International Journal of

Molecular Sciences

Review
CRISPR/Cas9 Technology for Enhancing Desirable Traits of Fish
Species in Aquaculture
Minli Zhu 1,2 , Sahr Lamin Sumana 1 , Mukhtar Muhammad Abdullateef 1 , Opeoluwa Christiana Falayi 1 ,
Yan Shui 3 , Chengfeng Zhang 3 , Jian Zhu 3 and Shengyan Su 1,2,3, *

1 Wuxi Fisheries College, Nanjing Agricultural University, Wuxi 214081, China; zhuminli1023@163.com (M.Z.);
sl5284sumana@gmail.com (S.L.S.); mukmuha1989@gmail.com (M.M.A.); opeoluwafalayi@gmail.com (O.C.F.)
2 National Demonstration Center for Experimental Fisheries Science Education, Shanghai Ocean University,
Shanghai 201306, China
3 Key Laboratory of Integrated Rice-Fish Farming Ecology, Ministry of Agriculture and Rural Affairs,
Freshwater Fisheries Research Center, Chinese Academy of Fishery Sciences, Wuxi 214081, China;
shuiy@ffrc.cn (Y.S.); zhangcf@ffrc.cn (C.Z.); zhuj@ffrc.cn (J.Z.)
* Correspondence: susy@ffrc.cn

Abstract: Aquaculture, the world’s fastest-growing food production sector, is critical for addressing
food security concerns because of its potential to deliver high-quality, nutrient-rich supplies by 2050.
This review assesses the effectiveness of CRISPR/Cas9 genome editing technology in enhancing
desirable traits in fish species, including growth rates, muscle quality, disease resistance, pigmenta-
tion, and more. It also focuses on the potential effectiveness of the technology in allowing precise
and targeted modifications of fish DNA to improve desirable characteristics. Many studies have
reported successful applications of CRISPR/Cas9, such as knocking out reproductive genes to control
reproduction and sex determination, enhancing feed conversion efficiency, and reducing off-target
effects. Additionally, this technology has contributed to environmental sustainability by reducing
nitrogen-rich waste and improving the nutritional composition of fish. However, the acceptance of
CRISPR/Cas9 modified fish by the public and consumers is hindered by concerns regarding public
perception, potential ecological impacts, and regulatory frameworks. To gain public approval and
consumer confidence, clear communication about the editing process, as well as data on the safety and
Citation: Zhu, M.; Sumana, S.L.;
environmental considerations of genetically modified fish, are essential. This review paper discusses
Abdullateef, M.M.; Falayi, O.C.; Shui,
Y.; Zhang, C.; Zhu, J.; Su, S.
these challenges, provides possible solutions, and recommends future research on the integration of
CRISPR/Cas9 Technology for CRISPR/Cas9 into sustainable aquaculture practices, focusing on the responsible management of
Enhancing Desirable Traits of Fish genetically modified fish to enable the creation of growth and disease-resistant strains. In conclusion,
Species in Aquaculture. Int. J. Mol. Sci. this review highlights the transformative potential of CRISPR/Cas9 technology in improving fish
2024, 25, 9299. https://doi.org/ traits, while also considering the challenges and ethical considerations associated with sustainable
10.3390/ijms25179299 and responsible practices in aquaculture.
Academic Editor: Nobuya Sasaki
Keywords: CRISPR/Cas9; desirable fish traits; aquaculture; public perceptions; environmental
Received: 4 July 2024 consideration
Revised: 18 August 2024
Accepted: 25 August 2024
Published: 27 August 2024
1. Introduction
Aquaculture is the fastest-growing sector in food production worldwide, addressing
Copyright: © 2024 by the authors.
challenges related to global food security caused by population growth, climate change,
Licensee MDPI, Basel, Switzerland.
and limited resources [1]. The projected global population of 9.7 billion by 2050 will require
This article is an open access article a 25 to 70 percent increase in food production. However, in order to meet these global
distributed under the terms and challenges, it is crucial to have high-quality, nutrient-rich food sources, which is where
conditions of the Creative Commons aquaculture comes in. In 2011, aquaculture was estimated to account for 85–89% of global
Attribution (CC BY) license (https:// production for household consumption, with 51% and 52%, respectively, representing
creativecommons.org/licenses/by/ the total population and undernourished population [2]. From the 1990s to 2022, human
4.0/). consumption of aquaculture and fisheries products has doubled from 81.6 to 164.6 million

Int. J. Mol. Sci. 2024, 25, 9299. https://doi.org/10.3390/ijms25179299 https://www.mdpi.com/journal/ijms

Int. J. Mol. Sci. 2024, 25, 9299 2 of 23

metric tonnes annually [3]. Aquaculture also plays a significant role in the food supply,
accounting for over 57% of aquatic animal foods in 2022 [3]. The long-term growth and
expansion of the aquaculture industry depend on protein-rich food products to meet the
needs of the growing global population. There are concerns about the rising demand for
aquatic products due to population growth, and the aquaculture sector seems to offer a
promising solution. This has been made possible through the relentless efforts of researchers
in advancing genetic engineering, particularly through the use of CRISPR/Cas9 technology
to address disease outbreaks, slow growth rates, and environmental stressors in various
fish species.
CRISPR/Cas9 is a groundbreaking genetic engineering tool that allows for precise
and targeted modifications of fish DNA to enhance desirable traits such as color pigmen-
tation, growth, muscle quality, and disease resistance [4–6]. This technology surpasses
traditional breeding techniques, offering a cheaper, easier, and more precise method for
genetic improvement [7–10]. It enables precise genome editing to improve essential traits,
including growth performance (such as increased body weight, length, and muscle fiber
development), muscle quality, disease resistance, and sex determination [11–17]. Addition-
ally, CRISPR/Cas9 technology offers a promising solution for enhancing disease resistance
in fish by targeting immune-related genes or pathogen recognition pathways, thereby
reducing reliance on antibiotics and chemical treatments [7]. Moreover, this technology
has revolutionized modern aquaculture by genetically enhancing key characteristics of fish
species. For example, researchers have successfully eliminated germ cells responsible for
reproductive cell sex differentiation in Atlantic salmon [18], improved feed conversion effi-
ciency for accelerated growth rates in yellow catfish [13], achieved efficient gene mutations
in tilapia, and minimized off-target effects [19].
The environment is vital for the sustainability of the aquaculture industry. This
technology not only enhances desirable traits in fish but also addresses environmental
sustainability and consumer preferences. Some authors have modified fish to produce
less nitrogen-rich waste, reducing the environmental impact of aquaculture. They have
also improved the nutritional composition of fish, increasing omega-3 fatty acid levels and
reducing undesirable compounds. The fatty acid desaturase fads2 gene in rainbow trout
has been edited to increase the production of omega-3 fatty acids, which are highly valued
by consumers.
However, the acceptance of this technology by the public and consumers is hindered
by factors such as public perception and potential ecological impacts [20] raised ethical
concerns about genetic engineering in animals, particularly through gene knockout and
knock-in methods. The commercialization of CRISPR/Cas9 products requires risk analysis,
regulatory approval, and public acceptance. In addition, public approval and consumer
confidence in genetically modified fish are influenced by the reliability of health, safety,
and environmental considerations [21]. To gain public approval and consumer confi-
dence, it is crucial to provide clear explanations about the process of editing fish traits, as
well as regulatory frameworks and data on genetically modified fish (GMF) to address
these concerns.
CRISPR/Cas9 technology has garnered interest in the field of aquaculture in recent
years. However, there is currently a lack of a comprehensive review that encompasses its
various applications and the challenges it poses in modifying key desirable traits in fish.
Previous studies have primarily concentrated on explaining the principles of the technology,
its advantages, and its utilization in enhancing economically significant traits [22].
Therefore, this review paper evaluates the effectiveness of CRISPR/Cas9 technology
in improving traits in aquaculture fish species, including growth rates, muscle quality,
disease resistance, and environmental stress tolerance. It also discusses the challenges and
ethical considerations involved in sustainable and responsible practices.
 Int. J. Mol. Sci. 2024, 25, x FOR PEER REVIEW 3 of 23

