International Journal of Aquaculture, 2025, Vol.15, No.2, 45-56 http://www.aquapublisher.com/index.php/ija 49 4.1.2 CRISPR/Cas9 targets the white spot virus (WSSV) receptor gene to improve disease resistance In addition to breeding, disease-resistant breeding is another most attractive application direction for gene editing. In the shrimp farming industry, white spot disease caused by the white spot syndrome virus (WSSV) is one of the most harmful diseases. Regarding WSSV, studies have pointed out that the Rab7 protein on the host cell membrane binds to the viral encapsulation protein VP28, which is an important way for WSSV to enter cells (Sritunyalucksana et al., 2006). In addition, some transcription factors (such as YinYang1, YY1) in shrimp are suspected to be involved in the viral replication cycle. Based on these understandings, multiple scientific research teams have tried to use CRISPR technology to knock out related genes to verify the anti-disease effect. In his master's study, Bermúdez et al. reported the attempt to target mutations of WSSV receptor genes in vannabinoid shrimp sperm cells using CRISPR/Cas9, hoping to cultivate anti-WSSV offspring by obtaining mutation-bearing sperm. Although this idea faces technical challenges (such as how to effectively edit germ cells), preliminary results show that the binding ability of CRISPR-treated semen cells to leukoplakia virus particles is reduced, suggesting that receptor gene function has been affected. 4.2 Selection and design strategies of target genes Successful gene editing experiments are inseparable from reasonable target gene selection and careful experimental design. In the application of shrimp CRISPR, researchers generally select genes closely related to the target trait and have clear functions as editing objects based on the aforementioned genomic and transcriptome studies. For example, in experiments that set to improve disease resistance, host genes that are proven to be necessary for pathogen invasion or replication will be preferred. For example, in terms of trait improvement, endocrine hormone genes and growth inhibitor genes have become the first target of choice because they have a significant impact on growth and reproduction (Miao et al., 2023). The ideal target gene should be non-essential and trait-specific so that its knockout is not fatal but can lead to expected phenotypic changes. In terms of design, 2 to 3 sgRNAs are usually designed for the functional key regions of the target gene (such as catalytic domain or ligand binding sites) to increase the probability of mutation hitting the critical position. sgRNA design requires the help of shrimp genome sequences to avoid complete matches of more than 3 to 4 bases with other sequences in the genome, thereby reducing the risk of off-targeting. Some specialized software and tools can help predict potential off-target sites on the shrimp genome, and even if shrimp are not a model species, basic evaluation can be performed as long as the genome sequence is provided. In addition to sequence design, the delivery strategy of editing tools is also an important part of the design process. In shrimp, the most commonly used method is to mix the mRNA or protein of Cas9 with sgRNA synthesized in vitro and inject it into the fertilized egg or single-cell embryo through microinjection. Microinjection requires an appropriate period of time: fertilized eggs of penaeid shrimp possess a chorion and initiate cleavage shortly after fertilization, so it is generally chosen to complete the injection before the first cleavage. There are also some explorations that try to inject CRISPR reagent through the semen to edit germ cells or pass them into shrimp oocytes through gene guns, electroporation, etc., but the efficiency still needs to be improved. 4.3 Editing efficiency and off-target effect issues The application of CRISPR technology in shrimps is still an emerging field, and a prominent challenge is the low editing efficiency. Compared with the high-efficiency mutation acquisition rate of more than 50% of mammalian and insect-mode species, shrimp gene editing often faces problems such as low injection survival rate and high chimera ratio. In the early study of Chi4 knockout in the spinal tail white shrimp, although hundreds of fertilized eggs were injected, only a handful of larvae that successfully grew into and had mutations. Crustaceans such as vannabinoid shrimp develop rapidly and are sensitive to operation and are prone to injury or infection during microinjection (Xu et al., 2019), thereby reducing survival. Even if the larvae hatch, the mutations introduced by CRISPR are often in a chimeric state (different cells carry different genotypes), resulting in a non-obvious phenotype and cannot be directly and stably passed on to offspring. All of these make screening and breeding of gene-edited shrimp more difficult.
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