# Defining epistatic interactions among Coxiella burnetii effector proteins using a CRISPRi approach

> **NIH NIH R21** · YALE UNIVERSITY · 2023 · $209,375

## Abstract

Project Summary/Abstract
Coxiella burnetii is an obligate intracellular bacterial pathogen of mammals, and the causative agent of the
zoonotic disease Q fever. Upon entry into mammalian cells, C. burnetii passively traffics through the
endolysosomal pathway before directing the establishment of a spacious, lysosome-derived organelle called
the Coxiella-containing vacuole (CCV) that allows bacterial replication. The process of CCV biogenesis and
maturation is driven by the collective activity of the roughly 130 effector proteins that are translocated by the
bacterial Dot/Icm type IVB secretion system into the host cell cytosol during infection. Despite several years of
intense research, the cellular targets, biochemical activities, and functional relationships among each other
remain unknown for most of the Dot/Icm effector proteins encoded by C. burnetii. Genetic manipulations in C.
burnetii, such as the generation of targeted gene deletion mutants, have proven to be a challenging, lengthy,
and highly inefficient undertaking. In this project we propose to develop an inducible and reversible CRISPR
interference (CRISPRi) platform to efficiently silence C. burnetii effector gene expression during intracellular
replication. In Aim 1, we will construct a comprehensive CRISPRi strain library targeting all genes that are
predicted to encode C. burnetii Dot/Icm effector proteins, individually. This targeted approach will allow us to
assess the contributions of individual effector proteins to vacuole biogenesis and intracellular replication. In
Aim 2, we will leverage the powerful advantage of CRISPRi to simultaneously reduce the expression of
multiple genes in the same strain. The goal is to begin teasing apart genetic interactions between effectors. We
will express pairwise combinations of sgRNAs targeting a set of 17 effector genes that have previously been
identified as important for CCV biogenesis and/or intracellular replication. Together, these aims will greatly
enhance the toolkit available for studying virulence genes involved in C. burnetii intracellular replication and
pathogenesis, and will start to shed light on epistatic relationships among effector proteins important for the
formation of the vacuolar niche.

## Key facts

- **NIH application ID:** 10629407
- **Project number:** 5R21AI171333-02
- **Recipient organization:** YALE UNIVERSITY
- **Principal Investigator:** Craig R. Roy
- **Activity code:** R21 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2023
- **Award amount:** $209,375
- **Award type:** 5
- **Project period:** 2022-06-01 → 2024-05-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10629407

## Citation

> US National Institutes of Health, RePORTER application 10629407, Defining epistatic interactions among Coxiella burnetii effector proteins using a CRISPRi approach (5R21AI171333-02). Retrieved via AI Analytics 2026-05-26 from https://api.ai-analytics.org/grant/nih/10629407. Licensed CC0.

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