The field of genome-editing technologies is rapidly evolving, and in the recent years, several genome-editing technologies have been developed. There are several genome-editing techniques that evolved such as the homing endonuclease system, a protein-based nuclease system like meganucleases, ZFNs, TALENs, and RNA-DNA system CRISPR/Cas (Khan, 2019). The core technologies commonly used in genome editing are (i) ZFNs (ii) TALENs (iii) CRISPR/Cas. ZFNs (zinc-finger nucleases), and TALENs (transcription activator–like effector nucleases), genome-editing technologies, utilize the dimer FokI nuclease for genome targeting. ZFNs are fusions between a custom-designed Cys2-His2 zinc-finger protein and a FokI endonuclease cleavage domain (Kim et al., 1996; Urnov et al., 2010). Each ZFN is typically composed of 3 or 4 zinc-finger domains, where each individual domain is composed of ∼30 amino acid residues. ZFNs function as dimers and the dimerization of the ZFN proteins is mediated by the FokI cleavage domain, which recognizes and cuts DNA within a 5–7 base pair spacer sequence (Smith et al., 2000). The major concern that prevents wider applications of ZFNs is off-target cleavage and undesired mutations (Pattanayak et al., 2011). Compared to ZFNs, TALENs exhibit reduced off-target effects and provides better specificity. TALENs are the fusions of transcription activator-like (TAL) proteins and a FokI nuclease and almost follow the same principle as ZFNs. TAL proteins are composed of 33–34 amino acid repeating motifs with two variable positions that have a strong recognition for specific nucleotides. TALENs recognize the target sites that consist of two TALE DNA-binding sites that flank a 12–20 base pair spacer sequence recognized by the FokI cleavage domain (Yang and Huang, 2019; Li et al., 2020). However, TALENs face major technical hurdles for cloning the repeat TALE arrays in the designing of the large-scale identical repeat sequences. Although ZFN and TALEN technologies have improved the capacity for genome editing, both of these techniques have limitations. Nevertheless, the newest developed CRISPR/Cas system employed a simple RNA-programmable method to edit the genome that can be used to generate gene knockouts (via insertion/deletion) or knockins (via HDR) (Gaj et al., 2016; Li et al., 2020). In contrast, ZFNs and TALENs use large fusion proteins consisting of a DNA-binding domain to recognize DNA and the FokI nuclease catalytic domain; the CRISPR/Cas system uses a single-guide RNA (sgRNA) that directs the Cas9 nuclease to a specific/targeted genomic location for disrupting the gene (Jinek et al., 2012; Mohapatra and Chakraborty, 2021). In addition, the protospacer adjacent motif (PAM, NGG) immediately downstream of the target sequence determines the specificity of this system. Moreover, CRISPR/Cas9 has the capacity to edit the multiple genes simultaneously, producing conditional alleles and generating tagged proteins with high efficiency. Thereafter, the CRISPR/Cas system shows superiority in convenience, efficiency, specificity, and versatility over ZFNs and TALENs; hence, it is widely utilized and revolutionized the genome editing (Khan, 2019; Mohapatra and Chakraborty, 2021). Very recently, a group of researchers from Kyushu University Japan have reported the pentatricopeptide repeat (PPR) protein–based genome editing technology (Yagi et al., 2013). Pentatricopeptide repeat (PPR) proteins constitute an interesting protein family and are distinguished by the presence of tandem-degenerate PPR motifs. The PPR technology can edit both DNA and RNA, which is very powerful, and in the future, this can revolutionize the gene editing technology. Although the PPR technology has not been used in aquaculture yet, it has a potential for future genome editing in the aquaculture field.

