-directed repair at targeted sequences. More recently,
the CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated protein 9)
system has rapidly emerged as a transformative genome-editing approach across multiple fields, including
agriculture, insect resistance management, environmental safety, human health, and industry. This article
provides an overview of various genome-editing techniques employed in insects, with a specific focus on the
application and future potential of the cutting-edge CRISPR/Cas system, which holds promise in surpassing
other genome-editing approaches.
Keywords: Genome editing, Sterile Insect Technique (SIT), Ribonucleic acid interference (RNAi), Zinc-Finger
Nucleases (ZFNs), Transcription Activator-Like Effector Nucleases (TALENs), Clustered Regularly Interspaced
Short Palindromic Repeats (CRISPR).
Background: Genome Editing in Insects
Recent advancements in insect genetics have
provided a wealth of genetic tools that can be em-
ployed for genome editing to tackle agricultural insect
pests, control vectors affecting human health, advance
biological or medical research, and address environ-
mental challenges. The introduction of engineered
traits into wild populations of insect pests could ad-
dress resistance development and issues with invasive
species. Furthermore, genetic control strategies target-
ing vector-borne diseases have made gene editing a
significant focus of current research (Xu et al., 2018).
For example, suppressing sex-determination pathway
genes that hinder vector competence, inducing lethal
recessive mutations (mutations that only manifest
Science Reviews - Biology, 2024, 3(3), 22-40 Muhammad Kashif Zahoor et al.
23
when two recessive alleles are present), or producing a
biased sex ratio in targeted insect populations could re-
duce the burden of vector-borne diseases (Ranian et al.,
2022; Zulhussnain et al., 2023).
The Sterile Insect Technique (SIT) is one of the
earliest and most successful genetic control strategies
used against insect pests. A major achievement in in-
sect pest management was the eradication of Cochli-
omyia hominivorax (screwworm) from North and Cen-
tral America. This pest endangered livestock, wildlife,
and human health, resulting in billions of dollars in
losses annually. These losses included reduced exports
of cattle and sheep hides, decreased meat and milk
production, and wastage of human resources (Scott et
al., 2017). The SIT approach involves mass-rearing tar-
get insect species, sterilizing them through radiation,
and releasing them to mate with wild-type popula-
tions. Sterile males produce no offspring, and a typical
release ratio of 1:10 sterile to normal males significantly
reduces the chances of normal males mating (Franz et
al., 2021). However, the need for mass-rearing and fre-
quent releases of sterile males remains a limiting factor
in the widespread use of SIT (Schliekelman et al., 2005).
Several genetic manipulation techniques have
since been developed in insects. For example, gene
function characterization, economic trait improvement,
and the production of recombinant proteins in silk-
worms have been achieved through various genome
editing methods such as RNAi, Zinc-Finger Nucleases
(ZFNs), Transcription Activator-Like Effector Nucle-
ases (TALENs), and the CRISPR/Cas9 system (Isobe et
al., 2004; Takasu et al., 2010; Xu et al., 2018; Chen et al.,
2023b). In the 1990s, RNAi was first introduced in Cae-
norhabditis elegans to silence endogenous mRNA
through the introduction of exogenous double-
stranded RNA (Fire et al., 1998; Belles, 2010; Terenius
et al., 2011).
RNAi has since been used to analyze gene
function, typically by injecting double-stranded
RNA (Jiang et al., 2021). However, RNAi-mediated
gene silencing in silkworms often shows low effi-
ciency, particularly for certain genes such as the
bilin-binding protein and pheromone-binding pro-
tein, which are expressed in larval epidermis and
pupal wing discs (Kobayashi et al., 2012). Similarly,
RNAi systems have exhibited low efficiency and
non-specificity in other Lepidoptera due to un-
known mechanisms (Daimon et al., 2014; Kol-
liopoulou and Swevers, 2014). By contrast, CRISPR-
based genome editing has demonstrated high and
stable knockdown efficiency in silkworms. For in-
stance, CRISPR/Cas13-mediated knockdown of the
homeobox gene Scr resulted in stunted growth, ab-
normal sex comb and salivary gland development
in larvae, and malformed head and pre-thoracic
segments in adults. This suggests that CRISPR-
RNAi editing systems may offer a more effective al-
ternative to RNAi in both silkworms and other Lep-
idopteran species (Huynh et al., 2020; Mahas et al.,
2019). Since the widespread use of RNAi in Drosoph-
ila in the 1990s, the CRISPR/Cas9 technique has
rapidly emerged over the past decade and is now
being successfully employed in various insects (Fig.