Int. J. Mol. Sci. 2024, 25, 9299 3 of 23

2. Major Fish Species Traits Improved by Genome Editing in Aquaculture

2. MajorAquaculture
Fish Species is vital forImproved
Traits global food security and
by Genome providing
Editing dietary protein. China
in Aquaculture
leads in genetic enhancement,
Aquaculture is vital for globalparticularly
food securityusingand the providing
CRISPR-Cas9 dietarysystem, to improve
protein. China
carp strains. This technology has revolutionized desirable
leads in genetic enhancement, particularly using the CRISPR-Cas9 system, to improve carp traits in aquaculture,
particularly
strains. in developing
This technology hascountries.
revolutionized desirable traits in aquaculture, particularly in
CRISPR-Cas9
developing countries. is used to target and modify specific genes in fish, leading to a range of
improvements.
CRISPR-Cas9 For example,
is used this and
to target technology has been
modify specific utilized
genes in fish,to leading
alter pigmentation
to a range
ofpathways, resulting
improvements. Forinexample,
enhanced color
this changes has
technology in ornamental
been utilized species
to alter like goldfish and
pigmentation
common carp
pathways, [23,24].
resulting Additionally,
in enhanced colorCRISPR-Cas9 has been applied
changes in ornamental speciesto likeincrease
goldfish growth
and
common carp [23,24]. Additionally, CRISPR-Cas9 has been applied to increase growthto
rates in economically important species such as tilapia and common carp, contributing
moreinefficient
rates production
economically systems
important [25,26].
species suchItasis tilapia
essential
andthat the area
common of improvement
carp, contributing to be
more efficient production systems [25,26]. It is essential that the area of improvement bein
disease resistance, as genetic modifications have enhanced the immune response
speciesresistance,
disease such as Atlantic
as geneticsalmon and rainbow
modifications trout, reducing
have enhanced the immune mortality
response fromincommon
species
aquaculture
such as Atlantic pathogens
salmon and [27]. In medaka,
rainbow CRISPR-Cas9
trout, reducing has from
mortality improved common feedaquaculture
conversion
efficiency,[27].
pathogens helping to lowerCRISPR-Cas9
In medaka, production costs and minimize
has improved environmental
feed conversion impacthelping
efficiency, [28].
to lowerTheproduction
nutritionalcosts quality
andofminimize
farmed fish has also seen
environmental improvements
impact [28]. through genome
editing. For instance,
The nutritional modifications
quality of farmed infish
Atlantic salmon
has also seenhave enhanced omega-3
improvements through fatty acid
genome
metabolism,
editing. increasingmodifications
For instance, their nutritional in value
Atlanticfor salmon
human consumption
have enhanced [29]. Furthermore,
omega-3 fatty
CRISPR-Cas9
acid metabolism, hasincreasing
been usedtheirto address
nutritionalreproductive
value forchallenges by controlling
human consumption sterility
[29]. Fur-
and fertility
thermore, in Nile tilapia
CRISPR-Cas9 hasand Atlantic
been used salmon,
to address aiding in better population
reproductive challengesmanagement
by control-
[30–33].
ling sterility and fertility in Nile tilapia and Atlantic salmon, aiding in better population
Moreover,
management sex control through genome editing has allowed for the manipulation of
[30–33].
sex-determination control
Moreover, sex genes in speciesgenome
through like Nile tilapia
editing hasand rainbow
allowed for thetrout, optimizing
manipulation
ofproduction efficiencygenes
sex-determination by controlling
in speciessex likeratios
Nile [29]. Theand
tilapia technology
rainbow has trout, also improved
optimizing
tolerance toefficiency
production abiotic stresses, such as sex
by controlling lowratios
oxygen levels
[29]. Theand increasedhas
technology salinity in Atlantic
also improved
salmon, enhancing
tolerance survival such
to abiotic stresses, ratesasin low
challenging environments
oxygen levels and increased[27]. salinity in Atlantic
salmon, Additionally, several researchers
enhancing survival have utilized
rates in challenging CRISPR-Cas9
environments [27]. to edit immune genes
in different fish species,
Additionally, several further enhancing
researchers diseaseCRISPR-Cas9
have utilized resistance and overall
to edit immune healthgenes
[34–36].
in
These advancements in genome editing have significantly contributed to reducing
different fish species, further enhancing disease resistance and overall health [34–36]. These
undesirable traits
advancements while enhancing
in genome editing have desirable ones, making
significantly aquaculture
contributed moreundesirable
to reducing efficient and
sustainable. With CRISPR-Cas9 technology, the aquaculture industry and
traits while enhancing desirable ones, making aquaculture more efficient can sustainable.
continue to
With
evolveCRISPR-Cas9
and meet the technology,
growingthe aquaculture
global demandindustry can continue
for nutritious to evolve and
and sustainably meet
produced
the growing global
protein food (Figure 1). demand for nutritious and sustainably produced protein food (Figure 1).

Figure1. 1.
Figure The
The conceptual
conceptual framework
framework outlines
outlines howCRISPR-Cas9
how the the CRISPR-Cas9
model canmodel can betoapplied
be applied enhanceto
enhance traits
desirable desirable traits
in fish, within the
fish,ultimate
with thegoal
ultimate goal of improving
of improving food availability.
food availability.
Int. J. Mol. Sci. 2024, 25, 9299 4 of 23

3. Disease Resistance
Disease resistance refers to a fish species’ ability to tolerate internal and external
factors that can disrupt its physiological or morphological functions. These factors include
infectious parasites, poor water quality (pollution), and climate change. Potts [14] provided
a helpful definition, stating that disease resistance is a broad term that covers a spectrum
ranging from increased tolerance to complete protection.
Disease poses a significant challenge in aquaculture development and negatively im-
pacts overall production yield, reducing fish farmers’ profits. This situation has prompted
genetic improvements in disease-resistant fish species, leading to new approaches for map-
ping loci that affect disease resistance. Studies have shown the potential of marker-assisted
selection, particularly genomic selection, in accelerating genetic gain in target traits [37].
CRISPR/Cas9-mediated genome editing has emerged as a powerful tool to enhance
disease resistance in fish. By integrating vector-engineered antimicrobial peptide genes
(AMGs), CRISPR/Cas9 can decrease bacterial colony-forming units in fish tissues, increase
post-infection survival rates, and alter the expression of immune-related genes [38]. This
technology allows for precise modifications in the fish genome, improving disease resis-
tance, growth, and reproduction [39].
One successful application of CRISPR/Cas9 is in salmon, where it has improved
resistance to infections such as infectious pancreatic necrosis (IPN), bacterial cold-water
disease, and viral infections. In grass carp cells, researchers have used CRISPR/Cas9
technology to knock out the JAM-A gene, which plays a role in viral entry into host cells,
conferring resistance against grass carp reovirus (GCRV) infection [39].
Furthermore, CRISPR/Cas9-based transgenesis allows for the site-directed knock-in
of foreign genes at multiple loci, enhancing disease resistance in combination with other
desirable traits such as fast growth and enriched fatty acid content [40]. For instance, this
approach has been used to integrate genes that confer resistance to specific pathogens
while simultaneously improving growth rates and nutritional quality in fish species such
as tilapia and catfish [41].
In tilapia, CRISPR/Cas9 has been employed to edit genes involved in the immune
response, leading to enhanced resistance against bacterial pathogens like Streptococcus
agalactiae and Aeromonas hydrophila [42]. Similarly, in catfish, CRISPR/Cas9 has been used to
target genes that regulate immune pathways, resulting in increased survival rates following
pathogen exposure [37,43].
In conclusion, CRISPR/Cas9 holds significant promise for modulating the innate
immune systems of fish and combating various pathogens. It is a valuable tool in aqua-
culture disease management, offering a path toward sustainable and resilient aquaculture
systems [37,44].