Genetic improvement programs may have multiple breeding goals; growth enhancement and a rapid growth rate are universally valued traits. The CRISPR genome-editing technique has been used for growth enhancement by targeting the myostatin (mstn) gene in several cultured fishes (Khalil et al., 2017; Kishimoto et al., 2018; Hallerman, 2021). Myostatin is a member of the transforming growth factor β (TGFβ) superfamily, and it is a key regulator of skeletal muscle growth (Sun et al., 2020b). The expression of mstn is linked with the double-muscled phenotype in cattle and other animals. CRISPR techniques have been used for growth enhancement by disabling the mstn gene in many fishes like the common carp (Cyprinus carpio) (Zhong et al., 2016), channel catfish (Ictalurus punctatus) (Khalil et al., 2017), red sea bream (Pagrus major) (Kishimoto et al., 2018), olive founder (Paralichthys olivaceus) (Kim et al., 2019), pacific oyster (Crassostrea gigas) (Yu et al., 2019), blunt snout bream (Megalobrama amblycephala) (Sun et al., 2020b), and Nile tilapia (Oreochromis niloticus) developed by the US-based company AquaBounty. The Mstn knockout red sea bream has been reported to significantly increase its muscle mass (16% increase of skeletal muscle), short body length, and better feed efficiency, resulting in better overall growth, which was not observed in other fishes (Kishimoto et al., 2018; Ohama et al., 2020). Recently, the CRISPR/Cas9 system was used for mstn-targeted disruption to obtain the first growing mud loach (Misgurnus anguillicaudatus) (Tao et al., 2021). Apart from the mstn gene, CRISPR is also used for growth-associated traits in the tiger puffer (Takifugu rubripe) by disrupting the leptin receptor gene that controls the appetite, causing fish to eat more (Kishimoto et al., 2018; Shimbun, 2021). Very recently, Japan has approved the sales of two CRISPR-edited fish, for example, tiger puffer and red sea bream, which grow faster than their counterpart. The growth hormone (GH)–insulin-like growth factor (IGF) is a positive regulator of growth, whereas IGF-binding proteins (IGFBP) binds to IGF and acts as a negative regulator of somatic growth. Hence, CRISPR/Cas9 was used to disrupt the IGFBP-2b gene in rainbow trout (Oncorhynchus mykiss) (Cleveland et al., 2018). To increase the per capita productivity via suppressing a cannibalistic behavior, we produced AVTR (alginin vasotosin receptor) V1a2 knockout chub mackerel (Scomber japonicus) (Ohga and Matsuyama, 2021) and found that, apart from the desired phenotype, they also showed docility and reduced oxygen demand (unpublished data). Such an alternative approach toward increasing productivity through genome editing might improve the situations of aquaculture sustainability in the future.

Disease resistance is another important trait for commercial production, and the genetic improvement of disease resistance remains a high breeding priority. Genome-editing experiments have already been investigated for addressing the improvement of immunity and disease resistance. A targeted disruption of the Toll-like receptor TLR22 gene has been done in farmed Indian major carp Rohu (Labeo rohita) that can be used as a model system to investigate the role of TLR22 against pathogenic dsRNA viruses, bacteria, and parasites, for example, fish lice (Chakrapani et al., 2016). As an effective approach to control the hemorrhagic disease induced by grass carp reovirus (GCRV), CRISPR/Cas9 was used to knock out the (Ctenopharyngodon idella) grass carp Junctional adhesion molecule-A (JAM-A) gene and in vitro resistance evaluated against GCRV (Ma et al., 2018). In the channel, catfish (Ictalurus punctatus) targeted the altered immunity toll/interleukin 1 receptor domain-containing adapter molecule (TICAM1) gene involved in the signaling pathway initiated by TLR3 and disease susceptibility–related gene rhamnose binding lectin (RBL) (Elaswad et al., 2018). Sun et al. (2020) demonstrated the function of the carotenoid isomerooxygenase (EcNinaB-X1) gene in the immune defense of Exopalaemon carinicauda by performing CRISPR/Cas9-mediated gene mutagenesis. EcNinaB-X1-Knock out prawns (E. carinicauda) had lower mortality than wild-type after the bacterial challenge. In another study, CRISPR/Cas9 HDR was used to integrate (knock-in) an exogenous alligator cathelicidin gene (which exhibits a broad-spectrum antimicrobial activity) into a targeted non-coding region of the channel catfish genome (Simora et al., 2020). Since many genes are involved in the complex traits like disease resistance and growth, many of the genes might require editing to attain the desired phenotypes; hence, problems arise in the genome editing. Significant work will be required for the application of CRISPR to determine which genes and versions should be targeted through editing.

