# Genome-wide CRISPR/Cas9-mediated loss-of-function screens to identify Toxoplasma genes that determine fitness in gamma interferon-stimulated human cells

> **NIH NIH R21** · UNIVERSITY OF CALIFORNIA AT DAVIS · 2020 · $235,500

## Abstract

The obligate intracellular protozoan parasite Toxoplasma gondii is a major cause of abortion in humans and
causes opportunistic infections in immunosuppressed patients such as AIDS patients. Toxoplasma co-opts the
host cell by secreting effector proteins into the host cell from specialized organelles called rhoptries (secreting
ROPs) and dense granules (secreting GRAs). The cytokine interferon gamma (IFNγ) induces a variety of
parasiticidal mechanisms in most cells of many species and is believed to be critical to control intracellular
pathogens, including Toxoplasma. In mice, IFNγ upregulates the immunity-related GTPases (IRGs), which can
destroy the vacuole Toxoplasma lives in and subsequently the parasite itself. However, humans lack IFNγ-
inducible IRGs. Indeed, the Toxoplasma secreted virulence factors ROP18 and ROP5, which cooperatively
inactivate murine IRGs, play no role in counteracting the human IFNγ response. Thus, despite Toxoplasma’s
enormous health implications, little is known about the parasite factors that determine pathogenesis in humans.
The goal of this proposal is to systematically identify Toxoplasma genes that contribute to its survival in IFNγ-
stimulated human cells. In our first aim we will perform genome-wide CRISPR/Cas9-mediated loss-of-function
screens to identify Toxoplasma genes that determine fitness in gamma interferon-stimulated human fibroblasts
and macrophages. We will generate a pool of loss-of-function mutants by transfecting a Toxoplasma strain
expressing Cas9 with a library of gRNAs targeting all Toxoplasma protein-coding genes. These mutants will be
used to infect unstimulated or IFNγ-stimulated human fibroblasts and human macrophages. We will determine
which mutants disappear in IFNγ-stimulated but not in unstimulated human cells. By repeating this screen
multiple times, we will provide the field with a list of Toxoplasma genes important for survival in IFNγ-stimulated
human cells. In our second aim we will confirm the top hits from the loss-of-function screens in Aim 1 by
generating individual knockout parasites and testing if these have a fitness defect specifically in IFNγ-
stimulated cells. Identification of novel Toxoplasma genes that determine fitness in IFNγ-stimulated human
fibroblasts and macrophages might eventually lead to the discovery of novel pathways of how human cells
detect and destroy Toxoplasma. Furthermore, if we know what Toxoplasma proteins are essential for its
survival in IFNγ-stimulated human cells we might be able to use this information to develop new therapies
against Toxoplasma.

## Key facts

- **NIH application ID:** 9872927
- **Project number:** 1R21AI149071-01
- **Recipient organization:** UNIVERSITY OF CALIFORNIA AT DAVIS
- **Principal Investigator:** JEROEN SAEIJ
- **Activity code:** R21 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $235,500
- **Award type:** 1
- **Project period:** 2020-02-18 → 2022-01-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9872927

## Citation

> US National Institutes of Health, RePORTER application 9872927, Genome-wide CRISPR/Cas9-mediated loss-of-function screens to identify Toxoplasma genes that determine fitness in gamma interferon-stimulated human cells (1R21AI149071-01). Retrieved via AI Analytics 2026-05-22 from https://api.ai-analytics.org/grant/nih/9872927. Licensed CC0.

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