* By Aquaculture Magazine Editorial Team
Aquaculture is a crucial sector for global food production and trade. Gene editing techniques, particularly clustered regularly interspaced short palindromic repeats-Cas9 (CRISPR/Cas9), are transforming the industry by enabling precise genetic modifications in fish species. These advancements enhance disease resistance, growth, and reproduction, making aquaculture more efficient and sustainable. Additionally, RNA interference (RNAi) is emerging as a powerful tool for gene silencing and functional genomics.
Introduction
Fish industry
Global fish production has become the fastest-growing food or in recent decades. Aquaculture now produces more fish biomass than beef and even capture fisheries (including non-edible species) (Edwards et al., 2019). In the 21st century, aquaculture and fisheries production have expanded significantly (Food Agriculture Organization, FAO, 2022). Aquaculture production surged from 5 million to 63 million tons, while capture fisheries increased from 69 million to 93 million tons over 30 years (FAO, 2014).
Food fish consumption grew at an annual rate of 1.4%, reaching 20.5 kg per capita in 2019. In 2020, aquaculture accounted for 178 million tons of food fish, with finfish (57.5 million tons), mollusks (17.7 million tons), and crustaceans (11.2 million tons) being the primary contributors. Asia led production (91.6%), with China dominating since 1991. Other key producers include Vietnam, Bangladesh, Egypt, Norway, and Chile. By 2050, with a projected global population of 10 billion, food production must become more efficient. Fish is an essential protein source, rich in omega-3 fatty acids, vitamins, and minerals (FAO, 2012).
The industry has seen a shift toward farming fed aquatic species, which has driven production growth and reduced fish prices. However, climate change poses a significant challenge, necessitating investments in sustainable practices and new technologies (FAO, 2022; Boyd et al., 2020). Genetic modification could support aquaculture expansion and improve fish health (World Bank, 2013).
Gene editing
Gene engineering techniques emerged in the late 20th century to modify genomes precisely (Perota et al., 2016). Advances in genetic engineering have significantly impacted medicine, particularly in gene therapy (Cathomen, 2008). Gene editing allows targeted deoxyribonucleic acid (DNA) modifications via engineered nucleases. It enables precise trait alterations, such as allele corrections or interspecies transfers (Malik, 2020; Gratacap, 2019). DNA repair mechanisms, primarily DNA repair mechanisms, primarily nonhomologous end-joining (NHEJ) and homology-directed repair (HDR), are crucial for postediting recovery (Takate, 1998; Lans, 2012).
Zinc Finger Nucleases (ZFNs)
ZFNs are engineered proteins used for gene editing (Choo, 1994). They consist of zinc-finger DNA-binding domains fused with the Fok I endonuclease. ZFNs introduce double-strand breaks (DSBs) at specific genome sites, triggering nonhomologous end joining (NHEJ) mediated repairs (Cathomen, 2008; Tang, 2015). Despite their precision, off-target effects remain a challenge (Carroll, 2014). This technique has been successfully applied to zebrafish and human cells (Palpant, 2013; Hockemeyer, 2009).
Transcription Activator-Like Effector Nucleases (TALENs)
TALENs, derived from Xanthomonas bacteria, employ DNA-binding proteins and Fok I nucleases to induce double-strand break (DSBs). They offer high specificity and efficiency but are labor-intensive and expensive (Joung, 2013; Lamb, 2013). Their applications include gene modification in model organisms, but the need for simpler alternatives has led to the development of more advanced techniques (Malik, 2020).
Clustered Regularly Interspaced Short Palindromic Repeats- Cas9 System (CRISPR/Cas9)
CRISPR/Cas9, discovered in 2012, revolutionized gene editing due to its simplicity, cost-effectiveness, and efficiency (Malik, 2020; Jinek, 2012). It uses guide RNA (sgRNA) to direct the Cas9 endonuclease to target DNA sequences, enabling precise edicts. The system is widely used in various fields, including biomedical and agricultural applications (Xu, 2020).
Gene silencing
RNA interference (RNAi) regulates gene expression by inhibiting mRNA translation (Singh, 2019; Hood; 2004). Dicer enzymes process doublestranded RNA (dsRNA) into small interfering RNA (siRNA), which guides the RNA-induced silencing complex (RISC) to degrade target mRNA (Singh, 2019; Sen, 2006). RNAi plays a role in immune defense and gene therapy innovations (Hood, 2004 Wang, 2007).
