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Background <p>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 <i>Penaeus monodon</i>. To address these limitations, this research has developed <i>Pm</i>MAD7, a codon-optimized CRISPR system specifically designed for <i>P. monodon</i>, which incorporates nuclear localization signals to improve editing efficiency and precision.</p> Results <p>This research successfully synthesized and delivered <i>Pm</i>MAD7 mRNA and crRNAs targeting the <i>ECH1</i> and <i>AQP4</i> genes into the hemocytes of <i>P. monodon</i>. Quantitative PCR analysis demonstrated that <i>Pm</i>MAD7 achieved significant gene silencing, reducing the expression levels of <i>ECH1</i> and <i>AQP4</i> 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 <i>ECH1</i> and 20.57% for <i>AQP4</i>, which were significantly higher than those obtained with <i>Lb</i>Cas12a (7.14% and 12.43%, respectively). Furthermore, functional analysis indicated that <i>ECH1</i> knockout resulted in increased cell volume and mortality, while <i>AQP4</i> 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 <i>Pm</i>MAD7 provide a versatile platform for gene editing in shrimp.</p> Conclusion <p><i>Pm</i>MAD7 constitutes an enhanced CRISPR editing tool specifically designed for <i>P. monodon</i>, 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.</p>

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

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2025年11月12日

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  • Sheng Huang,
  • Falin Zhou,
  • Ziyi Jiang,
  • Song Jiang,
  • Qibin Yang,
  • Lishi Yang,
  • Jianhua Huang,
  • Jianzhi Shi,
  • Yangyang Ding,
  • Erchao Li,
  • Yundong Li

摘要

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.