Lander Noelia, Chiurillo Miguel A, Storey Melissa, Vercesi Anibal E, Docampo Roberto
From the Departamento de Patologia Clínica, Faculdade de Ciências Médicas, Universidade Estadual de Campinas, Campinas, São Paulo 13083, Brazil and
From the Departamento de Patologia Clínica, Faculdade de Ciências Médicas, Universidade Estadual de Campinas, Campinas, São Paulo 13083, Brazil and.
J Biol Chem. 2016 Dec 2;291(49):25505-25515. doi: 10.1074/jbc.M116.749655. Epub 2016 Oct 28.
Methods for genetic manipulation of Trypanosoma cruzi, the etiologic agent of Chagas disease, have been highly inefficient, and no endogenous tagging of genes has been reported to date. We report here the use of the CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR-associated gene 9) system for endogenously tagging genes in this parasite. The utility of the method was established by tagging genes encoding proteins of known localization such as TcFCaBP (flagellar calcium binding protein) and TcVP1 (vacuolar proton pyrophosphatase), and two proteins of undefined or disputed localization, the TcMCU (mitochondrial calcium uniporter) and TcIPR (inositol 1,4,5-trisphosphate receptor). We confirmed the flagellar and acidocalcisome localization of TcFCaBP and TcVP1 by co-localization with antibodies to the flagellum and acidocalcisomes, respectively. As expected, TcMCU was co-localized with the voltage-dependent anion channel to the mitochondria. However, in contrast to previous reports and our own results using overexpressed TcIPR, endogenously tagged TcIPR showed co-localization with antibodies against VP1 to acidocalcisomes. These results are also in agreement with our previous reports on the localization of this channel to acidocalcisomes of Trypanosoma brucei and suggest that caution should be exercised when overexpression of tagged genes is done to localize proteins in T. cruzi.
恰加斯病的病原体克氏锥虫的基因操作方法一直效率低下，迄今为止尚无基因内源标记的报道。我们在此报告使用CRISPR（成簇规律间隔短回文重复序列）/Cas9（CRISPR相关基因9）系统对该寄生虫的基因进行内源标记。通过标记编码已知定位蛋白的基因，如鞭毛钙结合蛋白（TcFCaBP）和液泡质子焦磷酸酶（TcVP1），以及两种定位不明确或有争议的蛋白，线粒体钙单向转运体（TcMCU）和肌醇-1,4,5-三磷酸受体（TcIPR），确定了该方法的实用性。我们分别通过与抗鞭毛和酸性钙小体的抗体共定位，证实了TcFCaBP和TcVP1在鞭毛和酸性钙小体中的定位。正如预期的那样，TcMCU与电压依赖性阴离子通道在线粒体中共定位。然而，与之前的报道以及我们自己使用过表达的TcIPR的结果相反，内源标记的TcIPR与抗VP1抗体在酸性钙小体中共定位。这些结果也与我们之前关于该通道在布氏锥虫酸性钙小体中定位的报道一致，并表明在克氏锥虫中通过过表达标记基因来定位蛋白时应谨慎行事。