4. Fish Growth and Muscle Quality

CRISPR/Cas9-based genome editing has been extensively applied in various fish
species, particularly in freshwater fish used in aquaculture. This technology has been used
in species such as Nile tilapia [45], channel catfish [12], southern catfish, common carp [46],
rohu [47], grass carp [39], and rainbow trout.
Some of these genome-edited fish have demonstrated superior traits, such as improved
disease resistance or enhanced growth performance. For example, myostatin-knockout
channel catfish exhibited a 29.7% increase in mean body weight compared to control
individuals [12].
Fish growth and muscle quality are important traits for the development of aqua-
culture worldwide. Advances in genetic modification and biotechnology have allowed
researchers to target specific genes to improve these traits in different fish species. Nu-
merous studies have successfully modified genes associated with growth and muscle
development, producing promising results in species such as red sea bream, channel
catfish, and common carp [26].
Int. J. Mol. Sci. 2024, 25, 9299 5 of 23

One significant area of research focuses on manipulating growth hormone-related

genes. For example, alterations to the myostatin (mstn) gene, which negatively regulates
muscle growth, have been shown to enhance muscle growth and mass in various fish
species. In blunt snout bream, disrupting the mstn gene resulted in increased muscle
mass and body weight [48], while similar modifications in olive flounder led to significant
muscle mass enhancement [49]. Additionally, gene editing of mstn in Red Sea bream
has improved skeletal muscle mass and reduced body length, optimizing fish size for
commercial purposes [50].
In common carp, targeted genetic interventions have improved growth performance,
demonstrating the potential of genetic approaches to enhance aquaculture efficiency [51].
Beyond traditional genetic modification, CRISPR/Cas9 technology has emerged as a pow-
erful tool for precise genome editing. In zebrafish, CRISPR has been used to identify genes
related to the enteric nervous system, including those encoding opioid receptors, which
play a vital role in gut development [52].
CRISPR/Cas9 technology has also facilitated the study of developmental processes
and human diseases by allowing researchers to create gene knockouts and observe phe-
notypic outcomes in model organisms like zebrafish [53]. In aquaculture, this technology
holds great promise for improving fish growth, muscle quality, animal welfare, and farming
efficiency [54].
Moreover, transgenic approaches have been used to overexpress growth hormone
genes in species like Atlantic salmon, resulting in rapid growth rates and increased biomass
production [55]. Such genetic enhancements are crucial for meeting the growing global
demand for fish protein.
In addition to genetic modification, other factors such as nutrition, environmental
conditions, and selective breeding play important roles in optimizing fish growth and
muscle quality. Studies have shown that dietary supplements, such as amino acids and
omega-3 fatty acids, can improve muscle texture and nutritional value in aquaculture
species [56,57].

5. Off-Target Effects in CRISPR/Cas9 and Advances in Aquaculture

CRISPR/Cas9 technology can introduce off-target effects, affecting genomes in non-
target locations and potentially causing adverse effects on organisms, including fish species.
However, recent advancements have led to the development of high-fidelity Cas9 variants
that significantly reduce off-target activity. For example, the SpCas9-HF1 and eSpCas9
variants have shown reduced non-specific binding and cleavage compared to the wild-type
Cas9, thereby improving target specificity in fish species [58]. Additionally, the optimization
of guide RNA (gRNA) design has played a critical role in minimizing off-target effects.
Algorithms that predict potential off-target sites based on sequence similarity and binding
affinity have become more sophisticated. Tools like CRISPR-DO, CCTop, and others assist
researchers in designing more specific gRNAs [59].
Furthermore, new technologies such as base editors and prime editors offer precision
without causing double-strand breaks, thus further reducing the risk of off-target mutations.
Base editing allows for targeted nucleotide changes without introducing double-strand
breaks, while prime editing enables more precise insertions, deletions, or corrections [60,61].
In addition, advancements in delivery systems, including nanoparticles and viral vectors,
have improved the precision of CRISPR/Cas9 by enhancing gene editing accuracy at the
cellular level. These systems ensure more effective delivery of CRISPR/Cas9 components
to target cells while minimizing exposure to off-target sites [62,63].
Zebrafish are widely used as a model organism in CRISPR research due to their
transparent embryos and rapid development. Successful applications of CRISPR/Cas9
in zebrafish have demonstrated its effectiveness in gene knockout, gene knock-in, and
gene modification while minimizing off-target effects through the use of high-fidelity Cas9
variants and improved gRNA design [64] (see Figure 1).
Int. J. Mol. Sci. 2024, 25, x FOR PEER REVIEW 6 of 23

modification while minimizing off-target effects through the use of high-fidelity Cas9
Int. J. Mol. Sci. 2024, 25, 9299
variants and improved gRNA design [64] (see Figure 1). 6 of 23

In tilapia, CRISPR/Cas9 has been employed to enhance traits such as growth rate and
disease resistance. Recent studies have utilized improved Cas9 variants and optimized
gRNAsIn tilapia, CRISPR/Cas9
to achieve has been
precise genetic employed towith
modifications enhance traitsoff-target
reduced such as growth
effectsrate and
[25,65].
disease resistance. Recent studies have utilized improved Cas9 variants and optimized
CRISPR/Cas9 technology has also been applied to Atlantic salmon to improve traits such
gRNAs to achieve precise genetic modifications with reduced off-target effects [25,65].
as growth and disease resistance. Research in this area has focused on utilizing advanced
CRISPR/Cas9 technology has also been applied to Atlantic salmon to improve traits such
Cas9 variants and delivery methods to achieve precise editing with minimal off-target
as growth and disease resistance. Research in this area has focused on utilizing advanced
mutations [22]. The mechanism used by CRISPR/Cas9 in knockout genes in different fish
Cas9 variants and delivery methods to achieve precise editing with minimal off-target
species are indicated in Figure 2.
mutations [22]. The mechanism used by CRISPR/Cas9 in knockout genes in different fish
species are indicated in Figure 2.

Figure 2. The application of CRISPR/Cas9 in aquaculture involves several steps. First, a specific
Figure 2. The application of CRISPR/Cas9 in aquaculture involves several steps. First, a specific
gRNA
gRNAisisdesigned
designedtoto
match thethe
match target gene
target sequence.
gene Then,
sequence. the Cas9
Then, protein
the Cas9 bindsbinds
protein to the to
target
the DNA,
target
causing a double-strand break. Finally, the break is repaired.
DNA, causing a double-strand break. Finally, the break is repaired.
6. Sex Determination
6. Sex Determination
The process of differentiating between male or female in sexually reproducing or-
ganismsTheisprocess
called sexof determination.
differentiating Sex between male or female
determination in fish isina sexually reproducing
complex process that
organisms is called sex determination. Sex determination in fish is a complex
involves genetic, environmental, and epigenetic factors [59]. CRISPR/Cas9 technology process thathas
involves genetic,
significantly environmental,
contributed and epigenetic
to the understanding offactors [59]. CRISPR/Cas9
sex determination technology has
and differentiation in
significantly
fish by usingcontributed to theand
various genetic understanding
environmental of sex determination
signals andbeing
[16,66]. Fish, differentiation
the largest in
fish by using various genetic and environmental signals [16,66]. Fish, being
vertebrates, are ideal for studying sex determination and differentiation due to their di- the largest
vertebrates,
verse aquaticare ideal reproductive
habitats, for studying techniques,
sex determination and differentiation
and sex characteristics. due to their
The gene-editing
technology, CRISPR/Cas9, has revolutionized research on fish genetic traits byThe
diverse aquatic habitats, reproductive techniques, and sex characteristics. gene-
enabling
editing technology, CRISPR/Cas9, has revolutionized
precise DNA alteration and opening up diverse applications. research on fish genetic traits by
enabling precise DNA alteration and opening up diverse applications.
Sex determination and differentiation in fish involve a balance between male-promoting
Sex determinationfactors
and female-promoting and differentiation
in somatic cells.inMutations
fish involve a balance
in genes between
that favor femalesmale- can
promoting
decrease and female-promoting
estrogen factorsproduction,
and increase androgen in somatic leading
cells. Mutations in genes that
to female-to-male favor
sex rever-
females
sal can Conversely,
[67–71]. decrease estrogen
mutationsand in
increase androgen
genes that favor production, leadingestrogen
males can increase to female-to-(E2)
male sex reversal [67–71]. Conversely, mutations in genes that favor males
production and decrease ketotestosterone (11-KT) production, resulting in male-to-female can increase
estrogen
sex (E2)
reversal production and decrease ketotestosterone (11-KT) production, resulting in
[71].
male-to-female
CRISPR/Cas9 sex has
reversal
been [71].
used in several studies to investigate sex determination genes
in fish by disrupting these genes and observing the resulting phenotypes. In Nile tilapia,
the anti-Müllerian hormone (amh) gene, which is involved in male sex determination,
was targeted using CRISPR/Cas9. Disrupting the amh gene led to the development of
Int. J. Mol. Sci. 2024, 25, 9299 7 of 23

phenotypic females, even among genetic males, demonstrating the gene’s significance in
tilapia sex determination [45,72].
Similarly, in medaka fish, the dmrt1 gene, a key regulator of male sex determination in
this species, was targeted using CRISPR/Cas9. Disrupting dmrt1 resulted in phenotypic
females developing from genetic males, highlighting the gene’s crucial role in medaka sex
determination [25,72].
In zebrafish, which have a polygenic sex determination system, CRISPR/Cas9 has
been utilized to investigate the roles of various candidate genes, such as dmrt1, sox9a, and
amhy. These studies have provided insight into the complex genetic architecture of sex
determination in zebrafish [73].
Furthermore, CRISPR/Cas9 has been employed to discover new sex determination
genes. For example, a genome-wide CRISPR/Cas9 screening in rainbow trout identified
the sdY gene as the master regulator of male sex determination in this species [74].