Applications in aquaculture
Aquaculture faces significant challenges from infectious pathogens. Gene modification techniques can improve disease resistance, enhance production efficiency, and support fish health (Gotesman, 2018). Fish serve as effective bioreactors for medical applications due to their short generation intervals and costeffective maintenance. Gene editing has been successfully applied in fish breeding, spawning, and disease management, offering promising advancements for the industry (Lucas, 2013).
Gene Editing in Fish Farm Species Using CRISPR/Cas9 and Other Gene Editing Tools
Over 70 aquatic fish genomes have been deciphered in recent decades. CRISPR/Cas9 and other gene editing tools are advancing aquaculture by enabling sterility, disease resistance, pigmentation, and growth improvement. These innovations offer solutions to significant challenges in aquaculture.
Gene editing in fishery science
Zebrafish, widely used as a model organism, has contributed to breakthroughs in genetic modifications, toxicology, and host-pathogen interactions. CRISPR/Cas9 has successfully modified genes in various species, including Atlantic salmon, medaka, and tilapia. In Nile tilapia, targeted gene mutations have been efficiently transmitted to offspring, demonstrating the high efficacy of CRISPR/ Cas9 in non-model species.
Gene editing in mono-sex population
Gene editing can create mono-sex fish populations, improving yield rates and preventing unwanted reproduction in the wild. In tilapia, genes determining female sex have been knocked out to influence sex differentiation. Traditional hormone treatments for sex reversal pose ecological risks, but gene editing offers a sustainable alternative. Medaka fish studies using TALEN demonstrated effective gene knockout for reproductive regulation.
Gene editing in sterility of fish
Sterility prevents ecological risks posed by escaped farmed fish. Using ZFN technology, sterile channel catfish were produced by disrupting the pituitary luteinizing hormone gene. In Atlantic salmon, CRISPR/Cas9 knockouts of the dead end (dnd) gene eliminated germ cells, preventing gene flow between farmed and wild populations. Pigmentation genes (slc45a2) have also been targeted, producing albino salmon.
Gene editing in reproduction
The kisspeptin-encoding gene—Kiss1 (Kiss1/Grp54) system regulates reproduction in vertebrates. In zebrafish, TALEN-engineered mutations in kiss2 genes showed that reproductive potential remained unaffected, suggesting differences between mammalian and fish reproductive strategies.
Gene editing in fast-growing fishes
Cold-water fish species often have slow growth rates due to genetic and environmental factors. CRISPR/ Cas9 has been used to disrupt myostatin, a muscle growth inhibitor, in common carp, leading to increased muscle mass. Similar approaches have been applied to slow-growing species like snow trout to enhance growth rates.
Gene editing in ornamental fishes
Targeted gene editing has facilitated the development of ornamental fish desired colors and pigmentation. ZFN, TALEN, and CRISPR/Cas9 techniques have been used to mutate pigmentation-related genes in zebrafish, resulting in inheritable light-colored eyes and loss-of-function phenotypes. The CRISPR/Cas9 system has enabled efficient gene knockouts, affecting multiple loci simultaneously (Figure 1).
Gene editing in pigmentation
TALEN gene editing in cavefish (Astyanax mexicanus) targeted pigmentation genes resulting in mosaicpatterned loss of melanin. A recent study in Nile tilapia demonstrated heritable red pigmentation through CRISPR/Cas9-mediated mutation in the slc45a2 gene, confirming its applicability in aquaculture (Figure 2).
Gene editing in growth
Growth enhancement via gene transfer has led to size increases of up to 300%. CRISPR/Cas9-mediated knockout of the myostatin (MSTN) gene in channel catfish increased muscle growth and body weight. This approach could significantly boost aquaculture productivity.
Gene editing in body configuration
Transgenic modifications have improved fish nutritional properties. Zebrafish engineered with salmon desaturase genes showed increased omega-3 fatty acid levels, a result also observed in carp and catfish. These modifications enhance the nutritional value of farmed fish.
