Development and validation of PmMAD7 for efficient gene editing in Penaeus monodon

Abstract

Background

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-based gene editing has become a promising approach for enhancing traits in aquaculture species. Nevertheless, traditional CRISPR-Cas systems encounter challenges, including significant off-target effects and strict protospacer adjacent motif (PAM) requirements, which constrain their use in crustaceans such as Penaeus monodon. To address these limitations, this research has developed PmMAD7, a codon-optimized CRISPR system specifically designed for P. monodon, which incorporates nuclear localization signals to improve editing efficiency and precision.

Results

This research successfully synthesized and delivered PmMAD7 mRNA and crRNAs targeting the ECH1 and AQP4 genes into the hemocytes of P. monodon. Quantitative PCR analysis demonstrated that PmMAD7 achieved significant gene silencing, reducing the expression levels of ECH1 and AQP4 by 81.5% and 78.33%, respectively. Next-generation sequencing confirmed targeted insertions and deletions at the gene loci, with knockout efficiencies of 14.81% for ECH1 and 20.57% for AQP4, which were significantly higher than those obtained with LbCas12a (7.14% and 12.43%, respectively). Furthermore, functional analysis indicated that ECH1 knockout resulted in increased cell volume and mortality, while AQP4 knockout led to decreased cell volume and reduced viability. These specific results highlight the first successful demonstration of MAD7-based genome editing in shrimp. The broader PAM compatibility and enhanced editing efficiency of PmMAD7 provide a versatile platform for gene editing in shrimp.

Conclusion

PmMAD7 constitutes an enhanced CRISPR editing tool specifically designed for P. monodon, exhibiting superior precision, expanded PAM compatibility, and enhanced editing efficacy relative to conventional Cas12a systems. These results lay the groundwork for the advancement of gene editing applications in crustaceans and contribute to sustainable genetic improvements in aquaculture.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12896-025-01060-7.

Background

The black tiger shrimp, Penaeus monodon, is a high-value aquaculture species globally, yet its genetic improvement is hindered by disease susceptibility and limited tolerance to environmental stress. Establishing efficient gene-editing tools is therefore crucial for the sustainable production of this species. Gene editing technology presents considerable potential for advancement in aquaculture by significantly enhancing the growth rate, disease resistance, and environmental adaptability of economically significant species [1]. With ongoing progress in genomics, molecular biology, and biotechnology, CRISPR-Cas systems have emerged as essential tools for genetic enhancement in aquaculture. This technology facilitates precise genome modifications in aquatic species, thereby improving traits such as reproductive capacity, disease resistance, farming efficiency, and adaptability to environmental changes [2]. Despite the notable advancements achieved with CRISPR-Cas9 technology in plants, animals, and microorganisms, its application in aquaculture remains limited, especially in species with complex genomes. Key challenges include high off-target effects, strict PAM requirements, and low transformation efficiency. These problems reduce accuracy and efficiency and restrict the broader use of CRISPR tools in shrimp [3]. Consequently, there is an imperative demand for the advancement of more precise and efficient gene editing technologies, especially for species characterized by complex genomic architectures, in order to surmount existing technological constraints and further enhance aquaculture practices [4].

MAD7, a commercially available nuclease from Inscripta, is an innovative member of the Cas12a (Cpf1) family [5]. In comparison to the extensively utilized CRISPR-Cas9 system, MAD7 presents several distinct advantages [6]. Firstly, MAD7 is characterized by reduced off-target effects and more lenient protospacer adjacent motif (PAM) sequence requirements, which enable it to target a wider array of genomic sites. For shrimp such as P. monodon, these advantages are particularly critical. Specifically, MAD7 recognizes more flexible PAM sites (YTTN), which greatly expands the range of editable genomic loci in the complex and AT-rich shrimp genome. In addition, MAD7 is distributed under a royalty-free license, lowering barriers for its application in aquaculture breeding programs. These features make MAD7 a promising choice for genome editing in shrimp. This attribute enhances editing precision and flexibility, particularly in the context of complex genomes [7]. Furthermore, MAD7 produces sticky ends during DNA cleavage, facilitating more precise DNA integration and thereby augmenting its potential for editing intricate genomes [8]. MAD7 has been effectively employed across various organisms, including Escherichia coli, yeast, and mammalian cell lines such as HEK293, demonstrating editing efficiencies that are comparable to or surpass those of Cas9 [9, 10]. In plant systems, MAD7 has achieved considerable success, contributing to the genetic enhancement of crops like rice and wheat [10, 11]. Importantly, in zebrafish models, MAD7 has enabled precise gene modifications, establishing a foundation for its application in other aquatic species [12]. These successful applications underscore the extensive applicability and significant potential of MAD7 as a highly efficient and precise gene editing tool for use in agriculture, aquaculture, and biomedical research [13].

The application of CRISPR-based gene editing in crustaceans, particularly in shrimp species such as P. monodon, presents distinct challenges that markedly differ from those encountered in fish. Shrimp possess a highly intricate and less comprehensively characterized genome, which complicates the design and implementation of effective gene editing strategies [12]. The conventional CRISPR-Cas9 systems are associated with high off-target effects and stringent protospacer adjacent motif (PAM) sequence requirements, often resulting in suboptimal editing outcomes in shrimp, thereby limiting their applicability in aquaculture [14]. Additionally, shrimp exhibit unique physiological adaptations, notably their capacity to regulate osmotic balance and lipid metabolism, which are critical for their survival across varying salinity conditions. These adaptations are governed by specific genetic pathways that may not be directly analogous to those in fish, further complicating the application of gene editing tools developed for other aquatic species [15, 16].