7. Effects of CRISPR/Cas9 Technology on Different Biological and

Environmental Aspects
The modern fish farming system faces several discouraging factors that result in low
production and impact sustainability. These factors include disease outbreaks, poor growth
rates, and environmental degradation [22,75,76]. Conventional breeding methods have
had limited success in addressing these issues. However, the emergence of CRISPR/Cas9
technology offers new possibilities for improving the sustainability of aquaculture.
CRISPR/Cas9, initially designed for precise genetic modification, has revolutionized
genome study and manipulation, enabling researchers to create genetically modified organ-
isms with desired traits [4], alter sex determination [45], model human diseases in animal
systems [77], and conduct high-throughput genetic screens to uncover gene functions [78].
This gene-editing technology, introduced as a revolutionary tool, has significantly impacted
various biological and environmental domains [79]. Its versatility and precision have seen
it expanded to various applications, including controlling invasive species, engineering mi-
croorganisms for bioremediation and other environmental cleanup efforts, and developing
genetically modified fish for sustainable aquaculture [22].
Genome editing, particularly with the CRISPR/Cas9 technique, is being explored as a
potential solution to the challenges faced by the aquaculture industry. The development
of more effective and affordable genome editing techniques has made this approach in-
creasingly viable. For example, the AquAdvantage salmon was developed in Canada in
2016 and approved for food production in the U.S. in 2019 [80]. This genetically modified
salmon was created by combining the growth hormone gene from Chinook salmon with
a promoter sequence from the antifreeze protein gene of an ocean pout, creating a gene
construct that was then inserted into an Atlantic salmon egg to produce AquAdvantage
salmon [81]. Similarly, Nile tilapia was genetically modified in Argentina in 2018 to enhance
growth rates and disease resistance [82]. Table 1 provides a summary of the traits most
commonly targeted for genome editing in fish aquaculture [83].

Table 1. Effects of CRISPR/Cas9 on Biological and Environmental Aspects of Fish Species.

Applicable Fields Impacts Ref.

It is used to reduce the viral hemorrhagic septicemia virus (VHSV) infection of olive
[84]
flounder hirame natural embryo (HINAE) cells.
It enables gene editing in fish species such as salmon, tilapia, and shrimp to increase their
[85,86]
resistance to diseases.
Disease resistance
It helps in the deletion of the JAM-A gene in grass carp cells, which significantly enhances
[39]
resistance to grass carp reovirus (GCRV) infection.
It helps enhance fish cell lines for host response and genetic resistance against infectious
[22]
diseases, using Atlantic salmon and rainbow trout as model systems in aquaculture.
Int. J. Mol. Sci. 2024, 25, 9299 8 of 23

Table 1. Cont.

Applicable Fields Impacts Ref.

Environmental It helps to edit genes in fish species, such as farmed salmon, to adapt to
[31,86]
adaptation changing environments.
It increases muscle growth by knocking out melanocortin (mc4r) receptor genes and has
[28,87]
been experimentally tried on channel catfish and medaka fish.
Improved growth rates It improved the growth rates and increased muscle mass of the channel catfish by
[12]
and muscles modifying the myostatin gene in channel catfish embryos.
It helps increase the muscle mass of blunt snout bream due to the disruption of the mstna
[48]
and mstnb genes.
It helps in myostatin gene disruption of genes, such as transcription factor sp7, causing bone
Bone development defects in common carp, and increases muscular cells, resulting in a more robust [46]
muscular phenotype.
It can be used to edit genes involved in pigmentation pathways, potentially leading to loss
of skin pigmentation, e.g., edited mutant of large-scale loach, causing skin pigmentation [19,68]
loss and black patch dispersion in the Oujiang color common carp.
Colour defects It helps identify and introduce mutations in genes responsible for pigmentation, such as
[68]
tyrosinase or mitf, which can lead to pigmentation defects in fish species like salmon.
It helps to reveal a recessive inheritance pattern for the white-albino phenotype, lacking
[88]
pigment-containing chromatophores, in rainbow trout.
It can be used to disrupt or modify the gonadal soma-derived factor (gsdf ) gene, which is a
Sex determination crucial gene in teleost fish. Disruption of genes such as dmrt1 and cyp19a1a can lead to sex [89]
reversal phenotypes in zebrafish.
It facilitates the integration of exogenous DNA into the zebrafish genome, but it may also
DNA integration cause additional genetic mutations or disruptions, depending on the editing conditions and [90]
precision of the technique.
kidney and gonads It helps disrupt the Wilms tumor 1 (wt1a) gene, which may lead to abnormal gonad and
[69]
development kidney development in Nile tilapia.
It has been used to knock out or edit genes in salmon fish. However, overexpressing
Immune genes
interferon (IFN) or inducing stimulated genes (ISGs) does not guarantee broad [36]
improvement
disease resistance.

8. Effects of Using CRISPR/Cas9 in Gene Editing on Different Fish Species

The CRISPR/Cas9 gene editing technology has shown significant potential for improv-
ing various traits in fish and aquaculture species. Researchers have used this powerful tool
to gain important insights into the genetic regulation of key physiological and production-
related characteristics. CRISPR/Cas9 has aided researchers in developing gene drives,
which are genetic engineering technologies that enhance the chances of a gene being in-
herited by the next generation. This allows for the more rapid spread of specific traits
throughout a population compared to traditional inheritance methods. Gene drives achieve
this by ensuring that the engineered gene is inherited at significantly higher frequencies,
reaching close to 100%, instead of the typical 50% inheritance rate.
A notable example is the work of Li et al. [25], who used CRISPR/Cas9 to create
sterile, all-male populations of Nile tilapia. Their study found that these genetically
modified tilapia showed significantly improved growth rates compared to normal mixed-
sex populations. Additionally, producing sterile fish helps mitigate the environmental risks
associated with escaped farm-raised tilapia, which can disrupt local ecosystems through
competition and interbreeding with wild populations. This study has been influential in
demonstrating the potential of CRISPR technology to enhance aquaculture productivity
while minimizing ecological impacts.
Similarly, Wargelius et al. [18] applied CRISPR/Cas9 gene editing to Atlantic salmon,
conferring resistance against significant viral pathogens such as infectious pancreatic
Int. J. Mol. Sci. 2024, 25, 9299 9 of 23

necrosis virus (IPNV) and salmon alphavirus (SAV). By disrupting or modifying host genes
that these viruses rely on for infection and replication, the researchers were able to produce
salmon lines with increased disease resistance. This advancement is significant, as viral
diseases can cause substantial losses in Atlantic salmon aquaculture.
Building on these successes, other studies have used CRISPR/Cas9 to engineer disease
resistance in various commercially important fish species. Godel (2015) reported developing
carp resistant to koi herpesvirus (KHV), a devastating pathogen that can decimate carp
populations. Ma et al. and Chakrapani et al. [39,47] have also explored CRISPR-based
approaches to enhance disease resistance in grass carp and common carp, respectively.
In addition to disease resistance, researchers have used CRISPR/Cas9 to modify
growth-related genes in several fish species. Zhong et al. [46] targeted myostatin, a negative
regulator of muscle growth, in common carp, resulting in increased body size and growth
rates. Similar studies have been conducted in channel catfish [12], tiger pufferfish, red
sea bream [50], and olive flounder [49], demonstrating the versatility of this technology in
optimizing commercially valuable production traits.
CRISPR/Cas9 has also enabled the development of novel phenotypes in aquatic
organisms beyond these production-focused applications. Segev-Hadar et al. [91] used
the technology to create true albino Nile tilapia with distinctive pink eyes, while Yu
et al. [19] modified the myostatin gene in Pacific oysters, resulting in altered growth and
muscle development. Furthermore, Gui et al. [92] reported successful genome editing of
the ridgetail shrimp, showcasing the broad applicability of CRISPR/Cas9 across diverse
aquatic species (Table 2).