Gene editing in oomycetes
Aphanomyces invadans causes epizootic ulcerative syndrome (EUS), a significant threat to fish populations. CRISPR/Cas9 targeted serine protease genes in this pathogen, effectively preventing virulence. Experimental fish exposed to gene-edited A. invadans showed no signs of infection, highlighting CRISPR/Cas9´s potential in disease management and drug development. These advantages demonstrate the transformative potential of gene editing in aquaculture, improving sustainability, productivity, and ecological balance.
Gene Silencing in Fish Medicine
The RNA interference (RNAi) tool has been widely used to analyze gene function in aquatic diseases and develop antiviral therapies for livestock and aquatic species. Most studies on RNAi in fish have been conducted on zebrafish (Danio rerio), a key model for aquaculture and biomedicine.
Gene silencing in viral disease of fish medicine
RNAi-based therapies have been used to inhibit viral replication. In one study, small interfering RNA (siRNA) targeted the nucleoprotein (N) and phosphoprotein (P) transcripts of the viraemia of carp virus (SVCV) in epithelioma papulosum cyprinid (EPC) cells, reducing replication. Another study used siRNA to inhibit cyprinid herpesvirus-3 (CyHV-3) in common carp brain (CCB) cells by targeting thymidine kinase (TK) and DNA polymerase (DP) genes. Multiple viral genes must be targeted for optimal inhibition.
Gene silencing for gene function studies in fish medicine
Short hairpin RNA (shRNA) has successfully inhibited zebrafish gene expression. Studies targeted wnt5b and zDisc1 genes, demonstrating effective knockdown. Another study used an in vivo-transcribed T7 plasmid system to silence the green fluorescent protein (gfp) and no tail (nt1) genes, confirming RNAi machinery activity in zebrafish cells.
Gene silencing in oomycetes
In Saprolegnia parasitica, an aquaculture pathogen, silencing the tyrosinase (SpTyr) gene reduced melanin production and altered cell morphology, demonstrating RNAi as a functional tool.
Gene silencing in crustaceans
Despite limited genomic data on crustaceans, RNAi has proven effective. Studied showed RNAi-mediated inhibition of white spot syndrome virus (WSSV), yellow head virus (YHV), and Taura syndrome virus (TSV) in shrimp (Penaeus monodon). Additionally, RNAi targeted the pmYRP65 receptor protein, preventing YHV entry.
In bacterial infections, silencing the prophenoloxidase (proPO) gene in freshwater crayfish (Pacifastacus leniusculus) impaired immune defenses, while silencing pacifastin enhanced immunity. RNAi also inhibited crustacean hyperglycemic hormone (CHH) in Litopenaeus schmitti, reducing glucose levels. RNAi continues to be a powerful tool in understanding aquatic diseases.
Conclusions
The ethical concerns surrounding CRISPR-Cas9 revolve around balancing benefits and risks. Off-target mutations, unintended genetic alterations, and potential cell death pose significant challenges. Efforts to improve accuracy through enzyme variants are ongoing. The high cost of tools and reagents further limits accessibility. Environmental risks include the unintended release of genetically modified (GM) organisms, potentially disrupting ecosystems through gene drive extinction. Offtarget mutations can amplify across generations, making control difficult. Additionally, concerns exist regarding health risks, biodiversity reduction, and the ethical implications of genetic modification.
Gene editing is transforming aquaculture, enhancing genetic traits through selective breeding and biotechnologies. CRISPR/Cas9 enables precise modifications in species like Atlantic salmon and zebrafish, improving disease resistance and growth. RNA interference (RNAi) also plays a role in gene expression silencing. Despite advancements, regulatory, economic, and ethical challenges remain, influencing public acceptance and industry growth.
This is a summarized version developed by the editorial team of Aquaculture Magazine based on the review article titled “REVIEW: RECENT APPLICATIONS OF GENE EDITING IN FISH SPECIES AND AQUATIC MEDICINE.” developed by: GUTÁSI, A., HAMMER, S., EL-MATBOULI, M., and SALEH, M. – University of Veterinary Medicine, Vienna, Austria. The original article, including tables and figures, was published on APRIL, 2023, through ANIMALS. The full version can be accessed online through this link: https://doi.org/10.3390/ani13071250
