Genetic improvement in edible fish: status, constraints, and prospects on CRISPR-based genome engineering

Abstract

Conventional selective breeding in aquaculture has been effective in genetically enhancing economic traits like growth and disease resistance. However, its advances are restricted by heritability, the extended period required to produce a strain with desirable traits, and the necessity to target multiple characteristics simultaneously in the breeding programs. Genome editing tools like zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) are promising for faster genetic improvement in fishes. CRISPR/Cas9 technology is the least expensive, most precise, and well compatible with multiplexing of all genome editing approaches, making it a productive and highly targeted approach for developing customized fish strains with specified characteristics. As a result, the use of CRISPR/Cas9 technology in aquaculture is rapidly growing, with the main traits researched being reproduction and development, growth, pigmentation, disease resistance, trans-GFP utilization, and omega-3 metabolism. However, technological obstacles, such as off-target effects, ancestral genome duplication, and mosaicism in founder population, need to be addressed to achieve sustainable fish production. Furthermore, present regulatory and risk assessment frameworks are inadequate to address the technical hurdles of CRISPR/Cas9, even though public and regulatory approval is critical to commercializing novel technology products. In this review, we examine the potential of CRISPR/Cas9 technology for the genetic improvement of edible fish, the technical, ethical, and socio-economic challenges to using it in fish species, and its future scope for sustainable fish production.

Keywords: Aquaculture; CRISPR/Cas9; Edible fish; Genetic improvement; Genome editing; Selective breeding.

Fig. 1

Fish genome editing in a targeted manner to produce superior strains with desirable features utilizing genome editing tools, such as ZFNs (Zinc finger nucleases), TALENs (Transcription activator-like effector nucleases), and CRISPR/Cas9 (CRISPR-mediated RNA-guided DNA endonucleases). A ZFNs comprise a DNA-binding zinc finger domain and a nuclease domain derived from the restriction enzyme FokI. Zinc fingers recognize triplets, and the associated FokI nuclease dimerizes and cuts in the spacer region between two unique zinc finger target sites. B TALEN differs from ZFN in that the binding array components recognize individual nucleotides, and the two distinct TALENs recognize and bind to specific locations on opposite DNA strands; the assembled FokI dimer cleaves target DNA precisely. C The CRISPR/Cas9 system comprises a single-guide RNA (sgRNA) and a nuclease termed Cas9. The DNA site is recognized in the CRISPR-Cas9 system by base complementarity between genomic DNA and sgRNA, connected with tracrRNA, and loaded into Cas9 nuclease, which accomplishes DNA cleavage. Cleavage occurs three bp upstream of the PAM (Protospacer adjacent motif) on both strands. D Nuclease (FokI or Cas9) breaks the target DNA sequence, causing a double-strand break (DSB) that can be repaired by non-homologous end joining (NHEJ) or homology-directed repair (HDR). Random insertions or deletions (indels) occur at the editing site in the NHEJ pathway, frequently resulting in gene disruption. A donor template containing sequences homologous to the DSB flanking areas can be inserted into genomic DNA, resulting in gene repair. Thus, the better strain of fish generated utilizing three separate genome editing tools, in which the fish developed using CRISPR/Cas9 technology was inexpensive, could target several genes simultaneously for diverse desirable qualities with high targeting efficiency and precision

Fig. 2

Developing a disease-resistant fish population with a high growth rate for potential commercial application: Comparison of conventional selective breeding and the novel CRISPR/Cas9 genome editing system. A For example, in conventional selective breeding, a donor strain of high disease resistance but relatively low growth rate crosses with a strain with a high growth rate but comparatively low disease resistance. The fish population developed in that way will ultimately be highly disease-resistant and have a high growth rate. However, the introgression of desirable traits into the selected fish requires successive backcrossing, followed by the laborious screening of subsequent generations for desirable traits, which requires much time and energy. In addition, undesirable genes from the donor strain will be incorporated along with the desired gene, and genetic dilution of the recipient commercial strain will occur. B In genome editing, the disease susceptibility gene will be disrupted in a strain with a high growth rate using the CRISPR/Cas9 system, and the resulting lines of the fish population will be disease-resistant and have a high growth rate. The homozygous mutant fish population could be produced with 1–2 breeding cycles. Thus, CRISPR/Cas9 technology enhances the speed of the fish breeding process through targeted and precise genome editing without requiring much time, and it avoids the genetic dilution that usually occurs during the conventional breeding process

Fig. 3

The general procedure for CRISPR/Cas9-based genome editing in farmed fishes. After selecting the target gene from the candidate species genome database, sgRNA needs to be generated with the sgRNA design tool, and then sgRNA oligo must be synthesized. The mixture sgRNA and Cas9 protein is delivered into one cell stage of the fish embryo via microinjection or electroporation. Fish mutants are screened using mutagenesis analysis methods, such as heteroduplex mobility assay (HMA), high-resolution melting curve analysis assay (HRMA), mismatch cleavage assay, or sequencing method. The final stage is off-target free mutant selection, crossing with wild populations and producing a specific mutant line, assessing CRISPR-induced mutation-associated phenotype, and generating new varieties with desired traits in aquaculture

Fig. 4

Genome edited fish production via CRISPR/Cas9 based germline mutation and surrogacy technology. The primordial germ stem cells (PGCs)/ germline stem cells (GSCs) from fish will be isolated, cultured, and edited using CRISPR/Cas9 technology. The genome-edited germ stem cell is screened for successful edits and then transplanted into a sterilized (germ cell-less or ablated) surrogate fish with a short generation time via microinjection. The resultant progeny will produce male and female gamete based on the sex of the surrogate fish. Mixing these gametes will generate a fish with improved values in a shorter period