Table 2. Applications of CRISPR/Cas9 in Various Fish Species and Their Impacts.

Fish Species Technological Impacts Ref.

It is used to produce sterile Nile tilapia populations, reducing the risk of environmental
Nile tilapia [11]
damage from escaped fish.
It helps in gene editing to create species that are highly resistant to viral infections,
Atlantic salmon [18]
e.g., salmon.
It allows scientists to study mutations and genetic variants in zebrafish.
It can be used to successfully integrate composite tags into zebrafish embryos, enabling
Zebrafish precise labeling and visualization of cellular structures or proteins. This offers potential for [93]
studying protein dynamics, gene expression, and other biological processes in this
model organism.
It has been shown to reduce the expression of the igfbp-2b gene in rainbow trout, influencing
Rainbow trout growth and development, but its impact on overall performance and the endocrine system [35]
remains unclear.
Atlantic salmon and It has been used to target unique genes associated with growth and immunity in Atlantic
[22]
Rainbow trout salmon, rainbow trout, and coho salmon cells.
It has the potential to increase muscle growth and body weight in farmed fish species such
Japanese medaka as medaka. However, further investigation is needed to determine its impact on production [28]
yield and fish health.
It can be used to disrupt the myostatin gene in olive flounder, potentially increasing body
Olive flounder weight and muscle tissue, but further research is needed to understand its effects on [49]
production efficiency and fish health.
It has been used to modify the myostatin gene in Channel catfish to improve muscle growth
Channel catfish [12]
and quality, but further research is needed to fully understand its effects.

9. Potential Socioeconomic Impacts of Widespread Adoption of CRISPR-Based

Disease-Resistant Aquaculture in Developing Regions
The rapid progress of gene-editing technologies like CRISPR/Cas9 has created new
opportunities for the aquaculture industry in developing regions. Disease-resistant aqua-
culture using CRISPR/Cas9 has the potential to greatly affect the availability, cost, and
Int. J. Mol. Sci. 2024, 25, 9299 10 of 23

nutritional quality of seafood, as well as the livelihoods of small-scale and subsistence-level

fish farmers [94]. However, the widespread use of this technology also raises important
socioeconomic issues that require careful examination.
Improving disease resistance and productivity in aquaculture species through ad-
vancements in this technology can significantly increase the availability and affordability of
nutritious seafood. This has the potential to enhance food security and nutritional outcomes,
particularly for vulnerable populations [95]. These improvements could also boost incomes
for small-scale and subsistence-level fish farmers, helping to lift them out of poverty and
enhance their economic resilience [94]. Furthermore, implementing CRISPR/Cas9-based
disease-resistant aquaculture could strengthen the overall industry in developing regions,
contributing to economic development and integration into global aquaculture markets.
However, several potential challenges must be addressed. The high initial costs and
intellectual property restrictions associated with CRISPR/Cas9 technology may limit access
for small-scale and resource-constrained producers, potentially worsening existing inequal-
ities [96]. To ensure equitable access, careful policies and support mechanisms are needed
to prevent the marginalization of vulnerable groups. The introduction of CRISPR/Cas9-
based aquaculture could disrupt traditional fishing and aquaculture practices, potentially
displacing communities that rely on these activities for their livelihoods [94]. Inclusive
stakeholder engagement and transition support are essential to mitigate these challenges
and ensure a just and sustainable transformation.
Additionally, the widespread adoption of CRISPR/Cas9-modified aquaculture species
could pose unforeseen ecological risks, such as disrupting local ecosystems or introducing
invasive species [96]. Robust environmental impact assessments and appropriate safe-
guards are necessary to minimize these risks. The introduction of novel biotechnologies
including CRISPR/Cas9 in aquaculture may also face resistance or skepticism from local
communities due to cultural, religious, or traditional beliefs.

10. CRISPR-Based Aquaculture on Global Aquatic Product Trade and Food Security in
Developing Regions
The successful adoption of CRISPR-based disease-resistant aquaculture has the poten-
tial to significantly increase the overall production and supply of certain aquatic species in
global markets [94]. Regions with access to CRISPR technology may gain a competitive
advantage in the global aquatic product trade, potentially outcompeting other producers
and altering the relative competitiveness of different aquaculture-producing countries.
Additionally, the introduction of CRISPR-based aquaculture technologies may lead to new
trade barriers, such as regulations or tariffs, aimed at protecting domestic aquatic product
industries in certain countries.
However, the patenting and licensing of CRISPR-based aquaculture technologies
could result in increased market concentration, with a few multinational corporations or
research institutions controlling the supply and distribution of these modified aquatic
species [96]). This concentration of power could have far-reaching implications for global
seafood trade, impacting prices, accessibility, and the bargaining power of small-scale
producers in developing regions.
On the positive side, enhanced productivity and reduced disease-related losses asso-
ciated with CRISPR-based aquaculture can make aquatic products more affordable and
accessible in developing regions, thereby contributing to improved food security and nutri-
tion [95]). Nevertheless, the introduction of CRISPR-modified aquaculture species presents
both benefits and challenges for vulnerable populations reliant on aquatic products for
essential nutrients.
The widespread implementation of this technology may disrupt traditional fishing
practices and local food systems in developing regions, leading to unintended conse-
quences such as the displacement of small-scale producers and the erosion of local food
cultures [94]). Moreover, the high costs and intellectual property barriers associated with
CRISPR technology may hinder access to disease-resistant aquaculture species for resource-
Int. J. Mol. Sci. 2024, 25, 9299 11 of 23

constrained producers and consumers in developing regions [96]. To address these issues,
careful policy interventions and support mechanisms are essential to ensure equitable
access and prevent the marginalization of vulnerable groups, including small-scale and
subsistence aquaculture producers, indigenous and local communities, and women and
marginalized genders.
The large-scale adoption of CRISPR-modified aquaculture species could also have
ecological repercussions, such as disrupting local ecosystems and introducing invasive
species [96]. Robust environmental impact assessments and appropriate safeguards are
necessary to mitigate these risks.
Governments and international development organizations can help address these
challenges by providing targeted subsidies and financial assistance to small-scale producers
and marginalized communities in developing regions. These subsidies can cover the costs of
CRISPR-based aquaculture technology acquisition, implementation, and capacity building,
enabling these resource-constrained groups to benefit from the productivity and disease
resistance offered by CRISPR-modified aquaculture species.
Policymakers can also work to establish fair and accessible licensing schemes for
CRISPR-based aquaculture technologies. This could involve negotiating reasonable royalty
rates, tiered pricing structures, or even compulsory licensing arrangements to ensure that
producers in developing regions can affordably access these transformative innovations.
Additionally, governments and international organizations can facilitate the trans-
fer of CRISPR expertise and technical knowledge to research institutions and extension
services in developing regions. This can be achieved through training programs, joint
research collaborations, and the establishment of technology demonstration centers to
build local capacity and empower small-scale producers to effectively utilize CRISPR-based
aquaculture technologies.
Comprehensive regulatory frameworks should be developed to ensure the safe and
responsible use of CRISPR-modified aquaculture species, including robust environmental
impact assessments and biosafety measures. These regulations should balance the need for
technological advancement with the protection of local ecosystems and traditional food
systems in developing regions.
Finally, policymakers and development agencies should actively engage with local
communities, small-scale producers, and other key stakeholders to understand their needs,
concerns, and priorities. This inclusive approach will help ensure that policy interventions
and support mechanisms are tailored to the specific contexts and vulnerabilities of marginal-
ized populations, promoting equitable access to CRISPR technology in aquaculture.