This research developed PmMAD7, an optimized MAD7 system for P. monodon. Codon optimization and nuclear localization signals improved its expression in shrimp cells, increasing editing efficiency and precision. To test its efficacy, PmMAD7 was used to disrupt the ECH1 and AQP4 genes, involved in fatty acid metabolism and water regulation. PmMAD7 achieved higher knockout efficiency than Cas9. This advancement not only offers a novel approach for genetic enhancement in P. monodon but also provides essential technical support for gene editing in other aquatic species, thereby fostering innovations in aquaculture genetic improvement.

The principal aim of this study was to develop and validate a CRISPR-based gene editing tool specifically optimized for P. monodon, addressing the limitations inherent in existing systems. By targeting key genes involved in metabolic and osmotic regulation, this research sought to demonstrate the potential of PmMAD7 for precise and efficient genome editing in shrimp. This research not only enhances our understanding of gene editing in crustaceans but also contributes to the advancement of sustainable aquaculture practices by facilitating the genetic improvement of economically significant species.

Results

Structural analysis of MAD7

Comparative analyses of CRISPR-associated proteins revealed that LbCas12a and MAD7, both members of the Cas12a family, presented distinct structural and enzymatic properties. LbCas12a consists of approximately 1,225 amino acids, whereas MAD7 is slightly larger, containing 1,287 amino acids, both of which are smaller than SpCas9, which comprises 1,368 amino acids. Despite having a modest 63% sequence identity with LbCas12a, MAD7 maintained an identical structural domain configuration, as revealed by the predictive models generated by AlphaFold and SWISS-MODEL. These models revealed a bilobed structure, comprising the REC and Nuc lobes, both critical for nuclease activity (Fig. 1B).

Fig. 1.

Structural analysis and homolog comparison of MAD7: (a) Phylogenetic tree analysis comparing MAD7 with the homologous family Cas12a; (b) Domain analysis of MAD7 structure and comparison with Cas12a structure; (c) Comparative domain analysis of amino acid structures among MAD7, Cas12a, and Cas9; (d) construction of the PmMAD7 vector; (e) mRNA structure of PmMAD7; (f) crRNA structure analysis of PmMAD7

Unlike Cas9, which utilizes the HNH and RuvC domains for strand-specific cleavage, both Cas12a proteins, including MAD7, lack the HNH domain. The cleavage mechanism was instead predicated upon a different configuration: the Nuc lobe comprised the RuvC, PI, and WED domains. The WED domain, which harbors the RNase site, was crucial for crRNA processing, whereas the DNase site responsible for DNA cleavage was strategically positioned at the interface between the Nuc and RuvC domains (Fig. 1C).

Construction and optimization of the MAD7 system

Nuclear localization signal (NLS) sequences derived from SV40 and nucleoplasmin were appended to both the N- and C-termini of MAD7. The MAD7 coding sequence was codon-optimized to align with the gene translation mechanisms of P. monodon. The resulting PmMAD7 construct was driven by a cytomegalovirus (CMV) promoter, and an expression plasmid for PmMAD7 was generated (Fig. 1D).

mRNA was used for transfection to increase the expression efficiency of PmMAD7 in primary P. monodon cell lines. T7 RNA polymerase synthesized abundant PmMAD7 RNA from a DNA template positioned downstream of a T7 promoter inserted into the transcription vector. To improve mRNA stability, the transcripts were synthesized in vitro with cotranscriptional analogs, generating a Cap1 structure at the 5’ end and a poly(A) tail containing 100 adenines at the 3’ end. During mRNA synthesis, N1-methyl pseudouridine (m1Ψ) replaces uridine at uracil sites. This optimized MAD7 system for P. monodon was named PmMAD7 (Fig. 1E).

The RNAfold web server was used for structural prediction in the design of crRNAs, ensuring compatibility with PmMAD7. The structural analysis guided the creation of a 20-nucleotide spacer, akin to those used in Cas9 systems. Additionally, sulfur and 2’-O-methyl modifications were introduced at the first three bases on both the 3’ and 5’ ends of the crRNA sequence to increase crRNA stability (Fig. 1F).

Transfection of hemocytes and verification of gene editing in P. monodon

Gene targeting of the ECH1 and AQP4 genes in P. monodon was performed via nanoliposome-mediated delivery of crRNA and PmMAD7 mRNA to assess the knockout efficiency of PmMAD7. After transfection, genomic DNA was extracted from the shrimp, and the targeted gene regions were amplified. Sanger sequencing revealed double peaks near the target sites of ECH1 and AQP4, indicating the presence of heterozygous mutations, whereas nontransfected control cells presented single peaks, confirming the absence of heterozygosity (Fig. 2A-B). Next-generation sequencing (NGS) was used to further characterize the heterozygous mutations. The results revealed single nucleotide polymorphisms (SNPs) and insertions/deletions (indels) at the targeted loci, with knockout efficiencies of 14.81% for ECH1 and 20.57% for AQP4 (Fig. 2C-D).

Fig. 2.

