Bernhard Nocht Institute for Tropical Medicine, D-20359 Hamburg, Germany.
Instituto de Medicina Tropical Alexander von Humboldt, Universidad Peruana Cayetano Heredia, Lima 15102, Peru.
Genes (Basel). 2020 Sep 30;11(10):1159. doi: 10.3390/genes11101159.
The protozoan parasite () ) is the main cause of human tegumentary leishmaniasis in the New World, a disease affecting the skin and/or mucosal tissues. Despite its importance, the study of the unique biology of through reverse genetics analyses has so far lagged behind in comparison with Old World spp. In this study, we successfully applied a cloning-free, PCR-based CRISPR-Cas9 technology in that was previously developed for Old World and New World species. As proof of principle, we demonstrate the targeted replacement of a transgene () and two single-copy genes ( and ). We obtained homozygous Cas9-free - and -null mutants in that matched the phenotypes reported previously for the respective null mutants. The function of is indeed conserved throughout the Trypanosomatida as null mutants could be complemented phenotypically with transgenes from a range of trypanosomatids. In summary, the feasibility of genetic manipulation of by CRISPR-Cas9-mediated gene editing sets the stage for testing the role of specific genes in that parasite's biology, including functional studies of virulence factors in relevant animal models to reveal novel therapeutic targets to combat American tegumentary leishmaniasis.
原生动物寄生虫（）是新世界人体皮肤利什曼病的主要病原体，这种疾病会影响皮肤和/或粘膜组织。尽管它很重要，但与旧世界利什曼原虫属相比，通过反向遗传学分析研究的独特生物学特性在很大程度上落后了。在这项研究中，我们成功地将一种无克隆、基于 PCR 的 CRISPR-Cas9 技术应用于旧世界和新世界的利什曼原虫属，该技术之前已为旧世界和新世界的物种开发。作为原理的证明，我们展示了靶向替换一个转基因（）和两个单拷贝基因（和）。我们获得了在中纯合 Cas9 缺失的 - 和 - 突变体，与各自的 - 缺失突变体先前报道的表型相匹配。在整个锥虫门中，的功能确实是保守的，因为来自各种锥虫门的转基因可以在表型上互补 缺失突变体。总之，通过 CRISPR-Cas9 介导的基因编辑对进行遗传操作的可行性为测试该寄生虫生物学中特定基因的功能奠定了基础，包括在相关动物模型中对毒力因子进行功能研究，以揭示针对美洲皮肤利什曼病的新治疗靶点。