11. Mitigation Strategies for the Sustainable Application of CRISPR/Cas9 in

Aquaculture: Identifying Environmental and Ecological Impacts
The application of CRISPR/Cas9 technology in aquaculture shows promise for im-
proving desirable traits in fish, such as growth rates, disease resistance, and stress tolerance.
However, there are concerns about potential environmental and ecological impacts. One
major concern is the possibility of gene flow from genetically modified fish to wild popula-
tions, which could lead to genetic pollution, reduced genetic diversity, and instability in
ecosystems. Gene flow has the potential to disrupt aquatic ecosystems and have unforeseen
ecological effects. CRISPR/Cas9 can also result in unintended off-target effects, where
genes other than the intended targets are modified, potentially causing unpredictable out-
comes [97]. To prevent unintended ecological consequences, it is crucial to assess off-target
effects and understand broader ecological interactions. Off-target effects can result in
unintended genetic mutations that disrupt gene function and impact cellular processes. In
ecological contexts, these effects can change the competition among fish species and the
dissemination of modified traits. Moreover, off-target effects give rise to safety concerns in
medical applications.
Effective containment strategies are essential for minimizing the risk of transgenic
fish escaping and causing gene flow to wild populations. Studies have demonstrated that
Int. J. Mol. Sci. 2024, 25, 9299 12 of 23

implementing rigorous physical containment measures, such as multiple barriers, can

significantly reduce the likelihood of transgenic fish escape. In fact, some strategies have
been shown to decrease the risk by up to 99.9% [98]. Genetic strategies, such as Genetic
Use Restriction Technologies (GURTs), can limit the spread of transgenes by approximately
99% [97]. These technologies aim to prevent genetically modified organisms from surviving
and reproducing in the wild. By implementing multiple, independent, and redundant
genetic safeguards, the probability of functional transgene escape can be reduced to less
than one in a million [99].
Environmental risk assessments are crucial for evaluating potential adverse impacts.
They can mitigate up to 80% of adverse environmental effects [100]. Incorporating ecologi-
cal modeling and field trials can improve prediction accuracy by up to 90%. Comprehensive
monitoring programs and adaptive management strategies are vital for identifying and
mitigating environmental impacts, reducing undetected impacts by up to 75% [98] and
mitigating long-term irreversible impacts by up to 80% [101].
To address the risks associated with CRISPR/Cas9 in aquaculture, it is important to
establish a comprehensive regulatory framework and rigorous biosafety protocols. These risks
include potential escape and interbreeding with wild populations, transferring engineered
traits, and altering the fitness and behavior of wild fish, potentially disrupting the balance of
ecosystems and causing cascading effects throughout the food web [102]. Non-target species
may also experience unpredictable ecological impacts, leading to potential biodiversity loss.
To start, implement specific containment strategies, such as biotechnological ap-
proaches like triploid induction, to produce fish that are sterile or have limited repro-
ductive capacity. Additionally, ensure rigorous physical containment by using secure
enclosures [97].
However, long-term monitoring programs that track ecological effects, including
changes in population dynamics, biodiversity, and ecosystem functions must be established.
Develop adaptive management strategies to respond quickly to unforeseen ecological
impacts, adjusting practices based on monitoring results and emerging scientific insights.
Moreover, involve diverse stakeholders in decision-making processes, including local
communities, scientists, policymakers, and industry representatives. Utilize effective com-
munication strategies to raise public awareness about the risks and benefits of CRISPR/Cas9
technology, fostering informed decision-making and building public trust [63].
Furthermore, establish global standards and guidelines for risk assessment and regu-
latory oversight to ensure consistency across countries. Facilitate the transfer of CRISPR
expertise and technical knowledge to research institutions and extension services in devel-
oping regions, building local capacity and promoting the responsible use of this technology.

12. Challenges and Limitations Genome Editing Technology in the Genetic

Improvement of Fish in Aquaculture
Diseases pose significant challenges for fish farmers, impacting investment plans and
increasing costs. Although CRISPR/Cas9 is a promising tool for genetic improvement in
aquaculture, it carries potential risks, such as the introduction of new traits that might affect
disease dynamics if GMF interbreed with wild populations. Genetic modification itself does
not inherently increase disease transmission, but altered genes can have unintended effects
on immune-related functions, potentially increasing disease susceptibility [103]. Concerns
have been raised about genetically modified Atlantic salmon potentially spreading diseases
to wild populations [104].
Aquaculture technology can impact biodiversity by modifying fish species for favor-
able traits, potentially outcompeting wild populations and altering ecological dynamics.
This may lead to reduced genetic diversity and changes in ecological interactions. Public
concerns about genetically modified organisms (GMOs) influence consumer behavior, af-
fecting market demand and price. Additionally, ethical issues related to animal welfare
and unforeseen outcomes associated with CRISPR/Cas9 in animal breeding emphasize the
need for appropriate regulations and oversight.
 potentially spreading diseases to wild populations [104].
Aquaculture technology can impact biodiversity by modifying fish species for
favorable traits, potentially outcompeting wild populations and altering ecological
dynamics. This may lead to reduced genetic diversity and changes in ecological
interactions. Public concerns about genetically modified organisms (GMOs) influence
Int. J. Mol. Sci. 2024, 25, 9299
consumer behavior, affecting market demand and price. Additionally, ethical issues 13 of 23

related to animal welfare and unforeseen outcomes associated with CRISPR/Cas9 in

animal breeding emphasize the need for appropriate regulations and oversight.
CRISPR/Cas9 technologyfaces
CRISPR/Cas9 technology faceschallenges
challengessuch suchasasoff-target
off-targeteffects,
effects,regulatory
regulatorybarriers,
barriers,
and public perception issues, which can affect environmental sustainability
and public perception issues, which can affect environmental sustainability and fish health. and fish health.
Efforts are underway to improve specificity and reduce off-target
Efforts are underway to improve specificity and reduce off-target effects. The cost and effects. The cost and
scalability of CRISPR/Cas9 applications are important considerations,
scalability of CRISPR/Cas9 applications are important considerations, alongside ongoing alongside ongoing
discussions about
discussions about ethical
ethicalissues
issuesand andtechnological
technological limitations.
limitations. Although
AlthoughCRISPR/Cas9
CRISPR/Cas9 has thehas
potential to revolutionize aquaculture by editing fish genomes, potential drawbacks include
the potential to revolutionize aquaculture by editing fish genomes, potential drawbacks
reducedreduced
include fertility, fertility,
increasedincreased
disease risk, and ecological
disease risk, and consequences if geneticallyifengineered
ecological consequences genetically
fish interbreed
engineered fishwith native populations
interbreed with native(see Figure 3). (see Figure 3).
populations
ManyCRISPR/Cas9
Many CRISPR/Cas9 applications
applicationsin inaquaculture
aquacultureremainremaintheoretical,
theoretical,requiring
requiringfurther
further
study to assess their risks and benefits. Challenges include limited genetic resources for
study to assess their risks and benefits. Challenges include limited genetic resources for
aquatic species, which complicates the identification of trait-related genes. Additionally,
aquatic species, which complicates the identification of trait-related genes. Additionally,
microinjectionsuccess
microinjection success rates
rates are
are low
low inin egg-laying
egg-laying fish fishdue
dueto tothe
thescarcity
scarcityofofegg
eggmembrane
membrane
material. There is no standard protocol for genome editing in aquatic organisms, with
material. There is no standard protocol for genome editing in aquatic organisms, with
successfulapplications
successful applicationslimited
limitedto tospecies
specieslikelikeslipper
slippermollusks,
mollusks,shrimp,
shrimp,and andoysters
oystersduedueto
to technical
technical constraints
constraints [19,105,106].
[19,105,106].
Further refinement
Further refinement of CRISPR/Cas9
CRISPR/Cas9 techniquestechniquesisisnecessary
necessarytotofullyfullyrealize
realizetheir
their
potentialininaquaculture.
potential aquaculture. Long
Long generation
generation intervals
intervals in aquatic
in aquatic speciesspecies
makemakegenome genome
editing
editing time-consuming,
time-consuming, but combiningbut combining it with technology
it with surrogacy surrogacy technology
may providemay provide
a solution a
[107].
solution [107]. Producing sterile organisms is the preferred strategy for commercial
Producing sterile organisms is the preferred strategy for commercial applications to protect
applicationsproperty
intellectual to protect and intellectual property and
prevent unintended prevent
genetic unintended
invasion. genetic
Evidence invasion.
suggests that
Evidence suggests that germ cells in Atlantic salmon can be manipulated to produce sterile
germ cells in Atlantic salmon can be manipulated to produce sterile offspring, mitigating
offspring,
risks mitigating
associated risks associated
with modified with modified
fish escaping into thefishwild escaping
[31]. into the wild [31].

