Comprehensive insights into molecular techniques and applications for genetic modification and targeted mutagenesis in zebrafish

1 Introduction

The zebrafish (ZF) is a member of the Teleostei infraclass and serves as an excellent disease model owing to its high levels of genomic, molecular, anatomical, and physiological similarities to mammals. For instance, research on the ZF heart development has significantly increased our understanding of human cardiovascular diseases (Nguyen et al., 2008). According to Lam and Gong (2006) and Jang et al. (2012), ZF livers can be used as models for liver carcinogenesis. Consequently, ZF can also be easily used to study the underlying causes of human diseases. ZF embryos are easy to work with and observe because they develop quickly outside of the mother and are optically transparent. Gastrulation takes 5 h to complete, whereas the blastula stage lasts only 3 h. At 18 h, the embryo is transparent and features a very well-developed brain, ears, eyes, and segmenting muscles. After 1 day, the majority of the primary organ systems are developed, and segmentation is complete. After 72 h, the embryo breaks out of the chorion, and 2 days later, it starts searching for food. The development of the embryo takes only 4 days. Therefore, genetic modification is easier and less complex than that in mice. The adult ZF matures quickly, taking only approximately 10 weeks to reach sexual maturity. Additionally, zebrafish exhibits a high reproductive capacity under laboratory conditions. When raised under ideal conditions, ZF can lay up to 200 eggs per week (Brand et al., 2002; Carpio et al., 2006). Because it can spawn continuously in high-density aquarium systems throughout the year, providing an abundance of offspring from selected pairs, the transparent ZF embryos and larval stages are an ideal candidate for large-scale genetic approaches to identify novel genes and uncover their specific functions in vertebrates (Pelegri, 2002). Furthermore, ZF makes it possible to image biological processes such as skeletal development and repair in real time. This facilitates the monitoring of skeletal development processes in specially created ZF models of bone diseases. Researchers have reportedly used ZF embryonic stages, which are thought to be among the most effective models for studying bone formation, to study the process of osteogenesis since the first calcified vertebrae appear only 5 days after the egg is fertilized (Carnovali et al., 2019).

ZF has an incredibly hardy nature and can be maintained easily under laboratory conditions. Furthermore, it requires much less space and cost, and reduced maintenance compared to animal facilities used for mammals. These advantages make this a desirable and experimental model organism for studies pertaining to development, toxicology, and transgenesis (Lele and Krone, 1996). Following the release of a complete gene set in 2013 by the ZF genome sequencing project, it was discovered that orthologs for approximately 70% of human genes are present in ZF, and the ZF genome shares significant similarities with the human genome (Howe et al., 2013). These features enable strong forward and reverse genetic approaches to be used to study the molecular and genetic bases of disease and development (Figure 1).

ZF has become much more useful for genetic modification and targeted mutagenesis owing to recent developments in molecular techniques. The CRISPR/Cas9 system has transformed genome editing by enabling gene knockdown and knock-in techniques in various fish species (Hwang et al., 2013; Varshney et al., 2015; Iqbal et al., 2025). Its use in in vivo screening and large-scale functional genomics has been further illustrated by recent research. For instance, genes related to zebrafish tissue regeneration have been found using CRISPR/Cas9-based F0 screening techniques (Lu et al., 2023). Furthermore, systematic gene function research at scale in zebrafish models has been made possible using high-throughput in vivo CRISPR screening tools such as MIC-Drop (Parvez et al., 2023). Moreover, next-generation methods such as base editing and prime editing have improved editing precision and decreased off-target effects by enabling precise nucleotide-level alterations without creating double-strand breaks (DSBs) (Anzalone et al., 2019; Rees and Liu, 2018). Furthermore, the discovery of CRISPR interference and activation (CRISPRi/a) systems has expanded the scope of functional genomics research by enabling targeted and reversible regulation of gene expression (Barrientos et al., 2024). Gene function analysis has been further expedited by high-throughput methods such as F₀ CRISPR screening, which enable quick phenotypic evaluation in the first generation (Davidson et al., 2025). Collectively, these advancements have significantly enhanced the precision, scalability, and applicability of ZF as a model for studying gene function, disease mechanisms, and translational research.

An updated overview of molecular techniques for targeted mutagenesis and genetic manipulation in ZF is given in this article. It covers cutting-edge genome-editing technologies, conventional mutagenesis techniques, and newly created CRISPR-based regulatory systems such as CRISPRi/a. It highlights current developments, such as F₀ CRISPR screening and multiplex genome editing, and connects them to their uses in aquaculture, disease modeling, and functional genomics.

2 Molecular techniques for zebrafish genetic modification

Mutagenesis, defined as the intentional or unintentional modification of DNA at a specific genomic location, is used by scientists to study the effects of mutations on the structure and function of organs (Figure 2). Mutagenesis is essential for understanding gene function and elucidating gene phenotype relationships by introducing controlled genetic variations (De Rienzo et al., 2012; Dos Santos et al., 2022). It has made it possible to do extensive functional genomics studies on ZF and identify genes related to both development and disease (Driever et al., 1996). The ability to produce both gain- and loss-of-function mutations, enable objective gene discovery, and facilitate precise genome alteration using cutting-edge techniques such as CRISPR/Cas9 are among the main benefits of mutagenesis (Hwang et al., 2013; Varshney et al., 2015). However, it also has drawbacks, such as the need for extensive screening in random mutagenesis techniques, the possibility of off-target effects, mosaicism in genome editing, and challenges in analyzing crucial genes because of embryonic lethality (Moens et al., 2008; Kettleborough et al., 2013). Many mutagenesis techniques, such as chemical, insertional, and radiation-induced mutagenesis, have been successfully used to analyze the functional genome of ZF. Some of the most crucial molecular tools for modifying the genetic makeup of ZF are listed in Table 1. Additionally, Table 2 summarizes disease model induction strategies and associated phenotypes in zebrafish-based studies.

