Background: Recent studies on CRISPR/Cas9-mediated gene editing in Schistosoma mansoni have shed new light on the study and control of this parasitic helminth. However, the gene editing efficiency in this parasite is modest. Methods: To improve the efficiency of CRISPR/Cas9 genome editing in schistosomes, we used lentivirus, which has been effectively used for gene editing in mammalian cells, to deliver plasmid DNA encoding Cas9 nuclease, a sgRNA targeting acetylcholinesterase (SmAChE) and a mCherry fluorescence marker into schistosomes. Results: MCherry fluorescence was observed in transduced eggs, schistosomula, and adult worms, indicating that the CRISPR components had been delivered into these parasite stages by lentivirus. In addition, clearly changed phenotypes were observed in SmAChE-edited parasites, including decreased SmAChE activity, reduced hatching ability of edited eggs, and altered behavior of miracidia hatched from edited eggs. Next-generation sequencing analysis demonstrated that the lentiviral transductionbased CRISPR/Cas9 gene modifications in SmAChE-edited schistosomes were homology-directed repair predominant but with much lower efficiency than that obtained using electroporation (data previously published by our laboratory) for the delivery of CRISPR components. Conclusion: Taken together, electroporation is more efficient than lentiviral transduction in the delivery of CRISPR/Cas9 into schistosomes for programmed genome editing. The exploration of tactics for enhancing CRISPR/Cas9 gene editing provides the basis for the future improvement of programmed genome editing in S. mansoni.

中文翻译：

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基于慢病毒转导的曼氏血吸虫乙酰胆碱酯酶 CRISPR/Cas9 编辑

背景：最近对曼氏血吸虫中 CRISPR/Cas9 介导的基因编辑的研究为这种寄生蠕虫的研究和控制提供了新的线索。然而，这种寄生虫的基因编辑效率并不高。方法：为了提高血吸虫中 CRISPR/Cas9 基因组编辑的效率，我们使用已有效用于哺乳动物细胞基因编辑的慢病毒来传递编码 Cas9 核酸酶、靶向乙酰胆碱酯酶 (SmAChE) 的 sgRNA 和 mCherry 荧光的质粒 DNA标记为血吸虫。结果：在转导的卵、血吸虫和成虫中观察到 MCherry 荧光，表明 CRISPR 成分已通过慢病毒传递到这些寄生虫阶段。此外，在 SmAChE 编辑的寄生虫中观察到明显改变的表型，包括 SmAChE 活性降低、编辑卵的孵化能力降低以及从编辑卵孵化的毛蚴行为改变。新一代测序分析表明，SmAChE 编辑的血吸虫中基于慢病毒转导的 CRISPR/Cas9 基因修饰以同源定向修复为主，但效率远低于使用电穿孔（我们实验室之前发布的数据）传递 CRISPR 组件所获得的效率。结论：总而言之，在将 CRISPR/Cas9 递送到血吸虫中进行程序化基因组编辑方面，电穿孔比慢病毒转导更有效。增强CRISPR/Cas9基因编辑策略的探索为未来改进曼氏酵母程序化基因组编辑奠定了基础。