Minet Cécile, Chantal Isabelle, Berthier David
CIRAD, UMR INTERTRYP, F-34398, Montpellier, France; INTERTRYP, Univ Montpellier, CIRAD, IRD, Montpellier, France.
CIRAD, UMR INTERTRYP, F-34398, Montpellier, France; INTERTRYP, Univ Montpellier, CIRAD, IRD, Montpellier, France.
Exp Parasitol. 2023 Sep;252:108589. doi: 10.1016/j.exppara.2023.108589. Epub 2023 Jul 28.
African Animal Trypanosomosis (AAT or Nagana) is a vector-borne disease caused by Trypanosomatidae, genus Trypanosoma. The disease is transmitted by the bite of infected hematophagous insects, mainly tsetse flies but also other blood-sucking insects including stomoxes and tabanids. Although many trypanosome species infect animals, the main agents responsible for this disease with a strong socio-economic and veterinary health impact are Trypanosoma congolense (T. congolense or Tc), Trypanosoma vivax (T.vivax), and to a lesser extent, Trypanosoma brucei brucei (T.brucei brucei or Tbb). These parasites mainly infect livestock, including cattle, in sub-Saharan Africa, with major repercussions in terms of animal productivity and poverty for populations which are often already very poor. As there is currently no vaccine, the fight against the disease is primarily based on diagnosis, treatment and vector control. To develop new tools (particularly therapeutic tools) to fight against the disease, we need to know both the biology and the genes involved in the pathogenicity and virulence of the parasites. To date, unlike for Trypanosoma brucei (T.brucei) or Trypanosoma cruzi (T.cruzi), genome editing tools has been relatively little used to study T. congolense. We present an efficient, reproducible and stable CRISPR-Cas9 genome editing system for use in Tc bloodstream forms (Tc-BSF). This plasmid-free system is based on transient expression of Cas9 protein and the use of a ribonucleoprotein formed by the Cas9 and sgRNA complex. This is the first proof of concept of genome editing using CRISPR-Cas9 ribonucleoproteins on Tc-BSF. This adapted protocol enriches the "toolbox" for the functional study of genes of interest in blood forms of the Trypanosoma congolense. This proof of concept is an important step for the scientific community working on the study of trypanosomes and opens up new perspectives for the control of and fight against animal trypanosomosis.
非洲动物锥虫病（AAT或那加那病）是一种由锥虫属的锥虫科寄生虫引起的媒介传播疾病。该疾病通过受感染的吸血昆虫叮咬传播，主要是采采蝇，但也包括其他吸血昆虫，如厩螫蝇和虻。尽管许多锥虫物种会感染动物，但对社会经济和兽医健康有重大影响的主要病原体是刚果锥虫（T. congolense或Tc）、活跃锥虫（T.vivax），以及在较小程度上的布氏锥虫（T.brucei brucei或Tbb）。这些寄生虫主要感染撒哈拉以南非洲的家畜，包括牛，对动物生产力和往往已经非常贫困的人口的贫困状况产生重大影响。由于目前尚无疫苗，对抗该疾病主要基于诊断、治疗和媒介控制。为了开发对抗该疾病的新工具（特别是治疗工具），我们需要了解寄生虫致病性和毒力所涉及的生物学特性和基因。迄今为止，与布氏锥虫（T.brucei）或克氏锥虫（T.cruzi）不同，基因组编辑工具在研究刚果锥虫方面的应用相对较少。我们展示了一种高效、可重复且稳定的CRISPR-Cas9基因组编辑系统，用于刚果锥虫血流形式（Tc-BSF）。这个无质粒系统基于Cas9蛋白的瞬时表达以及使用由Cas9和sgRNA复合物形成的核糖核蛋白。这是首次使用CRISPR-Cas9核糖核蛋白对Tc-BSF进行基因组编辑的概念验证。这种经过调整的方案丰富了用于刚果锥虫血液形式中感兴趣基因功能研究的“工具箱”。这一概念验证对于致力于锥虫研究的科学界来说是重要的一步，并为控制和对抗动物锥虫病开辟了新的前景。