Table 1. A timeline of significant technological advancements in the field of ZF research.

ZF, zebrafish.

Table 2. Overview of disease model induction strategies and phenotypes in ZF.

ZF, zebrafish.

3 Random mutagenesis approaches

3.1 Chemical-mediated mutagenesis

A chemical mutagen known as N-ethyl-N-nitrosourea (ENU) can be used for achieving random gene disruption in ZF. An alkylating agent, ENU, modifies single DNA bases to produce point mutations that are dispersed randomly and can be passed down to succeeding generations. By ethylating oxygen or nitrogen atoms in DNA bases, it causes mutations. A mutation can be either a missense mutation, which changes the meaning of the affected codon, or a nonsense mutation, which truncates the protein, depending on its presence either on the splice sites or inside an open reading frame (ORF). While the majority of these mutations result in null or hypomorphic alleles, it is known that these mutations can also produce neomorphic and hypermorphic alleles (Coghill et al., 2002). A number of knockout ZF models, including tp53 and apc mutants, have been successfully generated using this process (Berghmans et al., 2005; Feitsma et al., 2007; Hurlstone et al., 2003; Wienholds et al., 2002, 2003). ZF embryos are first given a low concentration of ENU during a specific developmental stage in order to perform random gene disruption. To ensure that mutations are induced without seriously impairing development, the timing and dosage of ENU treatment are essential. Following ENU treatment, the embryos are allowed to mature into adult fish, which are subsequently bred to generate a population carrying ENU-induced germline mutations. The offspring of the mutagenized population’s ZF are then examined for phenotypic anomalies. This may entail monitoring outward manifestations, behavioral shifts, or other distinguishable qualities. Genetic mapping methods can be used to find the location of the mutation in the genome once a phenotype of interest has been identified. Analyzing the genetic markers in the progeny by crossing mutant fish with a reference mapping strain can be one way to accomplish this task. Sequencing and characterization, which entail comparing the DNA sequence to the wild-type sequence, come next. Methods such as phenotypic rescue experiments, protein characterization, and gene expression analysis are used as the final functional analysis. This approach also supports the identification of growth and disease-resistance genes relevant to aquaculture species.

ZF ENU-derived mutagenesis was previously used to relate easily visible phenotypes to gene functions (Driever et al., 1996). The ZF genome is now almost fully annotated, making it possible for users to select any desired genomic sequence precisely. This enables the specific detection of point mutations caused by ENU in a selected location using targeting induced local lesions in genomes (TILLING). A reverse genetic approach, TILLING employs an endonuclease, specifically engineered to detect mismatches inside a DNA heteroduplex in conjunction with resequencing (Moens et al., 2008). The workflow involves i) ENU mutagenesis, ii) DNA extraction from F1/F2 families, iii) polymerase chain reaction (PCR) amplification of target loci, iv) heteroduplex formation, and v) mutation detection by enzymatic cleavage or sequencing (Figure 3). Allelic series can be identified using this method without first selecting a phenotype. However, there are certain drawbacks, such as the need for large mutagenized populations, labor-intensive screening, and reliance on available sequence information for target selection (Moens et al., 2008). The use of TILLING in ZF to identify a point mutation in the ortholog of psen1 has enabled the investigation of a putative Alzheimer’s disease (AD)-associated gene in an animal model. The γ-secretase complex is reliant on the highly conserved protein PSEN1. The cleavage of amyloid precursor protein requires the action of γ-secretase. Psen1 mutations have been detected in many cases of autosomal dominant familial AD, and mis-cleavage of the amyloid precursor protein is generally considered a potential cause of progressive dementia associated with AD. Psen1 null mice are not viable, despite numerous attempts to study psen1 function in the mouse system. The psen1hu2547/hu2547 mutant ZF allows for the long-term investigation of altered psen1 function in a vertebrate brain, as it develops nearly normally and can be raised to adulthood. A truncated protein variant that is represented by the ZF psen1 allele hu2547 lacks the proteolytic domain. These fish had dramatically different numbers of histaminergic neurons, which is probably related to the decrease in neuronal histamine seen in AD patients (Shen et al., 1997; Sundvik et al., 2013; Bateman et al., 2011).

High-throughput sequencing techniques combined with the currently available ZF genome sequence data accelerate the finding of targeted mutations. Short biotinylated RNA probes that are complementary to all ZF protein-coding sequences were created by Kettleborough et al. (2013). These probes were hybridized with ZF genomic DNA that had been modified by the ENU mutation, allowing them to enrich exon sequences by dragging them down with streptavidin beads. Their ultimate objective was to employ exome sequencing to detect alterations in each gene that codes for a protein in ZF (Kettleborough et al., 2013). Conversely, identifying a point mutation requires a comprehensive analysis utilizing sequencing techniques.