Validation of PmMAD7 knockout in P. monodon hemocytes: (a) Peak diagrams from sequencing at the ECH1 target site; (b) Peak diagrams from sequencing at the AQP4 target site; (c) Type and efficiency analysis of NGS mutations at the ECH1 target site; (d) Type and efficiency analysis of NGS mutations at the AQP4 target site. Experimental groups included: untreated control hemocytes, PmMAD7-targeted ECH1 knockout hemocytes, PmMAD7-targeted AQP4 knockout hemocytes, and LbCas12a-transfected hemocytes. The data are presented as the mean values ± standard errors, with different lowercase letters indicating significant differences between samples (ECH1, p = 0.0047; AQP4, p = 0.0061). Each panel represents results from three independent biological replicates (n = 7 per replicate). Abbreviations: NGS, next-generation sequencing; PmMAD7, codon-optimized MAD7 system for P. monodon

For comparison, the same targets were transfected with LbCas12a, another member of the Cas12a family, and analyzed via next-generation sequencing (NGS). LbCas12a-mediated targeting of ECH1 and AQP4 also produced SNPs and indels but with lower efficiencies of 7.14% and 12.43%, respectively. These results demonstrated that PmMAD7 achieved increased targeting efficiency in P. monodon, exceeding that of LbCas12a by 7.67% for ECH1 and 8.14% for AQP4.

Phenotypic analysis and evaluation posttargeting of the ECH1 and AQP4 genes

To further investigate the effects of PmMAD7 targeting in P. monodon cells, the phenotypes of cells after ECH1 and AQP4 were analyzed (Fig. 3A-B). Specifically, Fig. 3A shows representative microscopic images of cultured hemocytes following ECH1 or AQP4 knockout compared with wild-type cells, highlighting visible morphological alterations such as cell swelling in the ECH1 knockout group and shrinkage in the AQP4 knockout group. Figure 3B presents the trypan blue staining assay results, where stained (blue) cells indicate non-viable hemocytes, clearly demonstrating increased mortality in the ECH1 knockout group and reduced viability in the AQP4 knockout group compared with wild-type cells. Total RNA was extracted from cells successfully targeted at the ECH1 and AQP4 loci, and quantitative analysis was performed. The results revealed a significant reduction in expression; ECH1 and AQP4 expression decreased by 81.5% and 78.33%, respectively, compared with that in wild-type (WT) cells (Fig. 3C-D).

Fig. 3.

Phenotypic observation of hemocyte knockouts in P. monodon: (a) Phenotypic comparison of cultured knockout hemocytes; (b) Analysis of mortality rates via trypan blue staining in hemocytes; (c) Quantitative analysis of the ECH1 gene; (d) Quantitative analysis of the AQP4 gene; (e) Measurement of cell diameters; (f) Survival rate determination after 24 h of cell culture. Experimental groups included: untreated control hemocytes, PmMAD7-targeted ECH1 knockout hemocytes, PmMAD7-targeted AQP4 knockout hemocytes, LbCas12a-transfected hemocytes, and AQP4 inhibitor (TGN-020)-treated hemocytes. The data are presented as the mean values ± standard errors, with different lowercase letters indicating significant differences between samples (ECH1 expression, p = 0.0023; AQP4 expression, p = 0.0038; cell diameter ECH1, p = 0.0015; cell diameter AQP4, p = 0.0027; survival ECH1, p = 0.0041; survival AQP4, p = 0.0056). All data were obtained from three independent biological replicates (n = 7 per replicate). Abbreviations: WT, wild type; PmMAD7, codon-optimized MAD7 system for P. monodon

Phenotypic comparisons were then made with normal P. monodon hemocytes cultured in L15 medium. In L15 medium adjusted to a salinity of 30, cells with ECH1 knockout presented an increase in cell volume, with an average cell diameter of 17.88 μm, compared with 12.74 μm in the wild-type (WT), representing a 40.35% increase in diameter (Fig. 3E). However, after 24 h of culture, trypan blue staining revealed a high mortality rate of 85.33%. Conversely, cells with AQP4 knockout presented a reduction in cell volume, with an average diameter of 9.28 μm. The survival rate of the AQP4 knockout cells after 24 h was 25.33% (Fig. 3F). Phenotypic and survival analyses were performed only in the PmMAD7-targeted groups, as the lower editing efficiencies obtained with LbCas12a did not yield sufficient knockout cells for reliable assessment.

Discussion

MAD7 offers enhance precision and flexibility for shrimp genome editing

Cas12a (such as LbCas12a) and MAD7 differ significantly from Cas9 in their structural domains, which leads to distinctive characteristics in their gene editing functions [17]. First, with respect to recognition mechanisms and PAM sequences, Cas12a and MAD7 require recognition of PAM sequences to initiate DNA cleavage, typically recognizing YTTN (where Y represents A, C, or G and N represents any nucleotide). This recognition mechanism allows Cas12a and MAD7 to have a broader range of genomic targeting opportunities than Cas9, whose PAM sequence is NGG (where N represents any nucleotide). This restriction may limit Cas9 flexibility in some genomic targeting applications [18]. Furthermore, Cas12a and MAD7 require only a single crRNA for targeting and cleavage, whereas Cas9 needs both crRNA and tracrRNA (transactivating crRNA) to form a complex for targeting [19].

