Toxoplasma gondii has become a model for studying the phylum Apicomplexa, in 
part due to the availability of excellent genetic tools. Although reverse 
genetic tools are available in a few widely utilized laboratory strains, they 
rely on special genetic backgrounds that are not easily implemented in natural 
isolates. Recent progress in modifying CRISPR (clustered regularly interspaced 
short palindromic repeats), a system of DNA recognition used as a defense 
mechanism in bacteria and archaea, has led to extremely efficient gene 
disruption in a variety of organisms. Here we utilized a CRISPR/CAS9-based 
system with single guide RNAs to disrupt genes in T. gondii. CRISPR/CAS9 
provided an extremely efficient system for targeted gene disruption and for 
site-specific insertion of selectable markers through homologous recombination. 
CRISPR/CAS9 also facilitated site-specific insertion in the absence of homology, 
thus increasing the utility of this approach over existing technology. We then 
tested whether CRISPR/CAS9 would enable efficient transformation of a natural 
isolate. Using CRISPR/CAS9, we were able to rapidly generate both rop18 
knockouts and complemented lines in the type I GT1 strain, which has been used 
for forward genetic crosses but which remains refractory to reverse genetic 
approaches. Assessment of their phenotypes in vivo revealed that ROP18 
contributed a greater proportion to acute pathogenesis in GT1 than in the 
laboratory type I RH strain. Thus, CRISPR/CAS9 extends reverse genetic 
techniques to diverse isolates of T. gondii, allowing exploration of a much 
wider spectrum of biological diversity.
IMPORTANCE: Genetic approaches have proven very powerful for studying the 
biology of organisms, including microbes. However, ease of genetic manipulation 
varies widely among isolates, with common lab isolates often being the most 
amenable to such approaches. Unfortunately, such common lab isolates have also 
been passaged frequently in vitro and have thus lost many of the attributes of 
wild isolates, often affecting important traits, like virulence. On the other 
hand, wild isolates are often not amenable to standard genetic approaches, thus 
limiting inquiry about the genetic basis of biological diversity. Here we 
imported a new genetic system based on CRISPR/CAS9, which allows high efficiency 
of targeted gene disruption in natural isolates of T. gondii. This advance 
promises to bring the power of genetics to bear on the broad diversity of 
T. gondii strains that have been described recently.