3.2 Retroviral-mediated mutagenesis

The process, also known as insertional mutagenesis, is based on the mechanisms of the integration of retroviral vectors or transposable elements, which have the potential to introduce an interrupting cassette coding for a fluorescent protein or other readily detectable reporter into the ZF genome [for instance, an ORF encoding a green fluorescent protein (GFP) and a minimal promoter] (Amsterdam et al., 2011; Sanders et al., 2003; Prouty et al., 2003). By employing this construct as a molecular landmark, the hit locus can be mapped more easily by identifying the nearby genomic areas. Fluorescence can be utilized to monitor the effectiveness of such integration, and the name given to this fundamental concept is entrapment (Trinh and Fraser, 2013).

Tol2 insertions were used in a small-scale screen in ZF to determine the frequency of gene inactivation. Selected carriers exhibited insertions that produced spatially restricted GFP expression patterns (Sivasubbu et al., 2007). Data indicated that only a tiny fraction of Tol2 insertions (4%) changed gene function sufficiently to result in an early embryonic phenotype. Retroviruses and transposons usually attain a substantially lower overall mutagenic frequency compared to chemical mutagenesis (Amsterdam et al., 1999). This implies that, for screening, substantially larger libraries of fish with mutations are required. The desired locus can be rapidly identified using the insertional element as an amplification tag in an inverted or ligation-mediated PCR (Amsterdam et al., 2004).

Retroviral-mediated mutagenesis was originally the most effective approach to insertional mutagenesis in ZF. In the early 1990s, it was shown (Lin et al., 1994) that the Moloney murine leukemia virus (MoMLV), when injected into ZF embryos and pseudotyped with the vesicular stomatitis virus (VSV) envelope protein, could infect the embryos, and the integrated DNA would travel from the germline to the progeny of the first generation. A large-scale forward genetic screen was conducted, and more than 500 mutants were discovered at approximately 400 loci. Retroviral-mediated mutagenesis is more difficult to carry out than chemical mutagenesis; however, 335 out of 400 loci were identified, indicating that the affected loci can be identified using linker-mediated PCR (Sundvik et al., 2013; Trinh and Fraser, 2013). Most insertional screens conducted in model organisms utilized a forward genetic strategy, which screened for phenotypes before identifying the mutated genes. These screens were restricted in their ability to address gene redundancy or genes with weaker phenotypes, but they were efficient in identifying mutations with apparent abnormalities. With the availability of high-quality genome sequencing data, reverse genetics found that the MLV retrovirus was a useful tool for mutagenesis.

4 Transient gene-silencing approaches

4.1 Morpholinos

The introduction of morpholino, antisense oligonucleotides for temporary gene knockdown in loss-of-function studies in 2000 marked a turning point in ZF research (Noveroske et al., 2000; Nasevicius and Ekker, 2000). A hypomorphic phenotype can be produced by precisely suppressing the expression of the target gene in early embryos using tiny antisense oligonucleotides, termed morpholinos, as an alternative to chemical mutagenesis (Rosen et al., 2004). To achieve penetrant effects of gene knockdown, morpholinos (MOs), which have neutral, non-ribose backbones exhibiting the four DNA bases (Summerton, 1999; Summerton and Weller, 1997), base-pair with RNA but are resistant to degradation. To prevent splicing or the synthesis of proteins, MOs operate by binding to the target transcript to generate heteroduplexes. Morpholino-binding can prevent a particular messenger RNA (mRNA) from being translated or spliced, depending on the target sequence. This results in reduced or even eliminated synthesis of the corresponding gene product. Reverse genetics is a widely used technique to create intermediate phenotypes of different intensities or to investigate the function of genes that do not have a mutant allele (Nasevicius and Ekker, 2000). Although there are certain intrinsic off-target challenges, this simple approach enables researchers to halt translation from mRNAs early in the developmental process in ZF, and hence, the defects observed in the corresponding mutant alleles were recreated in the treated embryos. To achieve this, they specifically targeted several developmental genes’ translation start sites (Nasevicius and Ekker, 2000). By concentrating on splice site junctions in the pre-mRNA, exon skipping can be employed to interfere with zygotic transcripts and alter gene expression specifically (Draper et al., 2001). RT-PCR measurement of the RNA target can demonstrate the success of gene knockdown by the disruption of splicing, as opposed to MOs that target translation start sites, which necessitate using a particular antibody to assess variations in the level of protein expression (Figure 4). Concentrating on particular splice sites can also result in isoform-specific knockdown (Mably et al., 2003; Xu et al., 2002).

