title: A redesigned CRISPR/Cas9 system for marker-free genome editing in Plasmodium falciparum.
abstract: 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.
  
  
  RESULTS
  The suicide and rescue vectors were constructed and co-transfected into Pf3D7. This system worked as expected when used to disrupt the Pfset2 gene and to insert a green fluorescent protein-renilla luciferase (gfp-ruc) fusion gene cassette of 3334 base pairs (bp) into the Pf47 locus, demonstrating that the suicide vector actually induced double-strand breaks (DSBs) and that the rescue vector functioned without maintenance via drug selection.
  
  
  CONCLUSIONS
  The adapted marker-free CRISPR/Cas9 system with only a single episome-selectable marker performs well as the current systems for general gene editing which lays a solid foundation for further studies including consecutive gene manipulations and large gene knock-ins.
authors.affiliation: Laboratory of Pathogen Biology, State Key Laboratory of Respiratory Disease, Center for Infection and Immunity, Guangzhou Institutes of Biomedicine and Health (GIBH), Chinese Academy of Sciences, No. 190 Kaiyuan Avenue, Guangzhou Science Park, Guangzhou, 510530, Guangdong Province, China.
authors.forename: Junnan
authors.initials: J
authors.lastname: Lu
authors.affiliation: CAS Lamvac Biotech Co., Ltd, No. 3 Lanyue Road, Guangzhou Science Park, Guangzhou, 510530, Guangdong Province, China.
authors.forename: Ying
authors.initials: Y
authors.lastname: Tong
authors.affiliation: CAS Lamvac Biotech Co., Ltd, No. 3 Lanyue Road, Guangzhou Science Park, Guangzhou, 510530, Guangdong Province, China.
authors.forename: Jiaqiang
authors.initials: J
authors.lastname: Pan
authors.affiliation: Laboratory of Pathogen Biology, State Key Laboratory of Respiratory Disease, Center for Infection and Immunity, Guangzhou Institutes of Biomedicine and Health (GIBH), Chinese Academy of Sciences, No. 190 Kaiyuan Avenue, Guangzhou Science Park, Guangzhou, 510530, Guangdong Province, China.
authors.forename: Yijun
authors.initials: Y
authors.lastname: Yang
authors.affiliation: Laboratory of Pathogen Biology, State Key Laboratory of Respiratory Disease, Center for Infection and Immunity, Guangzhou Institutes of Biomedicine and Health (GIBH), Chinese Academy of Sciences, No. 190 Kaiyuan Avenue, Guangzhou Science Park, Guangzhou, 510530, Guangdong Province, China.
authors.forename: Quan
authors.initials: Q
authors.lastname: Liu
authors.affiliation: Laboratory of Pathogen Biology, State Key Laboratory of Respiratory Disease, Center for Infection and Immunity, Guangzhou Institutes of Biomedicine and Health (GIBH), Chinese Academy of Sciences, No. 190 Kaiyuan Avenue, Guangzhou Science Park, Guangzhou, 510530, Guangdong Province, China.
authors.forename: Xuefang
authors.initials: X
authors.lastname: Tan
authors.affiliation: Laboratory of Pathogen Biology, State Key Laboratory of Respiratory Disease, Center for Infection and Immunity, Guangzhou Institutes of Biomedicine and Health (GIBH), Chinese Academy of Sciences, No. 190 Kaiyuan Avenue, Guangzhou Science Park, Guangzhou, 510530, Guangdong Province, China.
authors.forename: Siting
authors.initials: S
authors.lastname: Zhao
authors.affiliation: Laboratory of Pathogen Biology, State Key Laboratory of Respiratory Disease, Center for Infection and Immunity, Guangzhou Institutes of Biomedicine and Health (GIBH), Chinese Academy of Sciences, No. 190 Kaiyuan Avenue, Guangzhou Science Park, Guangzhou, 510530, Guangdong Province, China. qin_li@gibh.ac.cn.
authors.forename: Li
authors.initials: L
authors.lastname: Qin
authors.affiliation: Laboratory of Pathogen Biology, State Key Laboratory of Respiratory Disease, Center for Infection and Immunity, Guangzhou Institutes of Biomedicine and Health (GIBH), Chinese Academy of Sciences, No. 190 Kaiyuan Avenue, Guangzhou Science Park, Guangzhou, 510530, Guangdong Province, China. chen_xiaoping@gibh.ac.cn.
authors.forename: Xiaoping
authors.initials: X
authors.lastname: Chen
mesh_terms: D000818:Animals; D064113:CRISPR-Cas Systems / Q000235:genetics*; D018390:Gene Targeting; D005822:Genetic Vectors; D018503:Genome, Protozoan; D010957:Plasmids; D010963:Plasmodium falciparum / Q000235:genetics*
keywords: CRISPR/Cas9; Marker-free; Plasmodium falciparum