**3. Conclusions**

*Synthetic Biology - New Interdisciplinary Science*

*dnd*, *tyr*, *slc24a5* (C)

[60, 61]

*lh* (Z) [71]

*sp7a/b*, *runx2*, *bmp2a*, *opg & mstn* (T & C) [73]

*mstn* (T & C) [109]

*dmy*, *dmc1*, *fshb*, *gnrh1* (T); *gsdf* (Z) [110–114]

*mstn* (C) [115]

*dmrt1*, *foxl2*, *cyp19ala* (T) [116]

*Ntl*, *dnd* (T & C) [118, 119]

*C, clustered regularly interspaced short palindromic repeats (CRISPR).*

Cavefish *oca2* (T) [70]

Medaka *dnd* (M); *fox13*,

Rohu *tlr22* (C) [117]

Atlantic salmon

Atlantic killifish

Channel catfish

Chinese lamprey

Common carp

Japanese anchovy

Red sea bream

Rice field eel

Starlet and sturgeon

**Table 3.**

**Fishes Genes (method) Fishes Genes (method)**

*slc24a5* (C) [72] Yellow

catfish

Sturgeon *dnd* (C) [62]

*ahr2* (C) [63] Tilapia *fox12a*, *cyp19a1a*, *dmrt1*, *nanos*, *gsdf*, *sf-1/nr5a*, *mstn*

*mstn* (Z) [12]

(C); *rspo1*, *fox12a*, *cyp19a1a* (T) [64–69]

Zebrafish *dnd* (M); *ntl*, *slc24a5*, *kdr1*, *prl*, (Z); *gria3a*, *hey2*,

*cyp19a1a* (C) [62, 74–108]

*cyp19a1a*, *ryr3*, *ryr1a*, *tbx6*, *slc24a*, *slc45a2*, *fsh*, *lh*, *fshr*, *ihcgr*, *pgr*, *rb1*, *bmp15*, *mesp*, *gnrh3*, *zap70*, *nrld1*, *leg1a*, *mstn*, *rnf213a*, *mpl*, *dmrt1*, *cyp17a1*, *stat3*, *kiss1/2 & kissr1/2(*T); *mitfa*, *ddx19*, *slc24a5*, *slc45a2*, *seta/b*, *nrg1-I*, *stxbp1*, *nERs*, *gspt11*, *fus*, *akt2*, *atp6v1h*,

Theoretically, ZFN is an ideal tool for inducing mutations at target DNA sites in any organisms [53]. However, its application has been constrained by limitations in zinc finger domain design and construction as well as low efficiency [54]. Compared with ZFN, the recently emerged TALEN provides us a more advanced approach for genome editing; it is much easier to construct plasmids for expressing TALE proteins, making this technology easily available to most molecular biology laboratories. Because of this and its high specificity and efficiency, TALEN has quickly replaced ZFN as a dominant platform for genome editing since its establishment in 2011 [55]. Unlike ZFN and TALEN, the nuclease Cas9 is guided towards the target DNA site by a small guide RNA followed by random cleavage of the DNA. Particularly, the rapid emergence of CRISPR/Cas9 caused a paradigm shift in the research community [56]. There is complementary usage of these two technologies in recent years, as CRISPR/Cas9 works as monomer, it consists of protein and RNA and produces blunt end, while TALEN works as dimer, it consists of protein only and produces cohesive ends [57]. Although each one has its associated pros and cons [58], TALENs and CRISPR technologies have comparatively high specificity and efficiency with low off target effect [59]. Not only the methodology, but selection of delivery methodology (microinjection, electroporation, etc.), target tissue, and host is critical for ensured success in

*M, morpholino; Z, zinc finger nuclease (ZFN); T, transcription activator-like effector nucleases (TALEN),* 

*Genome editing using ZFN, TALEN and CRISPR system in varies model and non-model fish species.*

**62**

With the continual growth of global aquaculture, fish production continues to grow globally and till date only a small proportion of the aquatic animals come from managed breeding especially through applied molecular cloning and genomics (**Table 4**). The molecular biology of aquatic organisms offers many opportunities for rapid genetic gains as new genetic techniques make the improvement feasible in a wider range of model and non-model species. The future of molecular biology in aquaculture is bright with the technologies mentioned above being cheaper than ever, widely available and easily applicable in laboratories. However, the results obtained from these methods should not be conclusive without additional information, such as clinical diagnosis, as the mere detection of a certain pathogen does not


#### **Table 4.**

*Summary of molecular biology application in fish.*

imply necessarily that it is responsible for or even involved in a disease. Effective use of these techniques will reduce economic losses as well as risk of infection among wild fish species. Taking advantage of the numerous tissue specific sequence information available in the database, predictions of gene function by bioinformatics tools such as *in silico* and *in vitro* can be employed to identify candidate genes responsible for diseases or disease resistance that will reduce labor and cost of diagnosis and treatment. *In silico* approaches use computational tools to analyze raw DNA sequence data to simulate and predict the function and structural features of protein. In addition, the use of *in vitro* organoid models that refer to growing stem cells in 3D to generate cellular units that mimic an organ in both structure and function, is advancing rapidly. This method can also be applied in fish to study organ development, reproductive enhancement, fast tract selective breeding, disease and drug interactions as well. The new diagnostic techniques like, droplet digital PCR, Hybrid fusion FISH might improve the credibility and cost effectiveness of disease diagnosis.

Genome editing though has the advantage over traditional selective breeding and a trait can be introduced in a single generation without disrupting a favorable genetic background. Many traits of great significance in aquaculture could be targets for improvement by genome editing, including growth and reproductive performance, disease resistance, feed conversion efficiency, and tolerance to environmental stressors (temperature, salinity and oxygen). Keeping the animal welfare issues of "genetically modified organisms" in mind, fish that carry more muscle mass have also been produced by the disruption of a single gene (Myostatin, an inhibitor of skeletal muscle growth) in Common carp, Tilapia, Red sea bream and Japanese anchovy [74, 75, 82, 91]. But still the key question is whether the precise natural genome modifications will find greater public acceptance and make a way to commercial aquaculture. The long-term impacts of these non-transgenic GMOs on wild biodiversity and environment are an uncharted area too. So, in the coming era, we must rethink to what extent we can and should use these molecular advancements for aquaculture betterment.