In terms of DNA cleavage mechanisms and protein structure, Cas12a and MAD7 exhibit streamlined and efficient characteristics. Both Cas12a and MAD7 generate “sticky ends” at the site of DNA cleavage, which is advantageous for precise gene insertion and deletion. In contrast, Cas9 creates “blunt ends” at double-strand breaks, making it more suitable for applications such as gene knockout. Structurally, Cas12a and MAD7 are simpler, possessing only a RuvC cleavage domain and lacking the HNH domain, whereas Cas9 contains two primary cleavage domains, RuvC and HNH, which independently cleave the two strands of target DNA. The more compact and simplified structure of Cas12a and MAD7 contributes to their enhanced efficiency and specificity in gene editing. Furthermore, their smaller protein size facilitates delivery into certain cell types or tissues. While Cas9 was the first widely adopted CRISPR tool, the unique structural and functional features of Cas12a and MAD7 make them preferable for specific gene editing applications. In the case of shrimp, MAD7 offers distinct benefits compared with Cas9 and conventional Cas12a. Its relaxed PAM requirement (YTTN) overcomes the targeting limitations of Cas9 (NGG), which is often restrictive in the shrimp genome. Its reduced off-target activity improves the reliability of functional studies in non-model crustaceans. Moreover, its royalty-free licensing makes it a practical and accessible tool for large-scale breeding initiatives.

Codon optimization and NLS enhance MAD7 performance in P. monodon

The application of MAD7 in P. monodon, including the addition of nuclear localization signals (NLSs) and codon optimization tailored to shrimp cells, requires targeted optimization to improve gene editing efficiency. Before these modifications, MAD7 exhibited limited expression and inconsistent editing outcomes. The introduction of NLS sequences improved nuclear localization, enhancing MAD7 access to genomic DNA, whereas codon optimization increased the translation efficiency of MAD7. These adjustments demonstrated the importance of adapting CRISPR tools to the specific cellular environments of different species, resulting in a more effective gene-editing system.

Compared with other studies in which Cas9 was used for gene editing in P. monodon, the optimized PmMAD7 system showed comparable reliability despite the inherent challenges of working with shrimp genomes. Previous studies have reported that Cas9-based systems can achieve editing efficiencies ranging from 4.8% to 15.04% in Exopalaemon carinicauda, depending on the target gene and transfection method [20]. Although the editing efficiency of PmMAD7 in P. monodon did not always surpass these levels, it provided advantages such as fewer off-target effects and the generation of sticky ends, which can facilitate precise DNA insertions. These characteristics make PmMAD7 a valuable alternative to Cas9, offering a balance between precision and versatility and expanding the available options for effective gene editing in P. monodon and similar aquatic species.

Additionally, the use of mRNA transfection methods, in combination with improvements in stability from chemical modifications, such as the introduction of N1-methyl pseudouridine (m1Ψ), served to enhance both mRNA stability and transfection efficiency. These advancements suggest that optimizing gene editing tools for distinct cellular environments can markedly improve their efficacy, offering valuable new insights and methodologies for advancing gene editing research [21].

In developing PmMAD7, circulating hemocytes provided the only P. monodon cell population that simultaneously fulfilled the requirements of accessibility, viability and molecular responsiveness necessary for a quantitative editing assay. Haemolymph can be withdrawn from live animals and, after gentle centrifugation, yields intact cells without the enzymatic digestion that damages muscle, hepatopancreatic, gill or gonadal tissues, while still supplying a heterogeneous mixture of hyalinocytes, semigranulocytes and granulocytes for physiologically relevant testing [22]. This tractability enabled optimisation of the plasmid-free mRNA–crRNA ribonucleoprotein delivery and allowed indel formation to be monitored within two days. Primary cultures from solid organs, by contrast, still suffer from low survival and poor transfection efficiency, and permanent shrimp cell lines have not yet been reported, leaving hemocytes as the only practical platform for initial validation of genome editing [23]. Because the same chemical reagents are independent of plasmid backbones, the workflow can in principle be re-parameterised for other primary cells when reliable culture methods become available, and it can already be introduced into one-cell embryos by microinjection, laying the groundwork for germ-line editing and precision breeding of disease-resistant shrimp for aquaculture.

PmMAD7 has greater editing efficiency than LbCas12a

A direct comparison between PmMAD7 and LbCas12a revealed that PmMAD7 achieved higher editing efficiencies for both ECH1 and AQP4 genes. While LbCas12a has been widely used in various organisms, its application in shrimp has been limited by suboptimal editing outcomes [7, 24]. In contrast, PmMAD7 demonstrated knockout efficiencies of 14.81% for ECH1 and 20.57% for AQP4, which were significantly greater than those achieved by LbCas12a (7.14% for ECH1 and 12.43% for AQP4). This study represents the first demonstration of MAD7-based genome editing in shrimp. Previous reports on crustacean editing have primarily employed Cas9, which often showed limited and variable efficiency. By establishing an optimized MAD7 system with codon adaptation and nuclear localization signals, our work not only achieved higher efficiency than the Cas12a family control tested here but also introduced a new genome editing platform that extends beyond Cas9-based approaches in shrimp research. This contribution underscores both the novelty and broader applicability of MAD7 in crustacean functional genomics. This superior performance can be attributed to the optimized expression and nuclear localization of PmMAD7, as well as its relaxed PAM requirements, which allow for more flexible targeting. These results underscore the potential of PmMAD7 as a preferred gene editing tool for shrimp and other aquatic species with complex genomes.

