CRISPR-Cas Genome Editing Technique for Fish Disease: Comparison
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CRISPR-Cas genome-editing technique can be the potential solution to preventing diseases for aquaculture sustainability. CRISPR-Cas is cheaper, easier, and more precise than the other existing genome-editing technologies and can be used as a new disease treatment tool to solve the far-reaching challenges in aquaculture. This technique may now be employed in novel ways, such as modifying a single nucleotide base or tagging a location in the DNA with a fluorescent protein.

  • CRISPR-Cas
  • fish
  • pathogens
  • phages
  • RNA

1. Introduction

Fish diseases are a serious barrier in the aquaculture sector, affecting more than a billion dollars yearly. Climate change and developing fish farming may influence the balance or imbalance of pathogen, host, and environmental interaction, with new infections being detected or identified annually and more known diseases arising in various global regions and species [1]. Pathogen evolution is thought to be accelerated in intensive farming systems due to the high density of vulnerable hosts, which promotes pathogen transmission and virulence [2]. Because of higher population densities and host–pathogen interactions, this aspect of the farming environment is expected to extend to biological interactions between pathogenic bacteria and their phages, viruses, and parasites. However, many of these diseases or infections have no proven or approved recommended treatments, vaccinations, or control strategies and remain a substantial barrier to the economic sustainability of aquaculture in specific regions and species [1].
The CRISPR-Cas9 system has been developed as a new elite genome engineering tool, even for organisms where genome editing would be challenging. With this promising new technology, it is possible to overcome several challenges that the aquaculture industry faces. Genomic editing using CRISPR can quickly introduce significant genome changes, making it useful for genetic improvements, disease resistance, and disease control in aquaculture [11,12][3][4].

2. Application of CRISPR-Cas in Fish Disease

Aquaculture industries worldwide face serious problems such as infectious and parasitic diseases, reduced viability, decreased fertility, poor development, environmental contamination by escapee fish, coastal conflicts, and disagreements over the patenting of research products [15,40][5][6]. Among these, disease outbreaks in aquaculture are a major issue that are one of the main reasons for the reduction in fish production. Many reputable shellfish farms report shellfish dying overnight because of viral assaults. In fish aquaculture, reproduction, and development [41[7][8],42], growth [43][9], pigment [44[10][11],45], disease resistance [46][12], trans-GFP usage in research [47][13], and omega-3 metabolism [48,49][14][15] are the qualities that are most often targeted for genetic engineering [50][16]. However, using molecular biological techniques to resolve diseases has become a core technology. Genomic editing (GE) has created several controls for aquatic diseases, and it will continue to do so in the future in a variety of different ways. Among these GE methods, CRISPR-Cas has been applied to modify several genes for targeting species-specific pathogens as modern technology. CRISPR-Cas has been applied in immunological studies in channel catfish (Ictalurus punctatus) according to several types of research [51,52][17][18]. It enhanced the resistance of channel catfish to many diseases by injecting the alligator cathelicidin gene into the fish [53][19]. Additionally, this technology enhances the fish body’s natural immunity, which works against bacterial diseases or other infectious diseases such as Edwardsiella ictalurid and Flavobacterium columnare [54][20]. The editing of disease-resistance genes in channel catfish is an additional application of CRISPR-Cas of commercial relevance [51,52,55][17][18][21]. In shrimp and prawns, the eyestalk neuroendocrine complex contains suppressing/inhibiting substances that always prevent breeding and spawning under captivity. These limiting elements also hinder the process of growth. These aquatic organisms’ immune systems have reportedly been weak, making viral and bacterial diseases highly likely to strike them. Certain marine shrimps have already had their gonad-inhibiting hormone (GIH) and molt-inhibiting hormone (MIH) genes evaluated [56,57,58][22][23][24]. Using CRISPR-Cas technology has been able to eliminate the harmful effects of hormones on growth and reproduction, which may open the way to developing a powerful substitute for eyestalk ablation that has a comparable effect. Some researchers have tried to delete the gene using this RNA interference method [59,60][25][26]. When working on Penaeus monodon (giant tiger prawn), Treerattrakool et al. used the method of RNA interference to induce maturity in both wild and captive shrimp and reported that shrimps injected with anti-GIH double-stranded (ds) RNA showed enhanced maturation [59][25]. According to Das et al., RNA interference was used to silence the gonad-inhibiting hormone gene in the eyestalk neuroendocrine complex of the P. monodon (tiger shrimp) [60][26]. They discovered a three–five times increase in the transcript of the androgenic gland hormone (AGH) in males but no alteration in the expression of vitellogenin in females. Additionally, CRISPR-Cas technology can be utilized to manage bacterial and viral infections, particularly in shrimp and prawns. The CRISPR-Cas process in shrimp and prawns may also function similarly to that of bacteria when viral DNA attacks them. For example, CRISPR-Cas can replicate and insert portions of the white spot syndrome virus (WSSV) DNA into shrimp genomes as “spacers” between the short DNA repeats in CRISPR when WSSV invades them. By providing a template for RNA molecules to rapidly recognize and target the same DNA sequence in the case of future viral infections, these spacers improve the immune response of shrimp. The RNA molecules redirect the CRISPR complex to an incoming sequence of foreign DNA if they recognize it. There, the Plasmid Cas proteins of the shrimp cut the invading gene and render it inactive. The shrimp may be shielded against contagious infections because of this [60][26]. Culturing commercial species in the aquatic environment, every year, significant losses are attributed to mass mortality, rejection of aquaculture species’ shipments due to a lack of quality standards, the impact of biotic and abiotic stresses on aquaculture species, and the absence of standardized disease control and pollution-impact methods or protocols [3][27]. 