Not exclusively protein-coding RNAs, miRNA can also be suppressed through MO-based suppression, either by interfering with the maturation of miRNAs (Flynt et al., 2007; Kloosterman et al., 2007) or miRNA–target 3′ UTR interactions (Choi et al., 2007). Due to their efficiency, MOs are now the method of choice for quickly analyzing gene function in the early stages of development. As a result, they have greatly advanced our knowledge of a variety of early developmental processes, including the migration of primordial germ cells (Doitsidou et al., 2002), hematopoietic stem cell genesis (North et al., 2009), and left–right asymmetry formation (Neugebauer et al., 2009). MOs have also been employed in moderate-scale functional genomic screens to investigate gene function at a systems level (Eckfeldt et al., 2005; Pickart et al., 2006). As per Robu et al. (2007), morpholinos have the ability to enhance the p53 apoptotic factor’s activity. Because of this, induced cell death typically masks the true phenotype. In order to mitigate the off-target effect, the co-administration of a morpholino specific to p53 is permissible; however, certain questions remain unanswered (Robu et al., 2007; Gerety and Wilkinson, 2011). One recent study, for example, elucidated the key differences between the suppression of PAK4, a serine-threonine protein kinase produced by morpholino and a pak4 null allele. When the pak4 gene is present, ZF develops normally, but when it is not, there is a severe developmental defect (Law and Sargent, 2014). However, if the specificity is sufficiently defined, morpholinos can still be a useful tool to study gene function (Eisen and Smith, 2008; Bill et al., 2009). Figure 5 provides a schematic representation of morpholino-mediated gene knockdown in zebrafish embryos. Antisense morpholino oligonucleotides are designed to bind specifically to target mRNA, blocking its translation. Following fertilization, morpholinos are microinjected into early-stage embryos. This targeted binding results in the suppression of gene knockdown, leading to altered developmental phenotypes in the resulting larvae (Figure 5).

4.2 RNA interference

RNA interference (RNAi) is a biological mechanism that modifies gene expression by breaking down particular mRNA molecules. It can be used to reduce a gene’s expression in ZF and track the phenotypic changes that occur. Short single-strand RNA fragments are removed after processing exogenous double-strand RNA because they attach to complementary-sequenced mRNA. This is managed by a highly conserved mechanism of proteins (Perrimon et al., 2010). Despite ZF having every element of this system, most attempts to create an effective RNA interference technique have failed due to malfunctions or unintended consequences.

However, it has been shown that the ZF RNAi machinery’s processing of endogenously genetically encoded miRNAs is conserved in other vertebrates and critical for development (Kelly and Hurlstone, 2011). As a result, utilizing the typical ZF miRNA structure as a template, a new RNA interference tool was created. The miRNA stem-loop was substituted by a short target sequence that matched the protein-coding region of the target mRNA. Stable transgenic (RNAi lines) were produced once these constructs were incorporated into the genome. The expression of these structures decreased the expression levels of the associated genes and resulted in symptoms similar to those of comparable loss-of-function mutants. It has previously been possible to achieve cell type-specific gene knockdowns by combining modified miRNAs with an inducible tissue-specific expression mechanism (Dong et al., 2009, 2013). These encouraging techniques suggest that presently, ZF has a dependable and flexible RNAi tool, and such ZF can be used again in the future.

4.3 Ectopic expression by mRNA

Injecting synthetic mRNA expressing the target protein into early embryonic stages (e.g., one-cell embryos) is a commonly utilized approach in the ZF. In the developing embryo, mRNA is equally delivered into each newly created cell. Therefore, as overexpression of mRNA is not cell type-specific, it provides a rapid and practical method for studying the worldwide expression of gain- and loss-of-function variations of a target gene in order to study functional genes. This method can only be used to define the early stages of embryogenesis, as the injected mRNA is only stable during these periods. The validation of a loss-of-function phenotype occurs frequently by rescue using mRNA injection as well (Finckbeiner et al., 2011).

5 Stable transgenesis approaches

5.1 Transgenesis

ZF embryos can also be injected with an expression plasmid rather than mRNA. Well-known promoters, specifically the CMV promoter, allow for the creation of the required protein in ZF (Rupp et al., 1994). To allow for cell type-specific expression, a number of well-researched ZF enhancer elements and an ORF encoding a fluorescent protein can be coupled. A multicistronic expression cassette, which allows for the simultaneous, concurrent (and in some cases equimolar) synthesis of many components, can also be found within a plasmid. Because ZF embryos are visible throughout their entire developmental process and the fact that fluorescence reporters play a significant role in ZF research, a wide range of cutting-edge genetically encoded fluorescent microscopy instruments have been established (Weber and Köster, 2013). Unlike injected mRNA, plasmid DNA distributes in a mosaic pattern, which means that only a tiny portion of the multicellular embryo’s cells will generate the target protein. Therefore, the behavior of particular small cell populations or even single cells, as well as the cell autonomy of gain-of-function and loss-of-function phenotypes, is often addressed via plasmid injection (Mumm et al., 2006). Unlike mRNA-mediated transgenic expression, transgene expression seldom spreads through the germline, although the effects of transgenesis are long-lasting.

The active integration of the relevant construct into the ZF genome is necessary for the formation of stable transgenics that are functional. ZF has several transposon-based tools for this aim, including Tol1, Tol2, and Sleeping Beauty. For transgenesis, transposons are preferred over retroviral-based vectors due to their high insertion efficiency, versatility, and ability to carry complex DNA transgenes. The Tol2 transposon derived from medaka has been extensively employed in ZF and has emerged as the principal genetic instrument for transgenesis in this species. Tol2 cis sequences are only 200 bp long; therefore, it is easy to integrate them into the flanking regions of any desirable transgene in the ZF genome. The Tol2-flanked transgene has a high chance of integrating into the germline when synthetic capped mRNA expressing Tol2 transposase is co-injected into the early embryo. Therefore, its integration becomes stable due to the fast turnover of Tol2 transposase-producing mRNA and proteins. However, issues such as location effects and transgene silencing could arise, as is typically the case with random transgenic integration techniques in other model species (Ni et al., 2008; Balciunas et al., 2006).