1).
Figure 1: Comparison of number of publications of RNAi and CRISPR
Muhammad Kashif Zahoor et al. Science Reviews - Biology, 2024, 3(3), 22-40
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Mosquitoes are well-documented vectors of
numerous microorganisms responsible for diseases
such as dengue, malaria, Zika, filariasis, and
chikungunya (Zahoor et al., 2019; Reegan et al.,
2016). For decades, synthetic pesticides have been
the primary method used to control insect pests and
vector mosquitoes worldwide. However, the exten-
sive use of these pesticides has led to significant en-
vironmental damage. Their indiscriminate applica-
tion affects non-target organisms, including hu-
mans, and has contributed to the development of
pesticide resistance in many species (Bayen, 2012).
This highlights the urgent need for safer, more en-
vironmentally friendly control strategies. In this
context, genetic control methods are increasingly
seen as a more sustainable alternative.
In addition to the successful use of the Sterile
Insect Technique (SIT) in agricultural fields, which
has shown significant results in Northern America,
RNA interference (RNAi) has also garnered sub-
stantial scientific attention. With further advance-
ments in genetic tools, techniques such as Tran-
scription Activator-Like Effector Nucleases
(TALENs) and Zinc-Finger Nucleases (ZFNs) have
been introduced and successfully used to target
genes of interest in various insect species, including
crickets and mosquitoes (Watanabe et al., 2016;
Awata et al., 2015; Aryan et al., 2013; Smidler et al.,
2013; Watanabe et al., 2012). ZFN and TALENs have
been employed to produce gene knockouts in hem-
imetabolous insects such as Gryllus bimaculatus.
Site-specific mutations have been created using Sur-
veyor (Cel-I) nuclease through microinjection of
ZFNs and TALENs to generate homozygous knock-
out crickets. TALENs are artificial nucleases that in-
duce double-strand breaks (DSB) at specific DNA
loci, and this knockout strategy has been suggested
for use in non-transgenic insect control (Watanabe
et al., 2012). TALENs have also been used for knock-
in genome editing in Gryllus bimaculatus (Watanabe
et al., 2016). Similarly, the silkworm (Bombyx mori)
has been widely employed for gene function char-
acterization, improving economically important
traits, and producing recombinant proteins using
genome editing techniques such as RNAi, ZFNs,
TALENs, and CRISPR-Cas9 (Chen et al., 2023).
The CRISPR-Cas system has emerged as a
powerful tool for genetic manipulation, allowing
precise edits to specific DNA sequences. This tool
has been instrumental in exploring biological
functions, dissecting signaling pathways, generat-
ing mutants for biological research, preventing dis-
ease, studying ecological interactions, and control-
ling agricultural pests. The CRISPR-Cas9 system,
where the Cas9 protein is guided by RNA to target
specific DNA sequences, is widely used for genome
editing in various insects. These include flies such
as fruit flies, mosquitoes (e.g., Anopheles, Culex, and
Aedes species), bees (e.g., honeybees and bumble-
bees), beetles (e.g., lantern beetles and stored grain
beetles), butterflies, moths, silkworms, crickets, and
grasshoppers. The application of CRISPR-Cas9 has
revolutionized functional genomics in insects, ad-
vancing research in pest control and resistance
management (Rosli et al., 2024; Zulhussnain et al.,
2023; Ranian et al., 2022; Zahoor et al., 2021; Martin
et al., 2020; Tong et al., 2018; Taning et al., 2017;
Chen et al., 2016; Reid and O’Brochta, 2016; Ma et
al., 2017).