The escape of fishes from aquaculture facilities may cause environmental, ecological, and genetic impacts, especially if they are a non-native species. Creating sterile animals for aquaculture is desirable to profit the industry to prevent the introgression with wild stock for conserving the genetic integrity and to avoid the negative production consequences of early maturation. Additionally, in a genetic improvement program, reproductive confinement protects the interest of the breeder investment. In this context, CRISPR/Cas9 was used to develop new sterility models in Atlantic salmon (Salmo salar) fish by knocking out a germ cell-specific candidate, dead end (dnd) gene, for producing germ cell-free salmon (Wargelius et al., 2016). Germ cell-free Atlantic salmon remained immature and did not undergo puberty (Kleppe et al., 2017). In another study, sterile channel catfish (Ictalurus punctatus) were produced by editing the luteinizing hormone (LH) gene using a modified ZNF technology with electroporation (Qin et al., 2016). Along with the genome editing technique for producing sterile fish in breeding programs, the knock-in technique also need to be developed, which could lead to the production of an inducible “on-off” system for sterility. Nevertheless, some prior knock-in work with model fish species medaka and zebrafish suggests that restoring fertility for breeding stocks might be possible (Zhang et al., 2015; Nagasawa et al., 2019). In a similar investigation, we, at Kyushu university, employed platinum TALEN to knockout the LH gene Japanense anchovy (Engraulis japonicus) and could reverse the LH-KO mediated “immature phenotype” effects by gonadotropin [HCG (human chorionic gonadotropin)] administration even at the F0 generation (unpublished data). A strategy for recovering the reproductive ability is needed for salmon lacking germ cells to be useful for breeding and aquaculture, and a rescue approach is reported for producing germ cells in dnd knockout fish (Güralp et al., 2020). Sterlet (Acipenser ruthenus), which has the fastest reproductive cycle, can be used for the surrogate production of critically endangered and late age-at-maturity sturgeon fish. CRISPR/Cas9 was used to the knockout dnd1 gene and prepares a sterile Sterlet host for the surrogate production of the late-maturing and large sturgeon species (Chen et al., 2018).

Fishes exhibit a diversity of sex-determination systems, and these are not well characterized in aquaculture species (Mank and Avise, 2009). In addition, fishes also exhibit sexual plasticity since multiple factors including the genetic makeup or environmental factors (such as temperature, light, hormones, and pH) regulate the process indicating the involvement of epigenetic regulation in sexual plasticity (Kikuchi et al., 2020; Zhou et al., 2021). Rapid advances of sequencing and the genome editing technology such as CRISPR/cas9 have accelerated the understanding of the steroidogenic pathway, hormonal regulation, and molecular mechanisms of fish sex determination and differentiation (Li and Wang, 2017). The CRISPR/cas9 gene editing of single- or multiple-gene mutation helps to elucidate the precise roles of the different paralogs in sex determination and differentiation. Until now, functional studies of steroidogenic enzymes and sex steroid receptors of different mutant lines of star1, star2, cyp11a1, cyp17a1, cyp17a2, cyp11c1, cyp19a1a, and cyp19a1b have been constructed by gene editing in model fish species like zebrafish, medaka, and tilapia (Tang et al., 2018; Yan et al., 2019; Alward et al., 2020; Shu et al., 2020; Zhang et al., 2020; Nishiike et al., 2021; Zhou et al., 2021). Genome-editing techniques were used for understanding the sex determination in tilapia as a good model for studying the fish sex determination/reproduction and have contributed toward the development of reproductively confined aquaculture stocks. CRISPR/Cas9 was employed to knock out the doublesex- and mab-3-related transcription factor (dmrt6 and dmrt1) and forkhead box L2 (foxl2), Nanos C2HC-Type Zinc Finger (nanos2 and nanos3), steroidogenic factor-1 (Sf- 1), gonadal soma-derived factor (Gsdf), Wilms tumor 1 (Wt1), and eukaryotic translation elongation factor 1A (eEF1A) gene in (Oreochromis niloticus) Nile tilapia (Li et al., 2014; Xie et al., 2016; Chen et al., 2017; Jiang et al., 2017). Very recently, CRISPR/Cas9 was employed to produce the all-female common carp (Cyprinus carpio L.) population through the knockout of the Cytochrome P450 17A1 (cyp17a1) gene (Zhai et al., 2022).