Conditional mutants are useful genetic tools for researching gene function in vivo because they allow researchers to temporarily or tissue-specifically inactivate gene function. Numerous gene-trap/protein-trap cassettes based on transposon technology have been released recently. Trinh and Fraser (2013) created the FlipTrap, a tool for conditional knockout that combines the capabilities of Flp-mediated targeted recombination, conditional mutagenesis mediated by Cre, and gene trapping. Bacteriophage P1 is the source of the site-specific recombination tool known as the Cre/loxP system. It makes use of Cre recombinase, which identifies loxP sites and mediates recombination between them. Depending on the orientation of loxP sites, this may lead to excision, inversion, or translocation (Sauer and Henderson, 1988). By surrounding crucial exons with loxP sites (also known as floxed alleles), this technique is frequently used in ZF for conditional gene knockout and lineage tracing. Usually, tissue-specific or inducible Cre drivers, such as CreERT2, are crossed with floxed ZF lines such that recombination can be temporally regulated using tamoxifen (Feil et al., 1996). This allows for spatially and temporally limited gene inactivation, which is especially helpful for researching genes that, when completely disrupted, result in embryonic death. Variable recombination efficiency, mosaic activity in some tissues, and reliance on verified Cre driver lines are among the limitations.

When the FlipTrap cassette is inserted into an intron, the gene’s promoter is used to produce a functional fusion protein (citrine tag), indicating native protein expression. When this cassette is subjected to Cre recombinase, it can generate a mutant allele and a mCherry-tagged gene trap. Flp-mediated recombination opens up the possibility of exchanging the cassette with other cassettes. The community has access to approximately 200 FlipTap lines, and the website, http://www.fliptrap.org, provides comprehensive information (JN et al., 2009). A protein trap was integrated into the most current iteration of a gene-breaking transposon, which linked a fluorescent reporter to naturally produced genes while also deactivating the gene activity.

With its lack of an initial methionine codon (ATG), the monomeric red fluorescent protein (RFP) produced by fusing an endogenous gene with a splice acceptor on the RP2 cassette depends on the endogenous gene for translation start. RP2 is an effective mutagen because it reduced endogenous gene expression to less than 1% of normal by trapping the endogenous transcript. Furthermore, the RP2 construct has a 3′ poly-adenylation trap with a GFP activated by the β-actin promoter. This overrides the endogenous promoter to drive GFP expression independent of the insertion site. The two loxP sites on either side of the RP2 cassette can be employed with Cre recombinase or morpholinos that target the trap’s splice acceptor to release the trapped gene (Nasevicius and Ekker, 2000). Another Tol2-based vector with a highly mutagenic, bi-directional gene trap on the FlEx cassette was developed. A “strong” splice acceptor with a mCherry reporter oriented in one direction is part of the design, which traps almost all of the transcripts and truncates the gene in a mutagenic way. Normal expression is possible because there is no functional trap in the opposite direction. This enables the Cre or Flp recombinase to reverse the entire construct in vivo. Gene traps are designed to inactivate genes, like that of the RP2 cassette trap, when they are introduced into a gene’s intron oriented in the same direction as the transcript. However, the FlEx cassette can be reversed by either expressing Cre or Flp in a tissue-specific manner, resulting in tissue-specific rescue, or injecting Cre/Flp mRNA into one-cell-stage embryos, resulting in global rescue. Once the insertion is oriented inversely in the designated gene, there will be no mutagenic effects. To accomplish the desired global or conditional gene knockout, the FlEx cassette can be inverted again because it contains FRT and loxP sites (Robu et al., 2007; Gerety and Wilkinson, 2011). To temporally express Cre or Flp, various tissue-specific drivers are required to optimize the value of conditional mutant alleles. Using a gene trap containing a reporter constructed of mCherry-Cre linked to the mutant human ligand-binding region of the estrogen receptor (mCherry-CreERT2), an attempt is being made to produce numerous Cre driver lines. With this, researchers can now control not only the location of Cre recombinase expression but also the temporal regulation of activity by introducing tamoxifen at the desired time. A database named CreZoo (https://www.zfin.org/ZDB-PUB-130610-72) was developed to assist the community in locating a particular driver that fits their preferences. There are already 47 Cre driver lines that express CreERT2 in various spatiotemporal patterns, but more lines will be added as part of an ongoing effort (Law and Sargent, 2014).