CLUSTERED REGULARLY INTERSPACED
SHORT PALINDROMIC REPEATS (CRISPR)
Over the past decade, the clustered regularly
interspaced short palindromic repeats (CRISPR)
gene-editing technique has emerged as a highly
successful genetic tool for inducing mutations and
creating genetically edited insects. CRISPR has be-
come a key technique employed across diverse
fields, including biological sciences, agriculture, en-
vironmental conservation, health sciences, and in-
dustry (Rosli et al., 2024; Cannon and Kiem, 2021;
Knott and Doudna, 2018; Hsu et al., 2014).
Initially discovered in bacteria and archaea,
CRISPR functions as a defense mechanism, provid-
ing adaptive immunity against invading phages
and foreign nucleic acids. This system consists of
two main components: a CRISPR-associated (Cas)
nuclease, which cleaves the target DNA sequence
and generates precise double-stranded breaks
(DSBs), and a single guide RNA (sgRNA), which di-
rects the nuclease to the target DNA site
(Wiedenheft et al., 2012). The sgRNA is formed by
combining two RNA moleculesCRISPR RNA
(crRNA) and trans-activating crRNA (tracrRNA)
that are expressed separately. In bacterial cells, the
Cas proteins process these RNA molecules to pro-
duce mature guide RNA (gRNA), which then forms
a complex with Cas9 to recognize and cleave DNA
sequences near a proto-spacer adjacent motif
Science Reviews - Biology, 2024, 3(3), 22-40 Muhammad Kashif Zahoor et al.
25
(PAM). This cleavage results in DSBs at the specific
target site, which are subsequently repaired via two
primary pathways: non-homologous end-joining
(NHEJ) or homology-directed repair (HDR) (Jinek
et al., 2012; Hsu et al., 2014; Sorek et al., 2013; Shen
et al., 2017; Wang and Doudna, 2023; Wang et al.,
2022; Sander and Joung, 2014; Salsman and Dellaire,
2017).
The NHEJ pathway typically introduces ran-
dom insertions or deletions (InDels) at the DSB site,
often leading to gene knockouts. These InDels dis-
rupt the reading frame of the target gene, resulting
in truncated or non-functional proteins. In contrast,
HDR allows for precise gene editing by introducing
specific mutations, resulting in gene knock-ins.
Both NHEJ and HDR pathways are widely utilized
to generate mutants, contributing to advancements
in functional genomics research across numerous
fields (Sun et al., 2017).
Moreover, the availability of genetic tools has
expanded considerably, thanks in part to the
Addgene repository, which offers a large number of
gRNA and Cas9 plasmid constructs that facilitate
gene editing in a wide range of insect species
(https://www.addgene.org/) (Rosli et al., 2024; Za-
hoor et al., 2021; Wang et al., 2022; Ranian et al.,
2022; Wang and Doudna, 2023; Zulhussnain et al.,
2023).
Figure 2: Mechanism of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-based genome editing (Source:
www.addgene.org/crispr/guide/).
The development of comprehensive genome
databases has significantly advanced the field of ge-
netic research, with vast amounts of data now avail-
able through resources such as NCBI BioProjects,
i5K, InsectBase, and IAS1000. Additionally, tran-
scriptome data for many insect species can be ac-
cessed, which is essential for accelerating research
in genetics and genome editing. This wealth of in-
formation has enabled researchers to delve into var-
ious areas such as functional genetics, genetic
screening, and the identification of genes involved
in reproduction, sex determination, and insecticide
resistance. Furthermore, these resources support
studies on critical signaling pathways that regulate
metabolic functions, the genetic mechanisms be-
hind gene drives, and gene silencing techniques
used for genetic control (Qian and Wan, 2018; Li et
al., 2019).
The availability of these databases has been a
boon for the scientific community, providing novel
molecular tools to enhance gene editing research.