The hemocyte proof-of-concept establishes PmMAD7 as a practical entry point for trait improvement in shrimp aquaculture. Because the workflow relies on a plasmid-free mRNA–crRNA complex delivered by nanoliposome transfection, only minor adjustments to carrier composition or electrical pulse are required to extend it to other primary tissues such as hepatopancreas and muscle, and, once reliable ex-ovo culture protocols are available, to early embryos as well. This versatility will permit rapid validation and introgression of alleles that enhance resistance to white-spot syndrome virus, Enterocytozoon hepatopenaei or vibriosis, while simultaneously enabling functional interrogation of genes governing reproduction, larval robustness, growth rate and feed conversion without the delay of lengthy selection cycles. The reagents are lyophilisable, temperature-stable and distributed under a royalty-free licence, making on-site transfection in commercial hatcheries both technically and economically feasible. Taken together, these attributes position PmMAD7 not only as a research tool but also as a scalable platform for precision breeding and sustainable intensification of P. monodon production.

ECH1 and AQP4 knockout reveals critical roles in shrimp metabolism and osmotic regulation

In this study, an initial quantitative analysis was performed on the total RNA extracted from P. monodon cells, specifically focusing on the ECH1 and AQP4 genes. This analysis entailed quantifying the RNA sample concentrations to ensure the precision and reliability of the experimental data. The findings demonstrated a reduction in the expression levels of ECH1 and AQP4, suggesting that the targeting strategy effectively diminished the transcription levels of these genes, thereby establishing a foundation for subsequent phenotypic observations.

Following the targeting of the ECH1 gene, a notable enlargement in cell volume was observed. ECH1 serves as a crucial enzyme in the beta-oxidation pathway of fatty acids. A deficiency in this enzyme may result in disruptions to lipid metabolism, leading to the accumulation of fatty acids within the cell and consequently causing an increase in cell volume [23]. Such alterations in cell volume have the potential to significantly affect cellular function and viability. In particular, the stability and permeability of the cell membrane may be compromised, resulting in the excessive accumulation of intracellular fluids. In the current study, cells cultured in L15 media with a salinity level of 30 exhibited a significantly larger diameter compared to control cells. This increase in cell volume was correlated with variations in mortality rates after 24 h of culture, as assessed by trypan blue staining, indicating that abnormal cell volume expansion may adversely impact cell survival [24]. ECH1 homologs in crustaceans and other taxa have been associated with fatty acid β-oxidation and mitochondrial energy metabolism [15, 25]. The enlarged cell volume and reduced viability observed here are consistent with perturbations in lipid homeostasis and energy balance, supporting a role for ECH1 in maintaining cellular lipid metabolism and bioenergetic function in shrimp.

In experiments targeting the AQP4 gene, a significant reduction in cell volume was observed. AQP4, a water channel protein located on the cell membrane, plays a crucial role in regulating the water balance between the intracellular and extracellular environments [25]. The knockout of AQP4 resulted in decreased membrane permeability to water, leading to a loss of intracellular water and consequently a reduction in cell volume. This observation aligns with findings from cells treated with an AQP4 inhibitor, further corroborating the role of AQP4 in cell volume regulation [26]. A decrease in cell volume can adversely affect normal cellular functions by compromising membrane stability, thereby increasing cell mortality. Observations of altered cell mortality rates after 24 h underscore the critical importance of AQP4 for cell survival. Furthermore, AQP4 is directly involved in facilitating water transport across the cell membrane, which is vital for maintaining osmotic balance during fluctuations in external salinity. The expression of AQP4 plays a crucial role in modulating the capacity of cells to rapidly regulate their water content, a function essential for cellular survival under conditions of high or low salinity. Aquaporins in crustaceans have been implicated in salinity adaptation and stress resilience [26]. The decreased cell volume and reduced survival observed here are consistent with impaired water transport capacity and may indicate broader physiological consequences at the organismal level, such as altered salinity tolerance or stress responsiveness.

In summary, the phenotypic alterations observed in cells following the targeting of the ECH1 and AQP4 genes underscore the essential roles these genes play in cellular metabolism and water homeostasis. The knockout of ECH1 was associated with an increase in cell volume, while the targeted knockout of AQP4 led to a decrease in cell volume. These phenotypic modifications not only confirmed the efficacy of PmMAD7 in gene targeting but also offered significant biological insights into the functional roles of these genes. Future studies employing more detailed mechanistic assays, such as TUNEL, will be valuable to elucidate the specific apoptotic pathways involved in the observed cell death following ECH1 and AQP4 knockout.

Conclusion

This study introduces PmMAD7 as a novel gene-editing tool specifically designed for the shrimp species P. monodon and evaluates its potential utility in the realm of aquatic genome editing. By optimizing PmMAD7 with tailored nuclear localization signals (NLS) and codon modifications, the system exhibited proficient gene-editing capabilities in shrimp. The ECH1 and AQP4 genes, which play crucial roles in lipid metabolism and water homeostasis, were effectively knocked out. Despite its divergence from traditional Cas12a systems, PmMAD7 enables precise genome editing and serves as a versatile alternative to Cas9, offering an expanded range of PAM recognition. This research not only confirms the effectiveness of PmMAD7 in P. monodon but also suggests its applicability in broader aquaculture contexts, thereby setting the stage for future advancements in the field.

