AUTHOR=Chen Yun , Liu Qi , Xue Jun-Xin , Zhang Man-Yu , Geng Xiao-Ling , Wang Quan , Jiang Wei 

TITLE=Genome-Wide CRISPR/Cas9 Screen Identifies New Genes Critical for Defense Against Oxidant Stress in Toxoplasma gondii

JOURNAL=Frontiers in Microbiology

VOLUME=12

YEAR=2021

URL=https://www.frontiersin.org/journals/microbiology/articles/10.3389/fmicb.2021.670705

DOI=10.3389/fmicb.2021.670705

ISSN=1664-302X

ABSTRACT=<p><italic>Toxoplasma gondii</italic> is one of the most widespread apicomplexans and can cause serious infections in humans and animals. Its antioxidant system plays an important role in defending against oxidant stress imposed by the host. Some genes encoding the antioxidant enzymes of <italic>T. gondii</italic> have been identified; however, critical genes that function in response to reactive oxygen species (ROS) stress are still poorly understood. Here, we performed genome-wide CRISPR/Cas9 loss-of-function screening in the <italic>T. gondii</italic> RH strain to identify potential genes contributing to the ROS stress response. Under hydrogen peroxide treatment, 30 single guide RNAs targeting high-confidence genes were identified, including some known important antioxidant genes such as catalase and peroxiredoxin PRX3. In addition, several previously uncharacterized genes were identified, among which five hypothetical protein-coding genes, namely, <italic>HP1</italic>–<italic>HP5</italic>, were selected for further functional characterization. Targeted deletion of <italic>HP1</italic> in <italic>T. gondii</italic> RH led to significant sensitivity to H<sub>2</sub>O<sub>2</sub>, suggesting that <italic>HP1</italic> is critical for oxidative stress management. Furthermore, loss of <italic>HP1</italic> led to decreased antioxidant capacity, invasion efficiency, and proliferation <italic>in vitro</italic>. <italic>In vivo</italic> results also revealed that the survival time of mice infected with the <italic>HP1</italic>-KO strain was significantly prolonged relative to that of mice infected with the wild-type strain. Altogether, these findings demonstrate that the CRISPR/Cas9 system can be used to identify potential genes critical for oxidative stress management. Furthermore, <italic>HP1</italic> may confer protection against oxidative damage and contributes to <italic>T. gondii</italic> virulence in mice.</p>