International Journal of Aquaculture, 2025, Vol.15, No.2, 45-56 http://www.aquapublisher.com/index.php/ija 46 cultivate varieties that are more resistant to disease and grow faster (Ferdous et al., 2022). In particular, CRISPR can replace traditional eye stalk removal and other measures to promote shrimp maturation and reproduction by directly knocking out or modifying endocrine regulatory genes. This study will systematically discuss the current status and potential of CRISPR gene editing technology in shrimps, and analyze the scientific and social challenges that need to be overcome to achieve this technology. 2 Overview of CRISPR Gene Editing Technology 2.1 Basic principles of CRISPR-Cas system The CRISPR-Cas system originates from the immune mechanism of prokaryotic organisms. Its basic principle is to use Cas endonuclease and guide RNA (sgRNA) to identify and cause breakage of genome specific sequences, thereby achieving insertion/deletion mutations or other editing at the target site. In application, researchers first designed crRNA that is complementary to the target gene sequence and formed sgRNA with tracrRNA, guiding the Cas9 protein to localize and cleave specific DNA sequences. Following DNA double-strand breaks, cells may undergo non-homologous end joining (NHEJ), resulting in base insertions or deletions that knock out gene function; alternatively, homology-directed repair (HDR) can be utilized to insert exogenous fragments for precise genome editing (Yu et al., 2021). Due to its simplicity of operation and strong targeting, CRISPR-Cas9 is widely used in genetic function research. In model animal zebrafish, CRISPR knockdown of specific genes has become a routine operation, providing an important means for analyzing developmental and disease mechanisms. In contrast, traditional zinc finger endonuclease (ZFN) and TALENs are complex and expensive to build, limiting large-scale applications. It should be noted that there are naturally many types of CRISPR-Cas systems, among which the most widely used is the Cas9 protein derived from Streptococcus, which belongs to the Type II CRISPR system. "Cas9 introduces blunt-ended double-strand breaks, which typically trigger knockout effects mediated by non-homologous end joining (NHEJ). Another commonly used nuclease, Cas12a (also known as Cpf1), generates 5' overhangs upon cleavage, which facilitate the insertion of exogenous sequences through homology-directed repair (HDR).". Cas12a's identification requirements for PAM sequences are different from Cas9, providing complementary options for gene editing (Hillary et al., 2021). 2.2 Comparison with other gene editing technologies (such as ZFNs, TALENs) Before the advent of CRISPR, site-directed genome modification mainly relied on artificial nuclease technologies such as ZFN and TALENs. ZFN combines zinc finger protein with FokI endonuclease to form a double-strand cleavage tool that recognizes specific DNA sequences. The disadvantage is that each new target is designed, the corresponding zinc finger protein needs to be screened, which has a large workload and limited target sequence. TALENs use the transcriptional activator (TALE) repeat module of plant pathogens to identify DNA sequences, and also play a role in combination with FokI enzymes. TALENs are more specific and more flexible in design than ZFN, but it is also more cumbersome to build TALE vectors containing a large number of repeat sequences (Yu et al., 2021). In contrast, CRISPR-Cas9 only needs to change the guide RNA sequence to target new targets, greatly simplifying the design process. Literature statistics show that the mutation construction cycle of CRISPR is usually less than half of the traditional methods and the cost is lower. In addition, in terms of editing multiple sites simultaneously (multiple gene knockout), CRISPR can only be achieved by introducing multiple sgRNAs at the same time, while ZFN/TALEN is almost unable to achieve effective multi-site editing. This makes CRISPR an irreplaceable advantage in functional genomics research. Of course, CRISPR is not without limitations. The first is the off-target effect problem, that is, Cas9 may cause unexpected mutations in other sites in the genome that are similar to the target sequence. Secondly, the transmission and expression efficiency of CRISPR differs in different species. For example, in mammalian cells, Cas9/sgRNA can be efficiently sent to mammalian cells through viral vectors, but in some aquatic invertebrates, the success rate of microinjection is relatively low (Fu et al., 2024). 2.3 Evolution and improvement of CRISPR system (such as Cas9, Cas12, genome precision editing) Since the advent of the classic Cas9, CRISPR technology has evolved new tools to expand its functions and
RkJQdWJsaXNoZXIy MjQ4ODYzNA==