A redesigned CRISPR/Cas9 system for marker-free genome editing in Plasmodium falciparum

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• 作者：Junnan Lu ; Ying Tong ; Jiaqiang Pan ; Yijun Yang ; Quan Liu…
• 关键词：Marker ; free ; CRISPR/Cas9 ; Plasmodium falciparum
• 刊名：Parasites & Vectors
• 出版年：2016
• 出版时间：December 2016
• 年：2016
• 卷：9
• 期：1
• 全文大小：1,548 KB
• 刊物主题：Parasitology; Infectious Diseases; Tropical Medicine; Entomology;
• 出版者：BioMed Central
• ISSN：1756-3305

文摘

Background A highly efficient CRISPR/Cas9-based marker-free genome editing system has been established in Plasmodium falciparum (Pf). However, with the current methods, two drug-selectable markers are needed for episome retention, which may present hurdles for consecutive genome manipulations due to the limited number of available selectable markers. The loading capacity of donor DNA is also unsatisfactory due to the large size of the Cas9 nuclease and sgRNA co-expression system, which limits the size of knock-in DNA fragments. Because of the inefficient end joining (EJ) DNA repair mechanism of Pf, a suicide-rescue approach could be used to address the challenges. Cas9 nuclease and sgRNA were co-expressed from a single plasmid (suicide vector) with one selectable marker, and the donor DNA was ligated into the other plasmid (rescue vector) containing only the ampicillin-resistance gene (AmpR) and a ColEl replication origin (ori). Nonetheless, whether this approach can mediate even the regular gene editing in Pf remains unknown. This study aimed to demonstrate the basic gene editing function of this Cas9-mediated suicide-rescue system.