Gene edition using the CRISPR/Cas9 system has been achieved in few crustaceans, but in penaeid shrimps, several hurdles exist, restraining the potential of CRISPR-Cas9 technology in shrimp Aquaculture. A key step for gene editing is the efficient introduction of the CRISPR-Cas9 system into the cells. Although microinjection is an effective method for CRISPR-Cas9 system delivery into zygotes, the successful gene edition in this commercially important taxon is hindered due to the challenges posed by this technique. Therefore, experimental approaches based on microinjection-free protocols are of scientific interest for Aquaculture researchers. In this work, we performed physical (electroporation) and chemical (polyethylenimine and cationic lipids) transfection methods in P. vannamei zygotes. For Cas9 delivery, we used three different cargoes: DNA plasmid, mRNA and a recombinant protein. Different ratios of sgRNA designed to recognize the PvCatL gene were used to prepare the CRISPR-Cas9 complexes. Treated shrimp zygotes were genotyped by HRM analysis and Sanger sequencing. Although high hatching rates were observed for most treatments, no irrefutable evidence of typical CRISPR-Cas9-induced gene edition was found; instead, an enrichment of gene variants was observed in treated organisms, which was detectable by HRM. Our effort is of interest to Aquaculture researchers working on this challenging topic, helping to improve their experimental design or as a reference to evaluate additional conditions to achieve the gene editing in penaeid shrimps.

中文翻译：

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在对虾基因编辑中传递 CRISPR-Cas9 组件的障碍

使用 CRISPR/Cas9 系统的基因编辑已经在少数甲壳类动物中实现，但在对虾中，存在一些障碍，限制了 CRISPR-Cas9 技术在对虾水产养殖中的潜力。基因编辑的一个关键步骤是将 CRISPR-Cas9 系统有效引入细胞。尽管显微注射是将 CRISPR-Cas9 系统递送到受精卵的有效方法，但由于该技术带来的挑战，在这个具有商业重要性的分类群中成功进行基因编辑受到阻碍。因此，基于无显微注射协议的实验方法对水产养殖研究人员具有科学意义。在这项工作中，我们在P 中进行了物理（电穿孔）和化学（聚乙烯亚胺和阳离子脂质）转染方法。南美白对虾合子。对于 Cas9 递送，我们使用了三种不同的货物：DNA 质粒、mRNA 和重组蛋白。设计用于识别 PvCatL 基因的不同比例的 sgRNA 用于制备 CRISPR-Cas9 复合物。处理过的虾受精卵通过 HRM 分析和 Sanger 测序进行基因分型。尽管在大多数处理中观察到了高孵化率，但没有发现典型的 CRISPR-Cas9 诱导基因编辑的无可辩驳的证据；相反，在处理过的生物体中观察到基因变异的富集，这可以通过 HRM 检测到。我们的努力对从事这一具有挑战性的课题的水产养殖研究人员感兴趣，有助于改进他们的实验设计或作为评估额外条件以实现对虾基因编辑的参考。