CRISPR-Cas
New diseases & Science &
Extinction Environment

Environ. Escape New strains

Public perception
development

Invasion Competition Health

Belief
Less Laws & Concern
demand Policies

Figure3.3.Depicts
Figure Depictsthethe summarized
summarized challenges
challenges of developing
of developing new strains
new strains using CRISPR/Cas9
using CRISPR/Cas9 technol-
technology. The main arrow pointing downwards represents the development process, while
ogy. The main arrow pointing downwards represents the development process, while smaller arrows
smaller arrows indicate the various challenges associated with accepting the new strain resulting
indicate the various challenges associated with accepting the new strain resulting from this technol-
ogy. In Figure 3, two major challenges to the acceptability of the new strain through CRISPR/Cas9
technology are illustrated. These challenges are: (1) the potential escape of the new strain into
the environment, which could lead to the invasion of indigenous strains, the introduction of new
diseases, and increased competition, and (2) the concern that modified new strains may lead to certain
public perceptions, such as health concerns, religious/personal beliefs, and environmental concerns.
However, it is important to note that public perception towards the development of new strains is
influenced by people’s beliefs, values, attitudes, needs, and interests [108]. Personal awareness of
stimuli also plays a significant role in shaping attitudes towards science and the environment.
Int. J. Mol. Sci. 2024, 25, 9299 14 of 23

13. Public Acceptability and Concern for Genetically Modified Fish

Public concern plays a crucial role in the advancement of genetic modification in
aquaculture. The acceptance of genetically modified fish, especially those altered using
CRISPR/Cas9, is influenced by cultural, ethical, and environmental factors [109,110]. It is
important to understand these concerns to successfully develop and implement genetic
modification technologies in aquaculture.
Consumers prioritize safety and quality when it comes to genetically modified fish.
They are concerned about potential risks, including toxicity, allergenicity, and changes in
nutritional value. Ethical considerations are also significant in the genetic modification de-
bate. Questions about the moral status of animals, the idea of naturalness, and the value of
species greatly affect public perception and acceptance [111]. Furthermore, environmental
impacts such as gene flow to wild populations and biodiversity changes raise significant
public concerns. Studies suggest that genetically modified fish may affect disease resistance,
behavior, and reproductive patterns, leading to ecological questions [112,113].
Cultural attitudes towards genetic modification differ across regions. For example,
Europeans have historically been skeptical of genetically modified foods compared to other
regions [114]. This skepticism is evident in the strict regulations imposed by the European
Union on GMOs, requiring rigorous risk assessments and labeling to ensure safety and
consumer trust.
On the other hand, the United States generally takes a more permissive approach to
GMOs, including genetically modified fish. Regulatory bodies like the FDA focus on evalu-
ating safety and environmental impact, but often face criticism for being too lenient [111].
In contrast, Japan adopts a cautious stance, taking public opinion into account and priori-
tizing safety and consumer preferences in its regulatory approach [109]. Canada follows a
science-based framework, having approved genetically modified salmon for production
and consumption while emphasizing transparency and public engagement [110].
In developing countries, policies vary widely and are often influenced by economic
needs, technological capacity, and international trade considerations. Public acceptance in
these regions tends to be lower, with significant concerns about environmental impacts and
food security.
Despite high public skepticism due to cultural and ethical considerations, the Euro-
pean Union maintains strict regulations for GMOs [115]. The United States takes a more
lenient stance on GMOs, prioritizing safety and consumer preferences. Japan, on the other
hand, approaches GMOs with caution, also emphasizing safety and consumer preferences.
According to Ishii and [109], public opinion plays a significant role in shaping Japan’s
more conservative regulatory stance. In Canada, a science-based regulatory framework
is utilized, with a focus on transparency and public engagement. As demonstrated by
the approval of genetically modified salmon [110]. In order to enhance public acceptance
of genome-editing technologies, it is important to consider cultural values and concerns,
establish trust with community leaders, and ensure transparent regulatory processes and
open dialogue. Education and awareness initiatives can effectively address fears and
misconceptions, while ongoing monitoring of ecosystem impacts and long-term effects is
crucial for maintaining public confidence. By implementing these measures, we can foster
a more informed and supportive public perspective on genome-editing technologies.
The acceptance of genetically modified fish by the public is a complex and ever-
evolving issue. Cultural, ethical, and environmental factors must be carefully considered.
The differences in public opinion and policy among countries underscore the importance of
tailored regulatory approaches and effective communication strategies. As technologies like
CRISPR/Cas9 continue to advance, ongoing dialogue and transparent governance will be
essential for addressing public concerns and promoting acceptance. The journey towards
wider acceptance of fish modified using CRISPR/Cas9 technology should involve the
active participation of the public, stakeholders, and experts in a constructive and inclusive
manner, as summarized in Table 3.
Int. J. Mol. Sci. 2024, 25, 9299 15 of 23

Table 3. Key Factors Influencing Public Perception and Acceptance of Genetically Modified Fish.

Public Concerns Impacts Ref.

Awareness and It helps to increase public understanding and awareness of GMOs, particularly
[10,102]
education CRISPR-modified fish, and can significantly impact their acceptance and safety.
It improved nutrition and reduced environmental impact. Emphasizing safety assessments [102,
Benefits and risks
and risk mitigation strategies can significantly influence public opinion. 116]
Ethical and Public acceptance of genetically modified fish may be influenced by ethical and
[116,
environmental environmental concerns. Clear communication, transparency, and engagement play an
117]
considerations essential role in gaining public trust.
Public participation, achieved through an open dialogue among various stakeholders such
Transparency and
as consumers, scientists, policymakers, and environmental organizations, will foster trust [102]
engagement
and acceptance.
Food labeling and Clear labeling and transparent information about modified fish products can assist [116,
consumer choice consumers in making informed decisions regarding food consumption and safety. 118]
It provides potential environmental and sustainable aquaculture benefits, such as reduced
Environmental
antibiotic use and improved resource efficiency, which could have a positive impact on [102]
sustainability
public acceptance.

14. Potential Solution to Public Perceptions and Concerns Regarding the Challenges of
Genome Editing Fish
The application of genome editing technologies, especially CRISPR/Cas9, in aquacul-
ture presents both opportunities and challenges. These technologies have the potential to
improve growth rates, disease resistance, and environmental adaptability in fish, thereby
enhancing aquaculture productivity and sustainability [119]. However, releasing genet-
ically modified fish into the wild raises significant environmental and ethical concerns,
requiring comprehensive environmental risk assessments [120].
Thorough environmental risk assessments are essential before introducing genetically
modified fish into natural ecosystems. These assessments aim to identify and mitigate
potential ecological risks, such as effects on biodiversity and ecosystem stability. By
addressing these concerns, researchers and stakeholders can ensure that the use of genome-
edited fish aligns with sustainable aquaculture practices [121] (see Table 4).
Effective governance relies on collaboration among academia, industry, regulatory
agencies, and policymakers to establish comprehensive regulations that promote responsi-
ble use of CRISPR/Cas9 technology in aquaculture. These frameworks must ensure that
ethical and safety standards are met while maximizing the benefits of genetic enhance-
ments [122]. Clear guidelines and transparent communication with the public are essential
for building trust and addressing societal concerns [123].
Monitoring genetically modified fish populations is vital to evaluate their long-term
impact on ecosystems and aquaculture sustainability. Developing robust monitoring meth-
ods can help detect unexpected effects and facilitate adaptive management strategies [55].
This approach ensures that any adverse outcomes can be addressed promptly, maintaining
ecological balance and public confidence in genome editing technologies.
In addition to CRISPR/Cas9, other gene editing techniques like base editing and prime
editing offer precise genomic modifications. These technologies provide additional tools
for achieving targeted genetic improvements in aquaculture species, broadening the scope
of potential enhancements [123,124]. Continued research and development in these areas
can diversify the genetic strategies available for sustainable aquaculture.
Ensuring fair access to CRISPR/Cas9 technology is crucial for fostering innovation and
preventing monopolization. Open-source platforms, collaborative research initiatives, and
affordable licensing models can facilitate broader utilization of genome editing tools among
researchers and industry stakeholders [125]. By promoting inclusivity and collaboration,
the aquaculture sector can maximize the potential of genome editing to address global food
security challenges.
Int. J. Mol. Sci. 2024, 25, 9299 16 of 23

Table 4. Advancements and Safety Measures in CRISPR/Cas9 Gene editing for Aquatic species.

