1 Introduction

CRISPR-Cas9, a revolutionary genome editing tool, has transformed the landscape of genetic engineering across various fields, including agriculture, biomedicine, and functional genomics. Originating from a bacterial immune system, CRISPR-Cas9 allows for precise, targeted modifications of DNA sequences through the use of a guide RNA (gRNA) that directs the Cas9 endonuclease to specific genomic loci (Arora and Narula, 2017; Li et al, 2021; Richardson et al., 2023). This technology has been employed to generate knockouts, insertions, deletions, and even single-base substitutions, making it a versatile tool for genetic manipulation (Wang et al., 2016; Borrelli et al., 2018). The ease of design, high efficiency, and cost-effectiveness of CRISPR-Cas9 have made it the preferred method for genome editing over other technologies such as zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) (Belhaj et al., 2015; Chen et al., 2017).

Tilapia is one of the most widely farmed fish species globally, known for its rapid growth, adaptability to various environmental conditions, and high nutritional value. It plays a crucial role in food security and the economy of many developing countries. The demand for tilapia continues to rise due to its affordability and the increasing global population (Ahmad et al., 2020). However, challenges such as slow growth rates and susceptibility to diseases pose significant threats to tilapia farming, necessitating innovative solutions to enhance productivity and sustainability in aquaculture (Barrangou and Doudna, 2016).

Improving growth rates and disease resistance in tilapia is essential for maximizing yield and ensuring the sustainability of aquaculture practices. Traditional breeding methods have had limited success in achieving these goals due to the complex genetic traits involved. CRISPR-Cas9 offers a promising alternative by enabling precise genetic modifications that can enhance desirable traits such as faster growth and increased resistance to pathogens (Chen et al., 2017; Borrelli et al., 2018). By targeting specific genes associated with these traits, CRISPR-Cas9 can accelerate the development of superior tilapia strains, thereby addressing the challenges faced by the aquaculture industry (Wang et al., 2017).

The aim of this study is to explore the application of CRISPR-Cas9 mediated gene editing in tilapia, with a focus on its potential to enhance growth rates and improve disease resistance. Specifically, this study aims to provide an overview of the current state of CRISPR-Cas9 technology and its recent advancements. It also highlights the significance of tilapia in global aquaculture, discussing the challenges this species faces. Furthermore, the study discusses the rationale behind utilizing CRISPR-Cas9 for improving both growth rates and disease resistance in tilapia. Additionally, it covers recent studies and developments concerning the use of CRISPR-Cas9 in tilapia and other aquaculture species. The study identifies future directions and potential challenges related to the implementation of CRISPR-Cas9 technology in tilapia farming.

2 CRISPR-Cas9 Mechanism

2.1 Fundamentals of CRISPR-Cas9 gene editing

The CRISPR-Cas9 system is a revolutionary tool for genome editing, offering precision, efficiency, and versatility. It consists of two key components: the Cas9 enzyme, which acts as molecular scissors to cut DNA, and a guide RNA (gRNA) that directs Cas9 to the specific location in the genome to be edited. The process begins with the design of a gRNA complementary to the target DNA sequence. Once the gRNA binds to the target DNA, the Cas9 enzyme induces a double-strand break. The cell’s natural repair mechanisms then kick in, either through non-homologous end joining (NHEJ) or homology-directed repair (HDR), leading to gene disruption or the insertion of new genetic material (Li et al., 2020; Roy et al., 2022). Studies comparing the efficiency of different U6 promoters in CRISPR-Cas9 editing have shown that using species-specific promoters, such as the tilapia U6 promoter, significantly enhances mutational efficiency compared to non-species-specific promoters (Figure 1) (Hamar and Kültz, 2020). This highlights the importance of selecting appropriate regulatory elements to optimize gene editing outcomes.

2.2 Specific applications in aquatic species

CRISPR-Cas9 has been widely adopted in aquaculture to enhance traits such as growth rates, disease resistance, and reproductive control. In tilapia (Oreochromis niloticus), CRISPR-Cas9 has been used to create mutants that exhibit desirable traits. For instance, targeted gene editing has been employed to study sex determination and improve growth rates, which are critical for commercial aquaculture (Li and Wang, 2017; Li et al., 2020). The technology has also been applied to other fish species, such as Atlantic salmon and red sea bream, to enhance disease resistance and growth, demonstrating its broad applicability in the aquaculture industry (Okoli et al., 2021; Roy et al., 2022).