Hemocyte isolation and preculture of P. monodon

Juvenile P. monodon were obtained from the Shenzhen Experimental Base of the South China Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences (CAFS). Hemolymph was drawn from 42 juvenile P. monodon shrimp (45 days post-hatch, 10.3 ± 0.6 g) via a citrate-based anticoagulant (27 mM sodium citrate dihydrate, 336 mM sodium chloride, 115 mM glucose monohydrate, and 9 mM EDTA, pH 7.0). Animals were chilled on ice for 3 min to minimize stress, and 23-gauge needles mounted on 1 mL syringes pre-loaded with 0.5 mL ice-cold anticoagulant (sterilised by 0.22 μm filtration) were inserted into the ventral sinus. The hemolymph and anticoagulant were mixed at a 1: 1 ratio immediately upon withdrawal by gentle inversion to prevent clotting. A 1 mL mixture was then centrifuged at 600 × g for 10 min at 4 °C. After the supernatant was removed, the pellet was resuspended in the anticoagulant, and the centrifugation was repeated under the same conditions. Cell yield averaged (1.7 ± 0.2) × 10⁶ hemocytes per shrimp (n = 6) with > 90% viability confirmed by trypan-blue exclusion (Sigma-Aldrich, USA). The hemocytes were then cultured in L-15 medium (Gibco, Thermo Fisher Scientific, USA) with a salinity of 12 ppt, sealed in plates, and incubated under sterile conditions at 28 °C for 12 h, with 5% BSA (Solarbio, China) added to the culture medium. Leibovitz-15 was osmotically adjusted to 760 mOsm kg^− 1 with NaCl, and supplemented with 100 U mL^− 1 penicillin, 100 µg mL^− 1 streptomycin (Gibco, Thermo Fisher Scientific, USA) and 2 mM CaCl₂. Cells were seeded at 2 × 10⁵ per well in 24-well plates, the lids were sealed with Parafilm (Bemis, USA) to limit evaporation, and cultures were gently rocked (40 rpm) for the first hour to prevent settling.

MAD7 nuclease structural analysis and sequence information

In this study, MAD7 refers to the wild-type nuclease, while PmMAD7 denotes the codon-optimized and NLS-fused version specifically designed for expression in P. monodon. Structural analysis of the MAD7 nuclease was conducted using AlphaFold3 (https://alphafold.ebi.ac.uk) and SWISS-MODEL (https://swissmodel.ExPASy.org) to predict its three-dimensional structure and compare it with those of other Cas12a family members, revealing key structural features and active sites. The MAD7 gene sequence was obtained from Inscripta MAD7 Nuclease Product Page, and the specific sequence can be found in Sequence S1 (MAD7 original sequence). The optimized sequence was confirmed using SnapGene software (https://www.snapgene.com) to verify sequence integrity and correct incorporation of all designed modifications, ensuring its suitability for effective application of the PmMAD7 system in P. monodon. For structural analysis, the AlphaFold DB model (A0A3E5ARN3.1.A) of Type V CRISPR-associated protein Cpf1, derived from Agathobacter rectalis, was utilized. This model had a confidence score of 98.73% and indicated key structural features and active sites. Additionally, the model was built using SWISS-MODEL (EM, monomer) with 5x MG: 2.2.

Optimization of MAD7 expression and mRNA synthesis for efficient gene editing

The MAD7 nuclease coding sequence was codon-optimized for the translational machinery of P. monodon. This optimization involved replacing less frequently used codons with those more frequently used in P. monodon cells (https://www.detaibio.com/sms2/codon_usage.html). The optimized MAD7 gene sequence was further modified by adding nuclear localization signals (NLSs) from the SV40 large T antigen and nucleoplasmin to the N- and C-termini to increase nuclear localization and editing activity within P. monodon cells. The optimized MAD7 gene sequence is provided in Sequence S2 (PmMAD7 sequence).

The optimized MAD7 gene sequence was cloned and inserted into an expression vector containing a T7 promoter (Thermo Fisher Scientific, USA), and mRNA was synthesized via in vitro transcription. During transcription, 5’ capping and 3’ polyadenylation (poly(A) tail) modifications were applied to increase mRNA translation efficiency and stability within cells. Specifically, a chemically modified 7-methylguanosine cap was added to the 5’ end to protect the mRNA from rapid degradation and to facilitate its recognition by the translation machinery. A poly(A) tail was added to the 3’ end to further increase mRNA stability and promote efficient export to the cytoplasm for translation.

Selection and CrRNA synthesis for CRISPR in P. monodon

Target genes were selected by analyzing the P. monodon genome via the NCBI database, with a focus on genes with potential roles in physiological functions and disease resistance. The selected targets needed to contain appropriate PAM sequences (YTTN) for recognition and binding by the MAD7 system. Target site design for the ECH1 gene (NCBI Accession No.: LOC119594195), and AQP4 gene (NCBI Accession No.: LOC119575040). Target site design for the ECH1 and AQP4 genes was performed via the tool (http://chopchop.cbu.uib.no/), selecting sites with minimal off-target effects and high recognition efficiency for subsequent crRNA synthesis [27]. CHOPCHOP tool was used to select target sites with off-target scores < 0.2, GC content between 40 and 60%, and sites located within functional domains. The designed target sequences are provided in Supplementary Table S1: Target Sequences. Potential off-target sites were evaluated in CHOPCHOP v3 using the Penaeus vannamei genome as the nearest annotated invertebrate reference, because a contiguous P. monodon genome suitable for CRISPR off-target prediction is not yet available; no off-target sites were identified for any guide under a ≤ 3-mismatch threshold.

