Mohring Franziska, Hart Melissa N, Patel Avnish, Baker David A, Moon Robert W
Faculty of Infectious and Tropical Diseases, London School of Hygiene & Tropical Medicine, London WC1E 7HT, United Kingdom.
Bio Protoc. 2020 Feb 20;10(4):e3522. doi: 10.21769/BioProtoc.3522.
is a zoonotic malaria parasite in Southeast Asia that can cause severe and fatal malaria in humans. The main hosts are Macaques, but modern diagnostic tools reveal increasing numbers of human infections. After is the only other malaria parasite capable of being maintained in long term culture with human red blood cells (RBCs). Its closer ancestry to other non-falciparum human malaria parasites, more balanced AT-content, larger merozoites and higher transfection efficiencies, gives some key advantages over for the study of malaria parasite cell/molecular biology. Here, we describe the generation of marker-free CRISPR gene-edited parasites, the fast and scalable production of transfection constructs and analysis of transfection efficiencies. Our protocol allows rapid, reliable and unlimited rounds of genome editing in requiring only a single recyclable selection marker.
是东南亚的一种人畜共患疟原虫，可导致人类严重和致命的疟疾。主要宿主是猕猴，但现代诊断工具显示人类感染数量在增加。是唯一能够与人类红细胞（RBC）进行长期培养的另一种疟原虫。它与其他非恶性疟原虫人类疟原虫的亲缘关系更近、AT含量更平衡、裂殖子更大且转染效率更高，这使其在疟原虫细胞/分子生物学研究方面比具有一些关键优势。在这里，我们描述了无标记CRISPR基因编辑疟原虫的产生、转染构建体的快速且可扩展生产以及转染效率分析。我们的方案允许在仅需单个可回收选择标记的情况下，对进行快速、可靠且无限轮次的基因组编辑。
