Toxoplasma CRISPR/Cas9 constructs are functional for gene disruption in Neospora caninum

• Published in: International journal for parasitology Vol. 48; no. 8; pp. 597 - 600
• Main Authors: Arranz-Solís, David, Regidor-Cerrillo, Javier, Lourido, Sebastian, Ortega-Mora, Luis Miguel, Saeij, Jeroen P.J.
• Format: Journal Article
• Language: English
• Published: England Elsevier Ltd 01.07.2018
• Subjects: CRISPR-Cas Systems; CRISPR/Cas9; DNA, Helminth - genetics; DNA, Protozoan - genetics; Gene disruption; Gene Editing - methods; Nc-Spain7; NcGRA7; Neospora - genetics; Neospora caninum; Plasmids; Toxoplasma - genetics; Transfection; NcGRA7; Transfection; CRISPR/Cas9; Gene disruption; Nc-Spain7; Neospora caninum
• ISSN: 0020-7519; 1879-0135
• DOI: 10.1016/j.ijpara.2018.03.002

[Display omitted] •Effective gene disruption can be achieved in Neospora by using CRISPR/Cas9.•Homology arms are not required to introduce DNA templates at the site of cut.•Toxoplasma Cas9 plasmids can be directly used in Neospora caninum. Herein we describe, to our knowledge for the first time the use of the clustered regularly interspaced short palindromic repeats/CRISPR-associated gene 9 (CRISPR/Cas9) system for genome editing of Neospora caninum, an apicomplexan parasite considered one of the main causes of abortion in cattle worldwide. By using plasmids containing the CRISPR/Cas9 components adapted to the closely related parasite Toxoplasma gondii, we successfully knocked out a green fluorescent protein (GFP) in an Nc-1 GFP-expressing strain, and efficiently disrupted the NcGRA7 gene in the Nc-Spain7 isolate by insertion of a pyrimethamine resistance cassette. The successful use of this technology in N. caninum lays the foundation for an efficient, targeted gene modification tool in this parasite.