Remedies Impact Ref.

It improves the precision of developing guide RNA molecules that are more efficient and
Improved precision [126]
accurate, minimizing off-target effects and enhancing gene editing specificity.
It enables scientists to develop new delivery methods, such as electroporation,
Enhanced delivery
microinjection, or transfection, to improve the efficiency of delivering CRISPR/Cas9 [127]
methods
components into fish embryos for gene editing in aquaculture.
CRISPR/Cas9 helps researchers to gene drive systems to quickly introduce advantageous
Gene drive systems [44,128]
traits, such as disease resistance or enhanced growth, into a fish population.
Secure laboratories and greenhouses are utilized to contain and minimize the risk of
Biosecurity measures [119]
accidental release of GMOs into the environment.
Educating the public improves understanding of CRISPR/Cas9, which in turn enables
Public engagement and
better comprehension of its mechanisms, benefits, and limitations in aquaculture and [102]
education
other applications.
CRISPR/Cas9 allows for the simultaneous targeting and modification of multiple genes or
Targeting complex traits [129]
genetic loci in fish species.
The ethical discussions regarding gene editing in aquaculture are important, as they [116,
Ethical considerations
consider animal welfare, environmental impact, and potential unintended consequences. 124]

15. Ethical Considerations of the Use of CRISPR Technology in Aquaculture and Its
Impact on Food Security
The emergence of CRISPR/Cas9 technology has revolutionized various fields, includ-
ing aquaculture, by providing the potential for precise genetic modifications in aquatic
species. These modifications offer the promise of improved growth rates, disease resistance,
and overall productivity. However, the use of CRISPR/Cas9 in aquaculture presents several
ethical and practical concerns that need to be addressed to fully maximize its benefits while
minimizing potential drawbacks.
One major ethical concern regarding CRISPR-based aquaculture technologies is ensur-
ing equitable access, especially for small-scale and marginalized producers in developing
countries. There is a risk that advanced technologies like CRISPR may predominantly be
accessible to large-scale producers in wealthier nations, potentially worsening existing
disparities in the aquaculture sector [130,131]. This could result in concentrated benefits
for the few, leaving vulnerable populations without access to the potential productivity
gains offered by these innovations. To address this issue, strategies need to be developed
to ensure that small-scale and marginalized producers can also benefit from CRISPR-based
advancements [77,132].
Ecological concerns also arise with the introduction of CRISPR-modified organisms.
There is a risk of unintended escape and proliferation of genetically modified species,
which could disrupt native ecosystems and lead to unforeseen long-term environmental
impacts [86,133]. Therefore, careful risk assessment and stringent regulation are necessary
to mitigate these potential environmental consequences [44,95].
Public resistance to consuming CRISPR-derived seafood is another significant consid-
eration. In regions where genetically modified organisms face skepticism, it is essential
to prioritize transparency, clear labeling, and addressing consumer concerns about food
safety and ethics to gain public acceptance [22,132]. Effective communication and address-
ing ethical concerns will play a crucial role in facilitating the adoption of CRISPR-edited
aquaculture products.
The introduction of CRISPR-based aquaculture also has sociocultural implications.
It has the potential to disrupt traditional fishing and aquaculture practices, alter local
food systems, and possibly undermine the livelihoods and food security of small-scale
producers [134,135]. Therefore, careful consideration needs to be given to the sociocultural
Int. J. Mol. Sci. 2024, 25, 9299 17 of 23

impact of integrating genetic technologies into traditional practices to avoid any adverse
effects on local communities [136,137].
CRISPR-based aquaculture has the potential to increase food security by improving
productivity and reducing costs of fish and seafood. This could make them more affordable
and accessible for low-income consumers in developing regions [90,131]. It would also en-
hance food and nutrition security by increasing the availability of affordable, nutrient-rich
animal protein. However, introducing CRISPR-modified products into global trade could
disrupt existing markets and supply chains. This could lead to trade barriers and uneven
distribution of benefits [130,131]. To address these risks and concerns, inclusive gover-
nance frameworks are needed. These frameworks should involve diverse stakeholders,
including small-scale producers, local communities, and consumers, in the development
and regulation of CRISPR-based aquaculture technologies [134,138,139].
In conclusion, while CRISPR-based aquaculture offers opportunities for enhancing
productivity and food security, it requires a balanced approach that considers ethical, eco-
logical, and sociocultural challenges. Steps such as ensuring equitable access, mitigating
environmental impacts, addressing public concerns, and considering sociocultural implica-
tions are essential to realize the full potential of CRISPR-based aquaculture technologies
while protecting the interests of all stakeholders involved.

16. Conclusions
The advancement of genetic improvement in fish species is crucial for the evolution of
aquaculture, especially in rapidly expanding regions like Asia. CRISPR/Cas9 technology
has emerged as a transformative tool in this field, allowing precise modifications to enhance
desirable traits such as disease resistance, growth rates, and feed conversion efficiency.
This gene-editing technology has the potential to revolutionize aquaculture by providing
solutions to long-standing challenges in disease management and genetic optimization.
However, the application of CRISPR/Cas9 in aquaculture also raises significant con-
cerns. Ecological impacts, such as the potential escape of genetically modified fish into
natural habitats and their effect on native species, as well as social and ethical considerations
regarding public perception and acceptance, need to be addressed.
To maximize the potential of CRISPR/Cas9 technology while mitigating associated
risks, future research should focus on several crucial areas. One key direction is the
development of advanced gene-editing techniques that ensure greater precision and min-
imize off-target effects. Improvements in delivery methods, such as enhanced vectors
and less invasive procedures, could enhance the efficiency and safety of gene editing in
aquatic species.
Additionally, long-term studies are essential to assess the ecological impacts of CRISPR-
modified fish on natural ecosystems. Comprehensive risk assessments and monitoring
programs will be crucial for understanding and managing the environmental implications.
Public acceptance and regulatory frameworks must adapt to address consumer concerns
and establish clear guidelines for the responsible use of gene-edited organisms.
Furthermore, future research should explore the integration of CRISPR/Cas9 tech-
nology into sustainable aquaculture practices. This involves developing strategies for the
responsible management of genetically modified fish in both controlled environments and
open systems. Advances in genomics and biotechnology could enable the creation of fish
strains that are not only optimized for growth and disease resistance but also contribute to
the overall sustainability of aquaculture operations.
In conclusion, CRISPR/Cas9 technology has immense potential to advance the aqua-
culture industry by improving fish genetics and promoting more sustainable practices. By
addressing current challenges and focusing on forward-looking research, the aquaculture
sector can harness the full capabilities of gene editing technologies to build a more resilient,
efficient, and environmentally responsible industry. Collaboration among researchers, reg-
ulators, and stakeholders will be crucial in realizing the long-term benefits of CRISPR/Cas9
technology in aquaculture as we navigate these developments.
Int. J. Mol. Sci. 2024, 25, 9299 18 of 23

Funding: This work was supported by grants from the “JBGS” Project of Seed Industry Revitalization
in Jiangsu Province (JBGS [2021]123), the Central Public-Interest Scientific Institution Basal Research
Fund (CAFS) (NO. 2022XT01), and the Central Public-Interest Scientific Institution Basal Research
Fund, Freshwater Fisheries Research Center, CAFS (2023TD39).
Conflicts of Interest: The authors report that they have no conflicts of interest. The authors alone are
responsible for the content and writing of the paper.

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