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利用 CRISPR-Cas9 核糖核蛋白在原生动物寄生虫中进行快速、无选择、高效的基因组编辑。

Rapid, Selection-Free, High-Efficiency Genome Editing in Protozoan Parasites Using CRISPR-Cas9 Ribonucleoproteins.

机构信息

Center for Tropical and Emerging Global Diseases and Department of Cellular Biology, University of Georgia, Athens, Georgia, USA.

Center for Tropical and Emerging Global Diseases and Department of Cellular Biology, University of Georgia, Athens, Georgia, USA

出版信息

mBio. 2017 Nov 7;8(6):e01788-17. doi: 10.1128/mBio.01788-17.


DOI:10.1128/mBio.01788-17
PMID:29114029
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC5676044/
Abstract

Trypanosomatids (order Kinetoplastida), including the human pathogens (agent of Chagas disease), , (African sleeping sickness), and (leishmaniasis), affect millions of people and animals globally. is considered one of the least studied and most poorly understood tropical disease-causing parasites, in part because of the relative lack of facile genetic engineering tools. This situation has improved recently through the application of clustered regularly interspaced short palindromic repeats-CRISPR-associated protein 9 (CRISPR-Cas9) technology, but a number of limitations remain, including the toxicity of continuous Cas9 expression and the long drug marker selection times. In this study, we show that the delivery of ribonucleoprotein (RNP) complexes composed of recombinant Cas9 from (SaCas9), but not from the more routinely used Cas9 (SpCas9), and -transcribed single guide RNAs (sgRNAs) results in rapid gene edits in and other kinetoplastids at frequencies approaching 100%. The highly efficient genome editing via SaCas9/sgRNA RNPs was obtained for both reporter and endogenous genes and observed in multiple parasite life cycle stages in various strains of , as well as in and RNP complex delivery was also used to successfully tag proteins at endogenous loci and to assess the biological functions of essential genes. Thus, the use of SaCas9 RNP complexes for gene editing in kinetoplastids provides a simple, rapid, and cloning- and selection-free method to assess gene function in these important human pathogens. Protozoan parasites remain some of the highest-impact human and animal pathogens, with very limited treatment and prevention options. The development of improved therapeutics and vaccines depends on a better understanding of the unique biology of these organisms, and understanding their biology, in turn, requires the ability to track and manipulate the products of genes. In this work, we describe new methods that are available to essentially any laboratory and applicable to any parasite isolate for easily and rapidly editing the genomes of kinetoplastid parasites. We demonstrate that these methods provide the means to quickly assess function, including that of the products of essential genes and potential targets of drugs, and to tag gene products at their endogenous loci. This is all achieved without gene cloning or drug selection. We expect this advance to enable investigations, especially in and spp., that have eluded investigators for decades.

摘要

锥体虫(动基体目),包括人类病原体(恰加斯病的病原体)、、(非洲昏睡病)和(利什曼病),影响着全球数以百万计的人和动物。被认为是研究最少、了解最不充分的热带致病寄生虫之一,部分原因是缺乏简便的遗传工程工具。这种情况最近通过应用成簇规律间隔短回文重复(CRISPR)相关蛋白 9(CRISPR-Cas9)技术得到了改善,但仍存在一些限制,包括持续 Cas9 表达的毒性和长的药物标记选择时间。在这项研究中,我们表明,由(SaCas9)而非更常用的 Cas9(SpCas9)组成的核糖核蛋白(RNP)复合物的递送,以及由 -转录的单指导 RNA(sgRNA),可导致在和其他动基体目寄生虫中以接近 100%的频率快速进行基因编辑。通过 SaCas9/sgRNA RNP 高效进行基因组编辑,可用于报告基因和内源性基因,并观察到各种 、和 菌株的寄生虫生命周期的多个阶段。RNP 复合物的递送还用于成功标记内源性基因座的蛋白质,并评估必需基因的生物学功能。因此,在动基体目寄生虫中使用 SaCas9 RNP 复合物进行基因编辑提供了一种简单、快速、无需克隆和选择的方法来评估这些重要人类病原体中的基因功能。原生动物寄生虫仍然是对人类和动物影响最大的病原体之一,其治疗和预防选择非常有限。改进治疗方法和疫苗的发展取决于对这些生物体独特生物学的更好理解,而了解它们的生物学反过来又需要能够跟踪和操纵基因产物。在这项工作中,我们描述了几乎任何实验室都可用且适用于任何寄生虫分离株的新方法,用于轻松快速编辑动基体目寄生虫的基因组。我们证明,这些方法提供了快速评估功能的手段,包括必需基因产物的功能和潜在药物靶点的功能,并标记内源基因座的基因产物。所有这些都无需基因克隆或药物选择。我们希望这一进展能够使研究人员能够对几十年来一直困扰着研究人员的 和 种进行研究。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/5f64/5676044/8d352e8b5d0b/mbo0051735660006.jpg
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https://cdn.ncbi.nlm.nih.gov/pmc/blobs/5f64/5676044/f6dc012b62a2/mbo0051735660001.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/5f64/5676044/534476eb9150/mbo0051735660002.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/5f64/5676044/f798986113a1/mbo0051735660003.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/5f64/5676044/c66269418419/mbo0051735660004.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/5f64/5676044/0ac91b27a1ca/mbo0051735660005.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/5f64/5676044/8d352e8b5d0b/mbo0051735660006.jpg

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本文引用的文献

[1]
A CRISPR Cas9 high-throughput genome editing toolkit for kinetoplastids.

R Soc Open Sci. 2017-5-3

[2]
Different Roles of Mitochondrial Calcium Uniporter Complex Subunits in Growth and Infectivity of .

mBio. 2017-5-9

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Int J Parasitol Drugs Drug Resist. 2015-12-11

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PLoS Negl Trop Dis. 2016-1-21

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