Departamento de Bioquímica e Imunologia, Universidade Federal de Minas Gerais, Belo Horizonte, Brazil.
Centro de Pesquisa Rene Rachou, Fundação Osvaldo Cruz, Minas Gerais, Brazil.
Adv Exp Med Biol. 2023;1429:111-125. doi: 10.1007/978-3-031-33325-5_7.
Chagas disease, caused by the protozoan parasite Trypanosoma cruzi, is an illness that affects 6-8 million people worldwide and is responsible for approximately 50,000 deaths per year. Despite intense research efforts on Chagas disease and its causative agent, there is still a lack of effective treatments or strategies for disease control. Although significant progress has been made toward the elucidation of molecular mechanisms involved in host-parasite interactions, particularly immune evasion mechanisms, a deeper understanding of these processes has been hindered by a lack of efficient genetic manipulation protocols. One major challenge is the fact that several parasite virulence factors are encoded by multigene families, which constitute a distinctive feature of the T. cruzi genome. The recent advent of the CRISPR/Cas9 technology represented an enormous breakthrough in the studies involving T. cruzi genetic manipulation compared to previous protocols that are poorly efficient and required a long generation time to develop parasite mutants. Since the first publication of CRISPR gene editing in T. cruzi, in 2014, different groups have used distinct protocols to generated knockout mutants, parasites overexpressing a protein or expressing proteins with sequence tags inserted in the endogenous gene. Importantly, CRISPR gene editing allowed generation of parasite mutants with gene disruption in multi-copy gene families. We described four main strategies used to edit the T. cruzi genome and summarized a large list of studies performed by different groups in the past 7 years that are addressing several mechanisms involved with parasite proliferation, differentiation, and survival strategies within its different hosts.
克氏锥虫病由原生动物寄生虫克氏锥虫引起，是一种影响全球 600 至 800 万人的疾病，每年导致约 5 万人死亡。尽管针对克氏锥虫病及其病原体进行了大量研究，但仍缺乏有效的治疗方法或疾病控制策略。尽管在阐明宿主-寄生虫相互作用涉及的分子机制方面取得了重大进展，特别是免疫逃避机制，但由于缺乏有效的遗传操作协议，对这些过程的深入了解受到了阻碍。一个主要的挑战是，寄生虫的几个毒力因子是由多基因家族编码的，这是 T. cruzi 基因组的一个显著特征。与之前效率低下且需要很长代时才能开发寄生虫突变体的协议相比，CRISPR/Cas9 技术的出现代表了涉及 T. cruzi 遗传操作研究的巨大突破。自 2014 年首次在 T. cruzi 中发表 CRISPR 基因编辑以来，不同的研究小组已经使用不同的协议来生成敲除突变体、过表达蛋白的寄生虫或在内源基因中插入序列标签表达蛋白的寄生虫。重要的是，CRISPR 基因编辑允许在多拷贝基因家族中对寄生虫进行基因破坏的突变体的生成。我们描述了编辑 T. cruzi 基因组的四种主要策略，并总结了过去 7 年中不同研究小组进行的大量研究，这些研究涉及寄生虫增殖、分化和在不同宿主中生存策略的几个机制。