The health benefits of fish oil omega-3 long chain (≥C20) polyunsaturated fatty acids (n-3 LC-PUFAs), particularly EPA (eicosapentaenoic acid, 20:5n-3) and DHA (docosahexaenoic acid, 22:6n-3), are well documented and established as good fatty acids for human health. Since humans have a limited capacity for the endogenous synthesis of LC-PUFA (EPA and DHA), dietary supplementation remains the best way to meet the requirements. Fishes including Atlantic salmon are the primary sources of omega-3 LC-PUFAs (EPA and DHA) for the human diet. The capacity for LC-PUFA synthesis in any species depends on the complementary activities of fatty acyl desaturases (Fads2) and elongases of very- long-chain fatty acid (Elovls). An understanding of endogenous synthesis and the regulation of LC-PUFAs is required in in vivo functional studies. Datsomor et al. (2019) used the CRISPR/Cas9 editing of Δ5 and Δ6 desaturases, which impairs Δ8-desaturation and docosahexaenoic acid synthesis in Atlantic salmon (Salmo salar). In a subsequent study, Datsomor et al. (2019) used the CRISPR/Cas9 editing of elovl2 in Atlantic salmon, which inhibits the elongation of PUFA, demonstrating the key roles of elovl2 in PUFA synthesis and also suggests that sterol regulatory element binding protein-1 (Srebp-1) is a main regulator of endogenous PUFA synthesis in Atlantic salmon.

Coloration and color patterns in fishes are one of the important considerations in some aquaculture species, especially in ornamental trade. In fish, 6 kinds of pigment cells were identified (Fujii, 2000), while different fish species may have different sets of pigment cell types, and many genes that affect fish pigmentation can also affect the color pattern development. With the recent development of genome-editing tools like CRISPR/Cas9 methods for studying, these pigmentation genes have enriched the understanding and more colors of mutant fish have been obtained (Kimura et al., 2014). In Nile tilapia (Oreochromis niloticus), 25 genes related to pigmentation were involved in melanogenesis, pteridine metabolism, and the carotenoid absorption, and cleavage pathways were edited via CRISPR/Cas9. Among them, 13 genes had a phenotype in both F0 and F2 generations. Four types of pigment cells were identified in wild-type tilapia and, together with various naturally and artificially induced color gene mutants, can serve tilapia as a model system for studying color patterns in teleost fishes (Wang et al., 2021). In some cases, removing genes for pigmentation is also used as a tool to trace successful genome editing, as those individuals that are genetically changed will be without pigment, rendering a white or yellow color. The CRISPR/Cas9 system was used to knock out two genes, tyrosinase (tyr) and solute carrier family 45, member 2 (slc45a2), involved in pigmentation in Atlantic salmon and observed a graded range of phenotypes, ranging from a complete lack of pigmentation (albino) to partial loss and normal pigmentation (Hege Straume et al.; Edvardsen et al., 2014). The CRISPR/Cas9 system was used to target the tyr gene, a key enzyme in melanin synthesis and pigmentation, in two economically important fish species in Asia, loach (Paramisgurnus dabryanus) (Xu et al., 2019) and white crucian carp (Carassius auratus cuvieri) (Liu et al., 2019), which showed different degrees of pigmentation to clear albino. CRISPR/Cas was used to disrupt the HDL receptor/Scavenger receptor B1 (Scarb1); Scarb1-like genes involved in carotenoid metabolism and Gch1 (GTP cyclohydrolase 1) gene in the pteridine pathway resulted in the regional red skin fading into white color in ornamental common carp (Du et al., 2021). In another study, two Agouti signaling protein genes (ASIP 1 and ASIP 2) were disrupted via the CRISPR/Cas9 system and by which, black patches disappeared in the Oujiang color common carp (Chen et al., 2019). Recently, there is an increasing market demand for red tilapia that are the hybrids of different tilapia species or the red variant of the highly inbred Nile tilapia, and this red-color phenotype is unstable, resulting in a non-uniform coloration in appearance or dark-red color blotches that reduce the market value. Hence, the CRISPR/Cas9 system was used to target the slc45a2 gene in melanin biosynthesis and produces an albino variant of Nile tilapia with red skin and eyes (Segev-Hadar et al., 2021). Additionally, researchers are utilizing slc family genes to improve the visual confirmation of knockout fish or strain, and in such a research, Pandey et al. (2021) showed that slc2415 mutants showed reduced melanin pigmentation in the retina and bodies of Kawakawa (Euthynus affinis), highlighting toward a future where the visual recognition of a genome-edited stock will be possible.

