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 molecules—CRISPR 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