Rapid, Selection-Free, High-Efficiency Genome Editing in Protozoan Parasites Using CRISPR-Cas9 Ribonucleoproteins.
Rapid, Selection-Free, High-Efficiency Genome Editing in Protozoan Parasites Using CRISPR-Cas9 Ribonucleoproteins.
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DOI：
10.1128/mbio.01788-17
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发表时间：
2017-11-07
期刊：
影响因子：
6.4
通讯作者：
Tarleton RL
中科院分区：
文献类型：
--
作者：
Soares Medeiros LC;South L;Peng D;Bustamante JM;Wang W;Bunkofske M;Perumal N;Sanchez-Valdez F;Tarleton RL
锥虫，包括人类病原体克氏锥虫(恰加斯病的病原体)、布氏锥虫(非洲昏睡病)和利什曼原虫(利什曼病)，影响全球数百万人和动物。托克氏疟原虫被认为是研究最少、了解最少的热带疾病致病寄生虫之一，部分原因是相对缺乏方便的基因工程工具。最近，通过应用簇状规则间隔短回文重复序列-CRISPR-相关蛋白9(CRISPR-Cas9)技术，这种情况有所改善，但仍然存在一些限制，包括持续表达Cas9的毒性和较长的药物标记物选择时间。在本研究中，我们发现由金黄色葡萄球菌重组Cas9(SaCas9)组成的核糖核蛋白(RNP)复合体(RNP)，而不是来自更常规使用的化脓性链球菌Cas9(SpCas9)的核糖核蛋白(RNP)复合体，以及在玻璃转录的单引导RNAs(SgRNAs)的传递，导致金黄色葡萄球菌和其他动质体中快速基因编辑的频率接近100%。通过SaCas9/sgRNA RNPs获得了报告基因和内源基因的高效基因组编辑，并在不同品系的不同寄生虫生活史阶段以及在布氏毛滴虫和大利什曼原虫中观察到。RNP复合体传递也被用来成功地标记内源基因上的蛋白质，并评估必要基因的生物学功能。因此，使用SaCas9 RNP复合体在动质体内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的人类病原体的基因功能。原生动物寄生虫仍然是一些对人类和动物影响最大的病原体，治疗和预防选择非常有限。改进疗法和疫苗的发展依赖于更好地了解这些有机体的独特生物学，而了解它们的生物学反过来需要跟踪和操纵基因产物的能力。在这项工作中，我们描述了基本上适用于任何实验室和适用于任何寄生虫分离物的新方法，以方便和快速地编辑动质体寄生虫的基因组。我们证明，这些方法提供了快速评估功能的方法，包括必要基因产物和药物潜在靶点的功能，并在其内源基因位点标记基因产物。这一切都是在没有基因克隆或药物选择的情况下实现的。我们预计这一进展将使调查成为可能，特别是对克鲁氏锥虫和利什曼原虫的调查，而这些调查人员几十年来一直在逃避。
Trypanosomatids (order Kinetoplastida), including the human pathogens Trypanosoma cruzi (agent of Chagas disease), Trypanosoma brucei, (African sleeping sickness), and Leishmania (leishmaniasis), affect millions of people and animals globally. T. cruzi 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 Staphylococcus aureus (SaCas9), but not from the more routinely used Streptococcus pyogenes Cas9 (SpCas9), and in vitro-transcribed single guide RNAs (sgRNAs) results in rapid gene edits in T. cruzi 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 T. cruzi, as well as in T. brucei and Leishmania major. 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 Trypanosoma cruzi and Leishmania spp., that have eluded investigators for decades.