3. Advances in Bioinformatics in CRISPR-Cas

Bioinformatics is a scientific field that generates methodologies and software tools for analyzing biological data. It has been applied in various applications such as in silico studies of biological questions utilizing computational and statistical tools. It is frequently used to find potential genes and single nucleotide polymorphisms (SNPs). Furthermore, a field of study known as proteomics in bioinformatics seeks to comprehend the organizing concepts found in nucleic acid and protein sequences [61][28]. The main effects of bioinformatics have been the automation of microbial genome sequencing, the creation of integrated databases accessible through the internet, and genome analysis to comprehend gene and genome function. Bioinformatics is now used for a wide variety of other significant tasks in addition to the analysis of gene variation and expression, the analysis and prediction of gene and protein structure, as well as the prediction and detection of gene regulatory networks. It can analyze data more quickly to enhance the accuracy of the findings and explain the causes and phenomena of diseases at the gene/pathway level. Bioinformatics can be used to locate and insert CRISPR-Cas into the targeted genome [3][27]. The choice of the target site is constrained by the possibility of off-target editing and variations in editing effectiveness. Numerous computational techniques have been created in recent years to assist researchers in choosing target sites for CRISPR knock-in/out experiments. In developing single-guide RNA (sgRNA) for CRISPR applications, these methods are likely to be helpful in both target site selection and sgRNA creation. The sgRNA design tools are specifically suitable for genetic screening and CRISPR-mediated gene regulation research has also been developed, resulting from the expansion of CRISPR applications. Computational tools have been created to analyze CRISPR genome-edited data produced by Next Generation Sequencing (NGS) systems and aid in sgRNA creation [62][29]. The CRISPR-Cas9 genome-editing technologies use programmable nucleases to accurately and frequently modify a particular section of the genome which may use RNA-guided nucleases [63,64,65][30][31][32]. CRISPR-Cas has successfully modified specific genomes in significant model species, such as zebrafish [66][33]. It modifies two RNAs—a transactivating CRISPR RNA (tracrRNA) that base pairs with the crRNA and a CRISPR RNA (crRNA) complementary to the targeted DNA sequence—that recruit Cas9 to the target site. The target sequence should be followed by a protospacer adjacent motif (PAM) sequence for recognition (nGG, where n can be any nucleotide). The crRNA and tracrRNA may be combined to form a single synthetic guide RNA (sgRNA) [66][33] that works efficiently with Cas9 to cause cleavage of the target site (~20 bp), which must come after the PAM sequence in the genome. Using in vitro transcription promoters such as T7, T3, or SP6 to create sgRNAs restricts the target sequence. Here, the CRISPR-Cas system was used to modify the Xenopus tropicalis (western clawed frog) genome, providing another tool for quick and effective targeted mutagenesis. Cas 9 is a CRISPR-related protein adapted from a naturally occurring genome-editing system and used here as a bacterial immune defense. Most genomic restriction nucleases require substantial and complex PAM sequences that would restrict them due to reduced genome size. Distinct PAMs in the SpCas9 system are used for genome manipulation, including target gene disruption and single base-pair mutations in various organisms and cells. Developing the SpCas9 to identify more PAMs would be an alternative approach to increasing PAM specificity. Although SpCas9 is the most well-known nuclease, Cas9 can also be obtained from many bacterial species. The fundamental difference between them is the PAM sequence required for the cleavage of Cas9 nucleases from different bacteria [67][34].

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