2.3 Challenges and limitations in applying CRISPR-Cas9 to aquatic genome editing

Despite its potential, the application of CRISPR-Cas9 in aquatic species faces several challenges. One major issue is the off-target effects, where unintended regions of the genome are edited, potentially leading to undesirable traits or health issues. Additionally, the identification and functional annotation of trait-related genes remain incomplete, complicating the precise targeting of beneficial traits. Regulatory and public acceptance also pose significant hurdles. Current regulatory frameworks are not fully equipped to address the unique challenges posed by CRISPR-Cas9, and there is ongoing debate about the ethical implications of genome editing in aquaculture (Okoli et al., 2021). Furthermore, technical challenges such as efficient delivery of the CRISPR components into fish embryos and the establishment of stable breeding lines need to be addressed to fully realize the potential of this technology (Li et al., 2020; Roy et al., 2022).

3 Growth Rate Enhancement through CRISPR-Cas9

3.1 Identification of growth-related genes in tilapia

The identification of growth-related genes in tilapia is a critical step in enhancing growth rates through CRISPR-Cas9 mediated gene editing. Recent advancements in genome sequencing and bioinformatics have facilitated the discovery of key genetic loci associated with growth traits. For instance, the Nile tilapia (Oreochromis niloticus) has been extensively studied for its genetic makeup, providing a robust model for gene editing applications. The identification process involves selecting target sites that are crucial for growth regulation, followed by in vitro RNA transcription and microinjection into one-cell stage embryos to create mutants (Li et al., 2020). This foundational work sets the stage for precise genetic modifications aimed at improving growth rates in tilapia.

3.2 Case studies: successful gene editing for growth rate improvement

Several case studies have demonstrated the successful application of CRISPR-Cas9 technology to enhance growth rates in aquaculture species, including tilapia. One notable example is the use of endogenous promoters to drive Cas9 expression in tilapia cell lines, achieving high mutational efficiencies (Hamar and Kültz, 2020). This approach has shown promise in manipulating genetic loci that are directly linked to growth traits. Additionally, the optimization of Cas9 nuclear localization in tilapia cells has further improved the efficiency of gene editing, making it a viable tool for enhancing growth rates (Villapando et al., 2020). These case studies highlight the potential of CRISPR-Cas9 to bring about significant improvements in growth performance, thereby contributing to more efficient aquaculture practices.

3.3 Potential impacts on aquaculture efficiency

The application of CRISPR-Cas9 mediated gene editing in tilapia has far-reaching implications for aquaculture efficiency. By targeting and modifying specific growth-related genes, it is possible to achieve faster growth rates, which can lead to shorter production cycles and increased yield (Roy et al., 2022). This not only enhances the economic viability of tilapia farming but also addresses the growing demand for high-quality protein sources. Moreover, the precision and efficiency of CRISPR-Cas9 technology reduce the time and cost associated with traditional breeding methods, making it a more sustainable option for genetic improvement (Tang et al., 2023). The successful implementation of this technology in tilapia could serve as a model for other aquaculture species, paving the way for widespread adoption and significant advancements in the industry.

4 Disease Resistance Improvement via CRISPR-Cas9

4.1 Key genes involved in disease resistance in tilapia

The application of CRISPR-Cas9 technology in tilapia has opened new avenues for enhancing disease resistance by targeting specific genes associated with immune responses. Key genes involved in disease resistance in tilapia include those encoding for cytokines, antimicrobial peptides, and other immune-related proteins. For instance, genes such as IFN-γ, lyzc, hsp70, and IL-1β have been identified as crucial for mounting an effective immune response against pathogens (Xia et al., 2018). These genes play significant roles in the regulation of immune responses, inflammation, and pathogen recognition, making them prime targets for CRISPR-Cas9 mediated gene editing to enhance disease resistance.

4.2 Case studies: enhancing resistance to common tilapia pathogens

Several case studies have demonstrated the potential of CRISPR-Cas9 in enhancing disease resistance in tilapia. One notable example is the integration of the alligator cathelicidin gene into the genome of channel catfish, which resulted in increased resistance to bacterial infections (Simora et al., 2020). Although this study was conducted in catfish, it provides a proof-of-concept that similar strategies could be applied to tilapia. Additionally, the use of probiotics such as Lactobacillus rhamnosus and Lactococcus lactis has been shown to enhance the immune response and disease resistance in juvenile Nile tilapia, indicating that genetic modifications to enhance the expression of similar immune-boosting genes could be beneficial (Xia et al., 2018). In the genome of channel catfish, researchers successfully inserted a foreign gene at a specific site on chromosome 1 using CRISPR/Cas9 technology, utilizing different promoters to drive the expression of the foreign gene (Figure 2). This strategy highlights the potential of gene editing in enhancing disease resistance in fish species (Simora et al., 2020).