Researchers can now efficiently explore and manip-
ulate genes, leading to innovations in the manage-
ment of insect resistance and the broader under-
standing of biological systems.
Muhammad Kashif Zahoor et al. Science Reviews - Biology, 2024, 3(3), 22-40
26
APPLICATION OF CRISPR/CAS IN INSECTS
The CRISPR system is regarded as one of the
most advanced and powerful genome editing tools
available today. It has been successfully applied to
approximately 40 insect species across seven differ-
ent insect orders, contributing significantly to a
broad range of biological research areas (Chen et al.,
2024). The versatility and precision of the CRISPR
system make it an essential tool in various fields, in-
cluding genetics, agriculture, and pest management.
Below, the diverse applications of CRISPR are dis-
cussed in different categories (Fig. 3).
1. AGRICULTURE AND ENVIRONMENT
With the increasing global population, the de-
mand for food continues to rise. However, agricul-
tural production is severely impacted by both biotic
and abiotic factors. It is estimated that insect pests
alone contribute to approximately 20% of annual
crop yield loss worldwide. This has led to the indis-
criminate use of pesticides, which poses significant
risks to human health and severely degrades the en-
vironment. Pesticide resistance is becoming a criti-
cal threat to agricultural productivity, further com-
plicating the control of vector-borne diseases.
Recent research has focused on the potential
of gene editing, particularly using CRISPR/Cas9
technology, to address insecticide resistance. Nota-
bly, studies on Drosophila melanogaster have pro-
vided key insights. The role of ATP-binding cassette
(ABC) transporters in pesticide resistance has been
identified, highlighting their significance in the de-
velopment of resistance mechanisms (Douris et al.,
2020). Both RNAi and CRISPR/Cas9 have made
substantial contributions to our understanding of
pesticide toxicity and resistance in insects, offering
promising avenues for the development of more
sustainable pest control strategies (Amezian et al.,
2024).
Figure 3: A brief summary of applications of CRISPR
RNAi targeting the BtACTB gene was in-
duced through the expression of double-stranded
RNA (dsRNA) in the whitefly, Bemisia tabaci, lead-
ing to the development of both nuclear transgenic
and transplastomic tobacco plants. The nuclear
transgenic plants were found to be more effective in
controlling the whitefly Bemisia tabaci (Rosli et al.,
2024; Dong et al., 2020), a pest that also affects major
crops like cotton. In addition, a plastid-mediated
RNAi (PM-RNAi) approach has been reported to
genetically control Bemisia tabaci (Li et al., 2023),
highlighting the potential for genetic control
strategies to help eradicate other sap-sucking insect
pests in the future (Dong et al., 2020).
The cutworm Spodoptera litura, a polyphagous
and highly destructive pest of key crops such as cot-
ton, fruit trees, tobacco, pulses, potatoes, sweet po-
tatoes, and various vegetables, poses a significant
agricultural threat (Chandel et al., 2022). CRISPR-
Cas9 technology has been applied to Spodoptera
litura, where the Slabd-A gene was targeted, result-
ing in abnormal body segmentation and pigmenta-
tion (Bi et al., 2016). Similarly, the Orco gene, which
regulates sex pheromone production and
Science Reviews - Biology, 2024, 3(3), 22-40 Muhammad Kashif Zahoor et al.
27
interaction with plant odors in Spodoptera littoralis,
was targeted using CRISPR-Cas9 to control the pest
(Koutroumpa et al., 2022; Sun et al., 2023).
Furthermore, CRISPR-Cas9 has been em-
ployed in other species, such as the silkworm
Bombyx mori, targeting the Orco gene (Liu et al.,
2017), the hawkmoth Manduca sexta (Fandino et al.,
2019), and the locust Locusta migratoria, where ho-
mozygous and heterozygous mutant lines were
produced to help genetically control locusts and
other orthopteran species (Li et al., 2016). CRISPR-
Cas9 has also been used in the fruit fly Bactrocera
dorsalis (Xu et al., 2024) and the American bollworm
Helicoverpa armigera (Fan et al., 2022). Moreover,
CRISPR-Cas9 has been applied to genetically con-
trol Spodoptera frugiperda, commonly known as the
fall armyworm, which is currently emerging as a
significant agricultural pest (Gouda et al., 2024).