Like P-elements in Drosophila, Tol2 transposons are remobilized to cause random excision-induced genomic deletions. To do this, ZF embryos can be injected with transposase mRNA at the one-cell stage. This technique created enhanced trap transgenic lines with up to 1.3-kb deletions. With so many Tol2-based transgenic lines at our disposal, it may be a different way to inactivate the previously identified transgenic lines’ gene functions (Eisen and Smith, 2008). To date, every method disclosed for manipulating the genome at random to find mutations or integrations has relied on this approach. Although the methods are effective, every random strategy has its own set of drawbacks. The ZF system has been modified using combinatorial genetics to improve the adaptability of driving transgenes and enhancer elements unique to particular cell types. Using an upstream activating sequence (UAS) is one method of controlling the synthesis of an ORF in response to a specific binding of Gal4, a transcription activator protein (Brand and Perrimon, 1993; Scheer and Campos-Ortega, 1999). Originally generated from yeast, the Gal4/UAS system is a bipartite transcriptional regulation system that consists of a responder line that carries the desired gene downstream of UAS elements and a driver line that expresses Gal4 under a tissue-specific or inducible promoter. Gal4 selectively binds to UAS sequences upon genetic crossing, thereby controlling the downstream effector gene’s transcription both spatially and temporally. The process involves developing stable transgenic driver and responder lines, which are then crossed to produce offspring with limited gene expression in certain organs or developmental phases. The flexible combinatorial regulation of gene expression is made possible by this modular architecture, which also makes it possible to quickly create several experimental circumstances with the same genetic components. Tissue-specific enhancers, such as elavl3, are commonly employed in ZF to limit Gal4 expression to neuronal populations, allowing for accurate in vivo functional research and disease modeling applications (Weber and Köster, 2013). A major advantage of the Gal4/UAS system is its high specificity, scalability, and ability to drive robust transgene expression in selected cell populations, making it highly suitable for developmental biology and functional genomics studies. For a systematic gene function study, it also enables the creation of numerous separate driver–effector combinations. Nevertheless, positional effects resulting from random genomic integration, differences in expression levels among separate transgenic lines, and occasional leaky or ectopic expression based on enhancer strength and chromosomal context are among the drawbacks. Despite these drawbacks, the Gal4/UAS system continues to be one of the most popular techniques for spatiotemporal gene regulation in ZF, including simulating human neurodegenerative disorders such as Alzheimer’s disease by expressing pathogenic proteins such as Tau in specific ways (Paquet et al., 2009).

Gal4-activated UAS elements can synthesize multiple ORFs to generate the corresponding gene products (Weber and Köster, 2013). There is also the possibility of producing quadruple and triple transgenic fish (Almeida et al., 2014). Other systems, such as tetracycline-inducible transcription and LexPR systems, are also available (Emelyanov and Parinov, 2008; Knopf et al., 2010), alongside tamoxifen-inducible Gal4 variants (Gerety et al., 2013).

6 Targeted genome-editing approaches

6.1 Genome editing

The field of ZF mutagenesis is currently experiencing another breakthrough in developing quick and effective targeted mutagenesis techniques. Endonucleases created especially for genome editing are used to manipulate specific sequences. Zinc finger nucleases (ZFNs) were the foundation for the first-generation method. Zinc fingers are small protein motifs that bind to individual DNA triplets. These motifs make up the DNA-binding domain of ZFNs. The FokI endonuclease domain completes the ZFN structure. It is possible to combine zinc fingers with different binding characteristics to construct a sequence-specific domain that targets the relevant genomic sequence. As a result, a DSB is introduced between the two ZFN-binding sites. DSBs are repaired via non-homologous end joining (NHEJ); however, this technique is error-prone and may result in minute deletions or insertions. Therefore, a disruption in gene function may result from a ZFN-induced DSB. ZF experiments in 2008 demonstrated the efficacy of ZFNs (Doyon et al., 2008; Meng et al., 2008). This was the first instance of targeted gene inactivation in ZF, and it potentially made gene knockout technology more accessible to ZF biologists. ZFNs were developed long ago and have been shown to function in ZF and several other types of model organisms. It is not always possible to predict the specificity of a zinc finger array, though, as zinc fingers can affect one another’s binding characteristics (Moscou and Bogdanove, 2009). Because binding property can be time-consuming and costly, genome editing using ZFNs has not yet become a widely accepted technique.

The main disadvantages of creating ZFNs via selection are the time-consuming and tedious procedures needed to create and construct ZFNs for a particular gene, and weeks of selection or cloning are required (Ramirez et al., 2008). Despite being the first readily available genome-editing tools, fewer than 30 ZF genes have been targeted using ZFNs to date in order to create loss-of-function mutations for phenotypic research.

ZFNs and Transcription Activator-Like Effector Nucleases (TALENs) are made up of a site-specific DNA-binding domain and the FokI endonuclease domain (Cermak et al., 2011). With the exception of two nearby residues, the short repeats that make up the DNA-binding domain’s sequence are extremely conserved. These repeat variable residues determine a single repeat’s binding specificity to one of the four DNA bases; they have no effect on each other’s binding properties. As a result, a target DNA sequence can be easily converted into a series of linear repeats with various repeat variable positions. According to Moscou and Bogdanove (2009) and Christian et al. (2010), TALENs that target the desired DNA sequence can be created using common cloning techniques in a matter of a few steps, making this technology accessible and affordable for anyone. For the purpose of targeting genes in ZF, TALENs were adapted in 2011 (Huang et al., 2011; Iqbal et al., 2023a). For large-scale targeted gene disruption, TALENs were a very appealing option to ZFNs due to their ease of assembly and comparatively low cost of reagents (Bedell et al., 2012; Iqbal et al., 2023b). The first vertebrate in which TALENs were tested was the ZF. Indeed, heritable mutations were produced by genomic DSBs caused by two TALENs and improper NHEJ (Huang et al., 2011). Furthermore, the integration of a donor construct with a homologous region for homologous recombination (HR)-directed DSB repair has proven effective (Zu et al., 2013).