4.3 Implications for aquaculture sustainability

The implications of CRISPR-Cas9 mediated gene editing for disease resistance in tilapia are profound for the sustainability of aquaculture. By reducing the incidence and severity of diseases, genetically enhanced tilapia can lead to higher survival rates, improved growth performance, and reduced reliance on antibiotics and other chemical treatments (Okoli et al., 2021; Roy et al., 2022). This not only enhances the productivity and profitability of aquaculture operations but also addresses environmental and public health concerns associated with the overuse of antibiotics. Furthermore, the development of disease-resistant tilapia strains can contribute to more stable and resilient aquaculture systems, capable of withstanding the challenges posed by emerging pathogens and changing environmental conditions (Elaswad and Dunham, 2018).

5 Environmental and Regulatory Considerations

5.1 Ecological implications of gene editing in aquatic species

The application of CRISPR-Cas9 in tilapia and other aquatic species holds significant promise for enhancing growth rates and disease resistance. However, it also raises ecological concerns. Gene editing can lead to unintended side effects, such as genetic introgression into wild populations, which may disrupt local ecosystems and biodiversity (Gutási et al., 2023; Robinson et al., 2023). The potential for edited genes to spread to wild populations through escapees from aquaculture facilities is a critical issue that needs thorough investigation and mitigation strategies. Additionally, the ecological impacts of gene-edited tilapia must be assessed on a case-by-case basis to ensure that the benefits outweigh the potential risks to the environment (Robinson et al., 2023).

5.2 Risk assessment of genetically modified tilapia in aquatic ecosystems

Risk assessment frameworks are essential for evaluating the safety and potential impacts of genetically modified tilapia in aquatic ecosystems. Current frameworks need to be adapted to address the unique challenges posed by CRISPR-Cas9 technology. These frameworks should incorporate considerations such as off-target effects, the stability of genetic modifications, and the potential for gene flow to wild populations (Okoli et al., 2021). Effective risk assessment must also include long-term ecological studies to monitor the impacts of gene-edited tilapia on local ecosystems and biodiversity (Roy et al., 2022; Robinson et al., 2023). Public and regulatory acceptance of genetically modified organisms (GMOs) in aquaculture will depend on the robustness of these risk assessments and the implementation of appropriate mitigation measures (Okoli et al., 2021).

5.3 Regulatory frameworks governing CRISPR-Cas9 in aquaculture

The regulatory landscape for CRISPR-Cas9 applications in aquaculture is still evolving. Current regulations often do not fully address the specific challenges and opportunities presented by gene editing technologies. For instance, the regulatory status of CRISPR-edited fish, such as the FLT-01 Nile tilapia developed by AquaBounty, varies by region and is not always classified under traditional GMO regulations (Roy et al., 2022). There is a need for updated regulatory frameworks that can accommodate the rapid advancements in gene editing technologies while ensuring safety and sustainability (Okoli et al., 2021). These frameworks should include clear guidelines for the approval, monitoring, and commercialization of gene-edited aquatic species, with a focus on transparency and public engagement (Roy et al., 2022; Robinson et al., 2023).

6 Future Perspectives

6.1 Advances in CRISPR-Cas9 technology for aquaculture

The CRISPR-Cas9 technology has revolutionized genetic engineering in aquaculture, offering precise and efficient genome editing capabilities. Recent advancements have focused on optimizing the CRISPR-Cas9 system for use in various fish species, including tilapia. For instance, detailed protocols have been developed for gene mutation in tilapia, covering target site selection, RNA transcription, and microinjection techniques, which facilitate the establishment of mutant lines (Li et al., 2020). Additionally, the technology has been applied to generate stable and heritable phenotypes, such as the solid-red germline in Nile tilapia, by targeting specific genes like slc45a2 (Segev-Hadar et al., 2021). These advancements not only enhance our understanding of fish genetics but also pave the way for broader applications in aquaculture, including improved growth rates and disease resistance (Gratacap et al., 2019; Gutási et al., 2023).