The synthesized crRNA sequences corresponding to the selected target sites are listed in Supplementary Table S2. These sequences were carefully designed to ensure precise 20-base pair matches surrounding the target regions to guide the Cas protein efficiently. CrRNA synthesis was carried out via standard in vitro transcription methods with T7 RNA polymerase from a double-stranded DNA template. The resulting crRNA was purified to remove incomplete products and template DNA. To increase crRNA stability and reduce intracellular degradation, chemical modifications such as 2’-O-methylation and 3’-end phosphorylation were introduced.

Transfection of P. monodon hemocytes

The precultured hemocytes of P. monodon were removed from the L15 medium and transferred to Opti-MEM™ reduced serum medium for transfection. A mixture of 36 nmol crRNA and 36 nmol PmMAD7 mRNA was prepared at a 1:1 ratio, and 3.75 µl of Lipofectamine™ 3000 (Invitrogen, Thermo Fisher Scientific, USA) was combined with 3.75 µl of Opti-MEM™ reduced serum medium to incubate with the mRNA mixture for 10 min. This transfection mixture was then added to 1.5 ml of the cell culture medium for coculture transfection. Transfection was carried out using lipofection. The hemocytes were incubated at 28 °C for 3 days. After 72 h, genomic DNA was extracted from hemocytes in each experimental group, including the untreated wild-type, the ECH1-targeted PmMAD7 transfection group, the AQP4-targeted PmMAD7 transfection group, and the LbCas12a transfection group. Each group included three independent biological replicates, with seven shrimp sampled per replicate, resulting in a total of 21 genomic DNA samples for each group. Survival rates were determined at 24 h using trypan blue exclusion.

Genomic DNA extraction, NGS adapter PCR, and sequencing

Genomic DNA was extracted from 21 hemocyte samples, with each experimental group repeated 3 times, and 7 samples were extracted per repeat. The DNA was extracted using the HiPure Mollusk DNA Kit (MGBio). For NGS, adapter sequences were added to the primers flanking the target genes via the OPC method. The adapters were as follows: read1: 5’-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG-3’; read2: 5’-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG-3’. PCR amplification was carried out with TransStart FastPfu DNA Polymerase, and the primers used are listed in Supplementary Table S3. After PCR, the amplicons were analyzed via 1% agarose gel electrophoresis. The samples were then sent to Tsingke Biotechnology for sequencing [28].

Sequencing was performed via the Illumina platform at Tsingke Biotechnology. Libraries were prepared via end-repair, A-tailing, and adapter ligation. A PCR-free library was created, followed by quantification via Qubit 2.0 and assessment of the insert size via the Agilent 2100 Bioanalyzer. The library effective concentration (3 nM) was quantified via qPCR. Sequencing was conducted according to the required data output. The raw sequence data were processed via CASAVA, resulting in FASTQ files containing both sequence and quality information. The raw reads, which included adapter sequences and low-quality data, were filtered to obtain clean reads. The quality control parameters for raw reads included removing reads with adapter sequences and low-quality bases (below a quality score of 20) using fastp v0.20.1. Mapping was performed using BWA v0.7.17 with default parameters, and alignment was verified using MAFFT v7 for multiple sequence alignment [29].

qPCR validation of crRNA-mediated gene knockout in P. monodon cells

Quantitative PCR (qPCR) was employed to validate the gene knockout in P. monodon hemocytes mediated by crRNA. Total RNA was extracted from hemocytes of the untreated wild-type, the ECH1-targeted PmMAD7 transfection group, the AQP4-targeted PmMAD7 transfection group, and the LbCas12a transfection groups, using TRIzol reagent. RNA integrity and purity were confirmed, and the RNA was reverse transcribed into cDNA with a commercial reverse transcription kit. Five serial dilutions of cDNA were used to generate standard curves to evaluate the amplification efficiency of each primer pair. The standard curves showed slopes ranging from − 3.28 to − 3.40 and R² values greater than 0.98 (Figure S1).

Two reference genes, β-actin (ACTB) and EF-1α, were used for normalization to ensure the stability and accuracy of gene expression analysis. Specific primers for the target genes and reference genes were designed for the qPCRs. Each reaction consisted of a cDNA template, specific primers, SYBR Green PCR Master Mix, and nuclease-free water, with a total reaction volume of 20 µL. The qPCR conditions included initial denaturation at 95 °C for 30 s, followed by 40 cycles of denaturation at 95 °C for 5 s and annealing/extension at 60 °C for 30 s. All reactions were performed in triplicate, and melting curve analysis was conducted to confirm amplification specificity. The relative expression levels of the target genes were calculated using the 2^‒ΔΔCt method to assess transcriptional changes after knockout. Primer sequences are listed in Supplementary Table S4.

