Genomics and Applied Biology 2024, Vol.15, No.2, 99-106 http://bioscipublisher.com/index.php/gab 100 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.
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