Since fertilization is internal, crustaceans are difficult to transform; however, the CRISPR/Cas technology has also been demonstrated in crustaceans. Two potential new techniques, the receptor-mediated ovary transduction of cargo (ReMOT control) and electroporation, can be used for delivering CRISPR/Cas9 components into cultured crustacean species like model crustaceans Daphnia pulex and the decapod Exopalaemon carinicauda (Xu et al., 2020). CRISPR/Cas9 editing has not yet been reported in one of the important groups of aquaculture species shrimp (Penaeus sp.) that may be partly due to practical limitations of internal fertilization. Nevertheless, the microinjection method was developed for the ridgetail white prawn Exopalaemon carinicauda and the CRISPR/Cas9 system was demonstrated to knock out the chitinase 4 (EcChi4) gene (Gui et al., 2016).

Government regulation and public acceptance are the greatest challenge for CRISPR/Cas genome editing in aquaculture. The CRISPR technology has the potential to improve aquaculture production; however, public and regulatory acceptance are the key to its potential being realized (Okoli et al., 2021). CRISPR/Cas-editing applications remain mostly at the research stage, and in aquaculture breeding programs, it is still limited. This occurs mainly due to the fact that the common public has not been convinced yet for the genome-edited products and the global regulatory environment on genome editing remains uncertain. CRISPR/as genome editing is different from the genetically modified organisms (GMOs). In GMOs, new-foreign genes combine into the original DNA, whereas CRISPR genome editing does not introduce any foreign DNA into genome of interest species. Rather, it is a small-controlled change to the original DNA that improves the targeted trait by removing the inferior alleles (Yang et al., 2022). This means that the GMOs product/fish is not a creature that would exist in nature, whereas CRISPR-edited product/fish where the original genes are partly altered similar to the selective breeding process, thus shortening the mutation evolution process that occurs in nature over many generations of breeding. However, still there is a debate going on whether genome-editing approaches should be considered separately than the GMOs. There are no specific and harmonized guidelines for regulatory acceptance yet available for the genome-edited products. Nevertheless, different regulatory authorities throughout the world including the EU, Argentina, Brazil, Australia, New Zealand, Canada, the United States, and Norway have started discussions on how to regulate products arising from the new CRISPR editing techniques (Eckerstorfer et al., 2019). The process of regulatory acceptance and guidelines may vary considerably in different countries where in some countries, genome-edited products are not considered as GMOs and hence bio-safety screening is not required, whereas some countries will consider for safety screening.