Insect Pest Control
The CRISPR-Cas9 technique has emerged as a
highly effective tool for controlling insect pests in
agriculture (Fig. 4). By employing various strategies
within the CRISPR system, researchers can modify
specific target DNA sequences to manage insecti-
cide resistance or introduce traits that restore sus-
ceptibility to pests. One such strategy involves the
release of gene-edited insects into the wild, which
carry genes that can reduce the population of re-
sistant individuals. This method has shown signifi-
cant potential in the global fight against insect pests.
Additionally, gene drive systems are being used to
accelerate the spread of these genetic traits through
populations (Ying et al., 2023). Insect pest control
strategies using CRISPR often complement other
methods, enhancing overall effectiveness (Zahoor
et al., 2021; Ying et al., 2023).
CRISPR-Based Gene Drive
Gene drives, designed to spread genetic ma-
terial rapidly through natural populations, have
been instrumental in pest control efforts worldwide
(Champer et al., 2016). By introducing beneficial ge-
netic traits into pest populations, CRISPR-based
gene drives can significantly modify reproductive
patterns and increase susceptibility to pesticides.
These selfish genetic elements use the natural sex-
ual reproduction process to transmit DNA se-
quences or genes across generations, operating
more swiftly than traditional Mendelian inheritance
(Bier, 2022). Gene drives typically rely on transpos-
able elementsnaturally occurring sequences in in-
sect genomes that can be harnessed for genetic
modification (Sinkins and Gould, 2006; Wang et al.,
2022a). Homing gene drives, in particular, utilize
homing endonuclease genes, which recognize spe-
cific DNA sequences and induce targeted genome
changes (Burt, 2003; Hillary et al., 2020).
Gene drives that exploit CRISPR-Cas9 operate
through both the non-homologous end joining
(NHEJ) and homology-directed repair (HDR) path-
ways. These systems introduce effector genes that
disrupt critical biological processes, such as fertility
or sex ratios, effectively reducing pest populations
over time (Gantz and Bier, 2015; Galizi et al., 2016).
The primary goal of gene drives is to modify popu-
lations by spreading genetic variants that eliminate
harmful traits without destroying the species en-
tirely (Champer et al., 2016; Raban et al., 2020, 2022;
Devos et al., 2022a). For instance, gene-edited in-
sects with traits like reduced fertility are bred in la-
boratories and then released into the wild. The in-
troduction of these traits can lead to lower fertility
rates, skewed sex ratios, or other traits that destabi-
lize pest populations (Deredec et al., 2008; Oye et al.,
2014). CRISPR-based gene drives have been suc-
cessfully reported in various species, including Dro-
sophila, beetles, moths, grasshoppers, and, most no-
tably, vector-borne mosquitoes (Scott et al., 2014;
Shukla and Palli, 2013).
Sex Distortion
Sex ratio distortion is another promising
CRISPR-based strategy, especially in mosquito pop-
ulations. Studies on Drosophila melanogaster have
demonstrated two mechanisms of CRISPR-induced
sex ratio distortion: X-shredding and X-poisoning.
X-shredding operates during the meiotic phase of
spermatogenesis, targeting the X chromosome to in-
duce male-biased sex ratios, though it is counter-
acted by NHEJ repair. X-poisoning targets the RpS6
gene, leading to reduced reproductive output
(Fasulo et al., 2020). These sex distortion approaches
hold potential for genetic control of insect popula-
tions in agriculture and public health, offering a
means to suppress populations of harmful pests
through genetic manipulation. Further research in
this area could yield significant advances in pest
control and disease prevention.
Insecticide Resistance
Diamide resistance has been identified in nu-
merous lepidopteran pests, which function as acti-
vators of Ryanodine Receptors (RyRs). Research on
three diamides - chlorantraniliprole, flubendiamide,