Efficient genome engineering of Toxoplasma gondii using CRISPR/Cas9

PLoS One. 2014 Jun 27;9(6):e100450. doi: 10.1371/journal.pone.0100450. eCollection 2014.


Toxoplasma gondii is a parasite of humans and animals, and a model for other apicomplexans including Plasmodium spp., the causative agents of malaria. Despite many advances, manipulating the T. gondii genome remains labor intensive, and is often restricted to lab-adapted strains or lines carrying mutations that enable selection. Here, we use the RNA-guided Cas9 nuclease to efficiently generate knockouts without selection, and to introduce point mutations and epitope tags into the T. gondii genome. These methods will streamline the functional analysis of parasite genes and enable high-throughput engineering of their genomes.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Antigens, Protozoan / chemistry
  • Antigens, Protozoan / genetics
  • Base Sequence
  • CRISPR-Associated Proteins*
  • CRISPR-Cas Systems
  • Cell Line
  • Clustered Regularly Interspaced Short Palindromic Repeats*
  • DNA End-Joining Repair
  • Gene Order
  • Gene Targeting / methods
  • Genetic Engineering*
  • Genetic Loci
  • Genome, Protozoan
  • Humans
  • Molecular Sequence Data
  • Plasmids / genetics
  • Protozoan Proteins / chemistry
  • Protozoan Proteins / genetics
  • Sequence Alignment
  • Toxoplasma / genetics*


  • Antigens, Protozoan
  • CRISPR-Associated Proteins
  • Protozoan Proteins
  • SAG1 antigen, Toxoplasma