RNA-guided clustered regularly interspaced short palindromic repeats CRISPR-Cas system, the most recent targeted genome-editing technology, is remarkably elegant and simple, employing standard RNA injection procedures in ZF; it does not even need the design and production of a DNA targeting domain (Blackburn et al., 2013; Ma et al., 2013; Xiao et al., 2013; Ding et al., 2013). A guide RNA is co-delivered with synthetic capped mRNA that codes for the Cas9 endonuclease. The target genomic sequence is complementary to the guide RNA (Iqbal et al., 2025). The Cas9 endonuclease binds to the target site through the guide RNA, causing a DSB (Le Cong et al., 2013). Overall, CRISPR/Cas is currently the most efficient and widely adopted system, TALENs remain useful for applications requiring higher specificity, and ZFNs are now largely limited to specialized applications (Doyon et al., 2008; Hwang et al., 2013; Kleinstiver et al., 2016).

According to Jao et al. (2013) and Shen et al. (2013), appropriate vectors are already optimized for ZF or other organisms and just need the unique design of the guide RNA. Ye et al. (2024) developed an efficient all-in-one vector-based CRISPR/Cas9 system for ZF cell lines using the native ZFU6 promoter, enabling reliable genome editing in both fish and mammalian cells. Hwang et al. (2013) demonstrated the effectiveness of CRISPR/Cas9 as a straightforward, targeted mutagenesis technique for ZF. Jao et al. (2013) showed that CRISPR/Cas9 simultaneously targets many genes and is adequate for phenotypes to be observed in embryos that have been implanted. It has revolutionized ZF research by making gene knockout quick and simple in practically any lab (Varshney and Burgess, 2014).

CRISPR/Cas9-based ZF models have developed into potent translational platforms for improving human medicine and early-stage vaccine research, going beyond simple functional genomics. In vivo dissection of conserved gene functions pertinent to human pathology is made possible by CRISPR-enabled ZF, which enables the quick creation of accurate disease models that replicate important aspects of human disorders, such as cancer, cardiovascular abnormalities, and metabolic and infectious diseases (Hwang et al., 2013; Irion et al., 2014; Varshney et al., 2015; Liu et al., 2017). Because whole-organism screening offers an integrated evaluation of efficacy, toxicity, and pharmacological responses in a physiologically relevant vertebrate system, these engineered models are being utilized more frequently in high-throughput drug discovery pipelines (MacRae and Peterson, 2015; Cornet et al., 2018). CRISPR-edited ZF provides special benefits for real-time visualization of host–pathogen interactions in infectious disease research, making it easier to identify immune regulatory networks and possible treatment targets (Meijer and Spaink, 2011). Furthermore, by connecting genetic disruptions to immunological responses in vivo, the functional disruption of immune-related genes using CRISPR/Cas9 has been crucial in validating host defense pathways and supporting reverse vaccinology techniques (Sellaththurai et al., 2023).

Taken together, CRISPR/Cas9 ZF models represent efficient, scalable systems bridging genome editing with translational medicine and vaccine research. Li et al. (2023) demonstrated that ZF with FTR42 knocked out via CRISPR/Cas9 had increased antiviral immunity and survival after viral infection by eliminating the protein and its inhibitory influence on the type I IFN pathway. Rubbini et al. (2020) demonstrated that combining CRISPR genome editing with ZF enables rapid, scalable in vivo functional screening to accelerate the discovery and validation of new therapeutic targets. Nadarajapillai et al. (2024) demonstrated that CRISPR/Cas9-mediated knockout of tnf-α1 in ZF reduces their disease resistance to Edwardsiella piscicida infection, confirming the essential role of tnf-α1 in antibacterial immunity. CRISPR/Cas9-induced knockout of tumor necrosis factor-alpha type I (tnf-α1) in ZF significantly increases susceptibility and mortality following viral hemorrhagic septicemia virus (VHSV) infection, highlighting the critical role of tnf-α1 in antiviral defense (Kalaichelvan et al., 2025). Yin et al. (2025) demonstrated that CRISPR/Cas9-mediated knockout of ehd3 in ZF significantly increases the muscle content of highly unsaturated fatty acids, offering a genetic strategy to enhance fish nutritional quality. Genome editing of FTR42 improves zebrafish survival against virus infection by enhancing IFN immunity (Qu et al., 2024). Davidson et al. (2025) developed a rapid F₀ CRISPR/Cas9 screening pipeline in ZF that efficiently identifies regulator genes controlling enteric nervous system neuronal development. Barrientos et al. (2024) developed optimized CRISPR interference (CRISPRi) and activation (CRISPRa) systems to enable systematic biological exploration in ZF by modulating gene expression without creating DNA breaks. They employed catalytically inactive dCas9 fused with transcriptional repressor or activator domains and designed sgRNAs targeting promoter regions of endogenous ZF genes to either repress or activate gene expression precisely. By microinjecting these CRISPRi/a components into ZF embryos, they achieved robust and controlled gene expression, which was validated through fluorescent reporter assays and endogenous gene targets. This approach demonstrated high specificity and temporal control, facilitating functional genomic studies in vivo while avoiding the limitations of permanent knockouts, thus providing a scalable and versatile tool for dissecting gene function in ZF developmental and disease models.