6.2 Integration of gene editing in commercial tilapia breeding programs

The integration of CRISPR-Cas9 mediated gene editing into commercial tilapia breeding programs holds significant promise for the aquaculture industry. By enabling precise genetic modifications, CRISPR-Cas9 can expedite the breeding process, allowing for the rapid introduction of favorable traits such as enhanced growth and disease resistance (Gratacap et al., 2019). The high fecundity and external fertilization of tilapia make them ideal candidates for large-scale genome editing applications. However, challenges remain, including technical limitations and regulatory hurdles that need to be addressed to fully realize the potential of this technology in commercial settings (Segev-Hadar et al., 2021). Despite these challenges, the successful commercialization of CRISPR-edited fish, such as the red sea bream and the FLT-01 Nile tilapia, demonstrates the feasibility and potential benefits of integrating gene editing into breeding programs (Roy et al., 2022).

6.3 Prospects for broader applications in other aquatic species

The success of CRISPR-Cas9 mediated gene editing in tilapia sets a precedent for its application in other aquatic species. The technology has already been applied to over 20 different aquaculture species, targeting traits such as growth, disease resistance, and reproduction (Roy et al., 2022). For example, genome editing has been used to create a new breed of red sea bream with increased skeletal muscle mass, demonstrating the potential for significant improvements in aquaculture productivity (Kishimoto et al., 2018). Furthermore, the development of gene editing protocols and optimization of Cas9 nuclear localization in fish cell lines will facilitate the application of CRISPR-Cas9 in a broader range of species (Villapando et al., 2020). As the technology continues to advance, it is expected to play a crucial role in addressing the challenges faced by the aquaculture industry, including disease outbreaks and environmental sustainability (Robinson et al., 2023).

7 Concluding Remarks

The application of CRISPR-Cas9 in tilapia has demonstrated significant potential in enhancing growth rates and disease resistance. This gene-editing technology allows for precise modifications in the tilapia genome, leading to the development of desirable traits such as increased growth, improved disease resistance, and controlled reproduction. For instance, CRISPR-Cas9 has been successfully used to create stable and heritable phenotypes, such as the solid-red germline in Nile tilapia, which addresses market demands for specific fish traits. Additionally, the technology has enabled the efficient targeting of non-coding sequences, which play crucial roles in regulating various biological processes. Overall, CRISPR-Cas9 offers a powerful and versatile tool for genetic improvement in tilapia, promising to enhance productivity and sustainability in aquaculture.

The long-term impacts of CRISPR-Cas9-mediated gene editing on the global aquaculture industry are profound. By enabling the rapid and precise development of desirable traits, CRISPR-Cas9 can significantly improve the efficiency and sustainability of fish farming. Enhanced growth rates and disease resistance in tilapia can lead to higher yields and reduced losses due to disease outbreaks, thereby increasing the overall productivity of aquaculture operations. Moreover, the ability to produce genetically stable and uniform fish populations can meet market demands more effectively, potentially leading to higher economic returns for fish farmers. However, the widespread adoption of CRISPR-Cas9 in aquaculture also necessitates addressing regulatory and biosafety concerns to ensure the responsible and sustainable use of this technology. As the technology matures and regulatory frameworks evolve, CRISPR-Cas9 is poised to become a game-changer in the aquaculture industry, driving innovation and growth.

The future of gene editing in aquatic species, particularly through the use of CRISPR-Cas9, is promising yet complex. While the technology offers unprecedented opportunities for genetic improvement, several challenges remain. Technical issues such as off-target effects and the need for efficient delivery systems must be addressed to fully realize the potential of CRISPR-Cas9. Additionally, public perception and regulatory acceptance are critical factors that will influence the adoption and commercialization of gene-edited fish. Despite these challenges, the successful application of CRISPR-Cas9 in tilapia and other fish species demonstrates the feasibility and potential benefits of this technology. As research continues to advance and regulatory frameworks adapt, CRISPR-Cas9 is likely to play a pivotal role in the future of aquaculture, contributing to the development of more resilient, productive, and sustainable fish populations.

Acknowledgments

We are grateful to our colleagues for critically reading the manuscript and providing valuable feedback that improved the clarity of the text.

Conflict of Interest Disclosure

The authors affirm that this research was conducted without any commercial or financial relationships that could be construed as a potential conflict of interest.

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