title: Development of CRISPR/Cas9 for Efficient Genome Editing in Toxoplasma gondii.
abstract: Efficient and site-specific alteration of the genome is key to decoding and altering the genomic information of an organism. Over the last couple of years, the RNA-guided Cas9 nucleases derived from the prokaryotic type 2 CRISPR (clustered regularly interspaced short palindromic repeats) systems have drastically improved our ability to engineer the genomes of a variety of organisms including Toxoplasma gondii. In this chapter, we describe detailed protocols for using the CRISPR/Cas9 system adapted from Streptococcus pyogenes to perform efficient genetic manipulations in T. gondii such as gene disruption, gene tagging and genetic complementation. The technical details of the strategy, including CRISPR plasmid construction, target construct generation, parasite transfection and positive clone identification are also provided. These methods are easy to customize to any gene of interest (GOI) and will greatly accelerate studies on this important pathogen.
authors.affiliation: State Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan, Hubei, 430070, China.
authors.forename: Bang
authors.initials: B
authors.lastname: Shen
authors.affiliation: Department of Molecular Microbiology, Washington University School of Medicine, 660 S. Euclid Ave, St. Louis, MO, 63110, USA.
authors.forename: Kevin
authors.initials: K
authors.lastname: Brown
authors.affiliation: Department of Molecular Microbiology, Washington University School of Medicine, 660 S. Euclid Ave, St. Louis, MO, 63110, USA.
authors.forename: Shaojun
authors.initials: S
authors.lastname: Long
authors.affiliation: Department of Molecular Microbiology, Washington University School of Medicine, 660 S. Euclid Ave, St. Louis, MO, 63110, USA. sibley@wustl.edu.
authors.forename: L David
authors.initials: LD
authors.lastname: Sibley
mesh_terms: D064130:CRISPR-Associated Proteins / Q000235:genetics; D064113:CRISPR-Cas Systems / Q000235:genetics*; D064112:Clustered Regularly Interspaced Short Palindromic Repeats / Q000235:genetics*; D004720:Endonucleases / Q000235:genetics; D000072669:Gene Editing / Q000379:methods; D018390:Gene Targeting / Q000379:methods; D018503:Genome, Protozoan / Q000235:genetics*; D010957:Plasmids / Q000235:genetics; D000094704:RNA, Guide, CRISPR-Cas Systems / Q000235:genetics; D014122:Toxoplasma / Q000235:genetics*; D014162:Transfection / Q000379:methods
chemical_list: D064130:CRISPR-Associated Proteins; D000094704:RNA, Guide, CRISPR-Cas Systems; D004720:Endonucleases
keywords: CRISPR/Cas9; Gene editing; Genetic transformation; Protozoan parasites; Selectable markers