ZF research has undergone a significant transformation due to targeted genome editing. One can create a stable mutant allele in accordance with their own preferences in a matter of weeks rather than searching the databases for mutants produced by chance gene disruption or challenging the specificity of morpholinos. Thus far, numerous options exist: A true null allele may occur from a frameshift or an early stop codon caused by DSBs pointing to the beginning of an ORF and incorrect NHEJ. Shorter or different splice variants of the transcript can be produced when directed to another gene region. A wide range of DNA sequences, such as a basic gene-disrupting cassette or a more intricate fluorescent reporter, can be introduced via HR-based DSB repair. The promoter of the targeted gene would then drive these sequences. One can incorporate built-in point mutations or tiny protein tags beforehand by utilizing the targeted locus’s sequence as the HR template for a DSB (Trinh et al., 2011; Campbell et al., 2013; Lin et al., 2014; Kimura et al., 2014). It is also feasible to carry out a rescue experiment by fixing an existing mutation using the wild-type DNA.

It has been proven that simultaneous Cas9 endonuclease targeting of two guide RNAs or two TALEN pairs to the same chromosome in ZF causes very large deletions or even chromosomal inversions, which may be useful in disrupting entire gene clusters. Furthermore, several particular mutations can be produced using the same procedure by combining the Cas9 endonuclease with various guide RNAs that match distinct locations (Jao et al., 2013). A few days after injection, an initial indication of the expected phenotype can be seen, as in some of these animals when both alleles are impacted. Additional possible uses include the precise control of gene expression by the binding of the inactive Cas9 endonuclease to inhibit transcription at particular genomic locations or the guidance of effector domains to a promoter region utilizing the TALEN repeat domain (Zhang et al., 2011; Qi et al., 2013). All new methods, though, have disadvantages. These are off-target consequences in genome editing that result in undesirable background mutations. As a result, the target sequences need to be carefully selected. Ideally, they should be as distinct across the entire genome as possible to minimize off-target effects. Optimizing Cas9 enzymes is another option, like Cas9 nickase, which only causes a single-strand break rather than a DSB and appears to lessen off-target effects significantly (Cho et al., 2014). Combining TALENs and CRISPR-Cas allows for targeted mutagenesis, resulting in tiny changes and even in-frame knock-ins to any desired chromosomal site, thus elevating reverse genetics of ZF and other genetically tractable creatures to a whole new level. The same genetic possibilities for ZF are now readily, inexpensively, and quickly available because of this method, which was previously only available for mice.

Despite significant advancements in ZF genome-editing techniques and mutagenesis, key limitations persist. Issues such as off-target effects, variable editing efficiency, and incomplete understanding of long-term phenotypic outcomes and genetic compensation reduce reliability. Furthermore, more recent methods such as base editing, prime editing, and CRISPRi/a need to be further optimized, and there are still no standardized high-throughput platforms for functional gene validation. Additionally, their use in translational fields such as disease resistance and aquaculture is still restricted, underscoring the necessity of ongoing methodological integration and improvement.

Future developments in mutagenesis research are likely to focus on improving target specificity, minimizing unintended genomic alterations, and enabling more precise spatiotemporal control of gene function in ZF. It is anticipated that more efficient CRISPR variants with improved fidelity and reduced off-target activity will improve genome-editing applications (Kleinstiver et al., 2016; Slaymaker et al., 2016). Systematic gene function study at a genome-wide scale will be made easier by the ongoing development of automated and high-throughput functional screening systems (Kettleborough et al., 2013). Furthermore, integrating genome editing with in vivo imaging and phenotypic screening is expected to strengthen the use of ZF as a model for translational research and precision medicine. When taken as a whole, these developments will increase the range of mutagenesis research in terms of comprehending intricate characteristics, disease mechanisms, and practical domains such as aquaculture.

7 Conclusion

Over the past decade, zebrafish genome manipulation has advanced rapidly, supported by a wide range of genetic and genomic tools. The discovery of genes linked to human diseases has been expedited by large-scale projects such as Genome-Wide Association Study (GWAS), the 1000 Genome-Wide Association Study, ExAC, and gnomAD; zebrafish offers a potent vertebrate system for their functional validation (1000 Genomes Project Consortium, 2015). Compared to more conventional methods such as morpholino knockdown, CRISPR/Cas9 technology has enabled effective, large-scale functional genomics and reduced the time and expense needed for mutant generation. Systematic gene knockout efforts continue to improve our understanding of disease mechanisms and gene function. Additionally, the application of genome-editing methods such as CRISPR/Cas systems in modern mutagenesis is becoming more and more important for managing illness and boosting aquaculture productivity. These technologies enable the rapid identification of traits linked to enhanced performance and disease resistance in aquatic species by precisely modifying genes relevant to immunity, development, and stress tolerance. By encouraging the development of improved fish strains with higher innate immunity, this lessens the need for antibiotics and restricts antimicrobial resistance. Furthermore, genome editing makes it easier to understand host–pathogen interactions and helps identify targets for vaccine and therapy development. These tactics are expected to significantly enhance sustainable aquaculture and global food security in the near future.

Generative AI statement

The authors declared that generative AI was not used in the creation of this manuscript.

Any alternative text (alt text) provided alongside figures in this article has been generated by Frontiers with the support of artificial intelligence and reasonable efforts have been made to ensure accuracy, including review by the authors wherever possible. If you identify any issues, please contact us.

Publisher’s note

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.

Footnotes

Edited by: D. K. Meena, Central Inland Fisheries Research Institute (ICAR), India

Reviewed by: Qiang Zhang, Tianjin University of Traditional Chinese Medicine, China

Kiran Piyushbhai Modi, Central Institute of Brackishwater Aquaculture (ICAR), India