Lentiviral Transduction-based CRISPR/Cas9 Editing of Schistosoma mansoni Acetylcholinesterase.

IF 16.4 1区 化学 Q1 CHEMISTRY, MULTIDISCIPLINARY Accounts of Chemical Research Pub Date : 2023-11-22 DOI:10.2174/1389202924666230823094608
Xiaofeng Du, Donald P McManus, Juliet D French, Haran Sivakumaran, Rebecca L Johnston, Olga Kondrashova, Conor E Fogarty, Malcolm K Jones, Hong You
{"title":"Lentiviral Transduction-based CRISPR/Cas9 Editing of <i>Schistosoma mansoni</i> Acetylcholinesterase.","authors":"Xiaofeng Du, Donald P McManus, Juliet D French, Haran Sivakumaran, Rebecca L Johnston, Olga Kondrashova, Conor E Fogarty, Malcolm K Jones, Hong You","doi":"10.2174/1389202924666230823094608","DOIUrl":null,"url":null,"abstract":"<p><strong>Background: </strong>Recent studies on CRISPR/Cas9-mediated gene editing in <i>Schistosoma mansoni</i> have shed new light on the study and control of this parasitic helminth. However, the gene editing efficiency in this parasite is modest.</p><p><strong>Methods: </strong>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 (<i>SmAChE</i>) and a mCherry fluorescence marker into schistosomes.</p><p><strong>Results: </strong>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 <i>SmAChE</i>-edited parasites, including decreased <i>SmAChE</i> activity, reduced hatching ability of edited eggs, and altered behavior of miracidia hatched from edited eggs. Next-generation sequencing analysis demonstrated that the lentiviral transduction-based CRISPR/Cas9 gene modifications in <i>SmAChE</i>-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.</p><p><strong>Conclusion: </strong>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 <i>S. mansoni</i>.</p>","PeriodicalId":1,"journal":{"name":"Accounts of Chemical Research","volume":null,"pages":null},"PeriodicalIF":16.4000,"publicationDate":"2023-11-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10761339/pdf/","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Accounts of Chemical Research","FirstCategoryId":"99","ListUrlMain":"https://doi.org/10.2174/1389202924666230823094608","RegionNum":1,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q1","JCRName":"CHEMISTRY, MULTIDISCIPLINARY","Score":null,"Total":0}
引用次数: 0

Abstract

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 transduction-based 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.

查看原文
分享 分享
微信好友 朋友圈 QQ好友 复制链接
本刊更多论文
基于慢病毒转导的CRISPR/Cas9编辑曼氏血吸虫乙酰胆碱酯酶
最近对曼氏血吸虫CRISPR/Cas9介导的基因编辑的研究为研究和控制这种寄生蠕虫提供了新的线索。然而,这种寄生虫的基因编辑效率并不高。为了提高血吸虫CRISPR/Cas9基因组编辑的效率,我们使用在哺乳动物细胞中有效用于基因编辑的慢病毒,将编码Cas9核酸酶、靶向乙酰胆碱酯酶的sgRNA(SmAChE)和mCherry荧光标记的质粒DNA递送到血吸虫中。在转导的卵子、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的表型变化,包括SmAChE活性降低,编辑卵的孵化能力降低,以及从编辑卵孵化出的奇迹虫的行为改变。下一代测序分析表明,在SmAChE编辑的血吸虫中,基于慢病毒转导的CRISPR/Cas9基因修饰是同源定向修复为主的,但其效率远低于使用电穿孔(我们实验室先前公布的数据)递送CRISPR组分的效率。总之,在将CRISPR/Cas9递送到血吸虫中进行程序基因组编辑方面,电穿孔比慢病毒转导更有效。对增强CRISPR/Cas9基因编辑策略的探索为曼氏假单胞菌程序基因组编辑的未来改进提供了基础。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
求助全文
约1分钟内获得全文 去求助
来源期刊
Accounts of Chemical Research
Accounts of Chemical Research 化学-化学综合
CiteScore
31.40
自引率
1.10%
发文量
312
审稿时长
2 months
期刊介绍: Accounts of Chemical Research presents short, concise and critical articles offering easy-to-read overviews of basic research and applications in all areas of chemistry and biochemistry. These short reviews focus on research from the author’s own laboratory and are designed to teach the reader about a research project. In addition, Accounts of Chemical Research publishes commentaries that give an informed opinion on a current research problem. Special Issues online are devoted to a single topic of unusual activity and significance. Accounts of Chemical Research replaces the traditional article abstract with an article "Conspectus." These entries synopsize the research affording the reader a closer look at the content and significance of an article. Through this provision of a more detailed description of the article contents, the Conspectus enhances the article's discoverability by search engines and the exposure for the research.
期刊最新文献
Management of Cholesteatoma: Hearing Rehabilitation. Congenital Cholesteatoma. Evaluation of Cholesteatoma. Management of Cholesteatoma: Extension Beyond Middle Ear/Mastoid. Recidivism and Recurrence.
×
引用
GB/T 7714-2015
复制
MLA
复制
APA
复制
导出至
BibTeX EndNote RefMan NoteFirst NoteExpress
×
×
提示
您的信息不完整,为了账户安全,请先补充。
现在去补充
×
提示
您因"违规操作"
具体请查看互助需知
我知道了
×
提示
现在去查看 取消
×
提示
确定
0
微信
客服QQ
Book学术公众号 扫码关注我们
反馈
×
意见反馈
请填写您的意见或建议
请填写您的手机或邮箱
已复制链接
已复制链接
快去分享给好友吧!
我知道了
×
扫码分享
扫码分享
Book学术官方微信
Book学术文献互助
Book学术文献互助群
群 号:481959085
Book学术
文献互助 智能选刊 最新文献 互助须知 联系我们:info@booksci.cn
Book学术提供免费学术资源搜索服务,方便国内外学者检索中英文文献。致力于提供最便捷和优质的服务体验。
Copyright © 2023 Book学术 All rights reserved.
ghs 京公网安备 11010802042870号 京ICP备2023020795号-1