CRISPR genome editing products will be relatively easy for the consumer acceptance compared to GMOs, since CRISPR/Cas technology-derived products are called “genome edited” rather than “genetically modified,” this changes in nomenclature itself will save many adverse response which happened earlier for the GMOs (Figure 4). For promoting the consumer acceptance, public must be communicated well how and why CRISPR/Cas was applied and as well as for its value system, for example, producing healthy and quality food and increasing animal welfare, its safety, and sustainability points (Hallerman, 2021).

The world’s first CRISPR/Cas genome-edited fish are now on the market for sale and reached to the consumer. Japan has approved the two CRISPR-edited fish: red sea bream and tiger puffer both developed by the Kyoto-based startup Regional Fish Institute with Kyoto University and Kindai University. Approval has been done from the Ministry of Health, Labour and Welfare (MHLW) and the Ministry of Agriculture, Forestry and Fisheries (MAFF) of Japan after completing the national application process (Japan embraces CRISPR-edited fish, 2022). The fishes were CRISPR edited to grow bigger and have more edible parts (1.2 times higher red sea bream and 1.9 times higher tiger puffer fish than the conventional counterpart). In Japan, GMO products/food that contain foreign genes must undergo safety screening, but CRISPR edited was exempt from this step since it was developed without the addition of foreign genes, so it is not genetically modified, but a change that can happen in nature. As a food, it is safe and has no negative impact on biodiversity. Another aquatic product has been submitted to regulatory review is FLT-01 Nile tilapia developed by the AquaBounty Company (Hallerman, 2021; https://www.fishfarmingexpert.com/article/aquabounty-gets-argentina-go-ahead-for-edited-tilapia/). According to AquaBounty, the CRISPR-edited Nile tilapia has led to increased muscle mass, a significant improvement in the fillet yield and feed conversion than its unedited counterpart. Since FLT-01 Nile tilapia fish does not contain any foreign DNA or a new combination of genetic material that would warrant the regulation as genetically modified in Argentina, unlike transgenic AquAdvantage salmon developed by the same company, it is not covered by the definition of a regulated article under Res. 763 under the Cartagena Protocol for Biosafety. Under Argentine Resolution 173/15-New Breeding Techniques, this fish is not classified as a GMO and Brazil made a similar determination for this genome-edited fish in 2019.

Genome editing technology offers great promise for functional genomics and gene therapy. The outcomes of CRISPR/Cas genome editing are promising and have potential to speed up the aquaculture breeding process. One thing is certain that majority of the expected genome-edited aquaculture products for commercialization will be developed through the CRISPR/Cas technology. However, its application for genetic improvement in aquaculture is just in its infancy. So far, the major research efforts in aquaculture are based on the traditional genetic improvement initiatives like growth, production, and reproduction. In the future, more precise and complex genome editing needs to be performed at multiple loci/chromosomal locations to improve the complex traits and there is also a need to include other economically important traits and diversify the aquaculture species. The continuous improvement of the CRISPR/Cas9 technique and its application in aquaculture as a new breeding technology will revolutionize the sector by increasing the production and quality. Moreover, many solutions in aquaculture are expected in the near future (disease resistance, sterile breeding and targeted therapy, etc.) that could not have been possible to solve before by the traditional ways. For CRISPR/Cas to play a bigger role in aquaculture as new breeding technology, there is a need for the identification and functional annotation of the trait-related genes and signal pathways. Nevertheless, substantial success has been observed in CRISPR/Cas editing in Atlantic salmon and tilapia fish species, and the derivable knowledge from this can be transferable to other fish species. Accordingly, Atlantic salmon and tilapia can be employed as aquaculture model fish species to initiate the optimization of aquatic protocols and assess the off-target effects. It is also relevant to ensure that the process of genome editing is sustainable with regard to environmental interests and animal welfare. At last, the key for CRISPR/Cas genome editing success in aquaculture relies on the subject to favorable regulatory, public, and consumer acceptance.