Observation of transfected cells and mortality analysis

Transfected P. monodon cells were observed and analyzed for viability via a trypan blue exclusion assay. After 24 h of transfection, the cells were transferred to L15 medium with a salinity of 30 for further culture. The wild-type of cells was derived from untransfected P. monodon hemocytes. Mutant cells for aqp4 and ech1 were observed, with aqp4 inhibitor TGN-020 (20 µM)-treated cells serving as a positive control [30]. Each group included 3 biological replicates, with the same number of samples in both control and experimental groups. Morphological changes were photographed at 0, 3, 6, 12, and 24 h via a Nikon Ti2 microscope. After 24 h of culture, cell diameters were measured using ImageJ software, with no fewer than 30 cells analyzed per group to calculate the average. The results were plotted via GraphPad Prism 9.0.

To assess cell viability, 4% trypan blue staining was used. The cells were stained for 1 min, washed with L15 medium, and photographed after excess stain was removed. The mortality rates of cells in different treatment groups were observed under a microscope and statistically analyzed to evaluate the impact of gene knockout on cell viability. Mortality was assessed at the 24-hour time point, when cell death was most pronounced; later time points were excluded due to increased confounding factors.

Comparison with the conventional Cas12a system

To evaluate the gene-editing efficiency of the PmMAD7 system in P. monodon, a comparative experiment was conducted using the conventional LbCas12a system. LbCas12a mRNA and crRNAs targeting the same loci (ECH1 and AQP4) as used in the PmMAD7 group were synthesized and transfected under identical conditions. Specifically, 36 nmol of LbCas12a mRNA and 36 nmol of crRNA were co-transfected into primary P. monodon hemocytes using Lipofectamine™ 3000, followed by incubation at 28 °C for 72 h.

Genomic DNA was extracted from the hemocytes post-transfection, and target regions were amplified via PCR using the same primers and conditions as those applied for the PmMAD7 system. The samples were then processed using next-generation sequencing (NGS), following standardized protocols for library preparation, quality control, and data analysis to ensure consistency. This experimental setup enabled a direct comparison of editing efficiency and off-target effects between the two systems, as both were subjected to identical primers, reaction conditions, and experimental protocols.

Data processing and statistical analysis

The experimental data were statistically analyzed via GraphPad Prism 9.0 software. Statistical analyses were performed separately for each experiment based on the data type and distribution. Comparisons among groups were made via one-way analysis of variance (ANOVA) followed by the Duncan multiple range test for normally distributed continuous variables. Percentage data such as mortality rates were arcsine square root transformed prior to statistical analysis to meet the assumptions of normality and homogeneity of variance. qPCR data were evaluated by one-way ANOVA with Tukey’s post-hoc test, indel frequencies by two-tailed unpaired Student’s t-test. All the data are presented as the mean ± standard deviation (SD). A P value less than 0.05 was considered to indicate statistical significance.

Supplementary Information

Below is the link to the electronic supplementary material.

Abbreviations

AQP4

Aquaporin 4

Cas

CRISPR-associated protein

crRNA

CRISPR RNA

ECH1

Enoyl-CoA hydratase 1

gDNA

Genomic DNA

LbCas12a

Lachnospiraceae bacterium Cas12a

m1Ψ

N1-methyl pseudouridine

mRNA

Messenger RNA

NGS

Next-generation sequencing

NLS

Nuclear localization signal

PAM

Protospacer adjacent motif

PmMAD7

Codon-optimized MAD7 system for Penaeus monodon

qPCR

Quantitative polymerase chain reaction

RNP

Ribonucleoprotein complex

WT

Wild type

Author contributions

S.H. and Y.D.L. conceived and designed the experiments. F.Z., S.J., Z.Y.J and J.H. performed the gene editing and molecular validation. Q.B.Y., L.S.Y., and J.Z.S. carried out the hemocyte culture and phenotypic analysis. Y.Y.D. and E.C.L. were responsible for the structural modeling and codon optimization of MAD7. S.H. and J.H. analyzed the sequencing data. F.Z. and Q.B.Y. drafted the manuscript. Y.D.L. and E.C.L. revised the manuscript critically for important intellectual content. All authors read and approved the final manuscript.

Funding

This research was funded by the National Key R&D Program of China(2022YFD2401900); Central Public-interest Scientific Institution Basal Research Fund, CAFS(2023TD34; 2025XT0703); China Agriculture Research System(CARS-48); Guangdong Basic and Applied Basic Research Foundation(2023A1515012410); Hainan Provincial Natural Science Foundation of China(323MS127); Earmarked fund for HNARS(HNARS-10-ZJ01); Research on breeding technology of candidate species for Guangdong modern marine ranching(2024-MRB-00-001); Central Public-interest Scientific Institution Basal Research Fund, South China Sea Fisheries Research Institute, CAFS(NO.2024RC06); Young Talent Support Project of Guangzhou Association for Science ang Technology(QT-2025-020); and Key Laboratory of Efficient Utilization and Processing of Marine Fishery Resources of Hainan Provinc(KLEU-2024-7).

Data availability

[https://www.ncbi.nlm.nih.gov/nuccore/PV588691](https:/www.ncbi.nlm.nih.gov/nuccore/PV588691); [https://www.ncbi.nlm.nih.gov/nuccore/PV588692](https:/www.ncbi.nlm.nih.gov/nuccore/PV588692).

Declarations

Ethics approval and consent to participate

All experimental procedures involving P. monodon were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of the South China Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences. The ethics approval number is nhdf2025-33. The shrimp were obtained from the Shenzhen Experimental Base of the institute. No privately owned animals were used; thus, informed consent from owners was not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.