Study of the cytotoxic effect of Cas9 expression in Toxoplasma gondii. Toxoplasma gondii RH strain:RH

Streptococcus pyogenes Cas9 has been widely adopted for genome engineering in a variety of experimental systems, and many promising therapeutic applications. Understanding the character of its off-target effects is an important consideration in all such applications. Off-target activities have been mainly considered as a byproduct of guide RNA miss-targeting. However, Dr. Sebastian Lourido’s work in Toxoplasma gondii suggests that there may be toxicity intrinsic to the presence of Cas9, which can be overcome by the expression of a guide RNA (Huet and Sidik, et al. 2016 Cell). The aim of the current project is to sequence parasite genomes exposed to Cas9 in the presence or absence of the guide RNA, to understand the specificity of this Cas9 toxicity. These efforts will help us understand whether the cellular context in which Cas9 is expressed can affect its off-target activity, information that will impact the diverse implementations of this important technology.