CRISPR Screen to Identify Neutrophil Regulators of Interferon-Gamma; Signaling in Acute Lung Injury

Primary Topic Area: Acute Lung Injury Secondary Topic Areas: Burn Pit Exposure, Lung Injury, Respiratory Health Areas of Encouragement: Research on the etiology and prevention of ALI caused by host immune system This proposal addresses a critical flaw in our understanding of acute lung injury (ALI) and acute respiratory distress syndrome (ARDS). Its results will have direct and immediate implications for other inflammatory lung diseases such as burn pit exposure, chronic obstructive pulmonary disease (COPD), pulmonary fibrosis, and long-term respiratory health. Acute lung injury can be caused by infections, trauma, blast injuries, and inhalation of toxic material such as burn pit fumes. Neutrophils are important white blood cells that fight infections but also contribute to tissue damage in acute lung injury. We need to understand how neutrophils work to make therapies that block lung damage while allowing cells to fight infection. Neutrophils are extremely difficult to study because they only live for about 1.5 days, which is too short for many experimental techniques that reveal what genes and proteins do. We need a way to rapidly and cheaply test the function of tens of thousands of neutrophil genes in a single experiment to see which ones play a role in acute lung injury. We propose to use a technique called CRISPR editing (clustered regularly interspersed short palindromic repeats) to make ~90,000 specific changes to DNA of mouse stem cells and transplant these cells back into irradiated mice, where they will form neutrophils. These changes will alter a single gene in each cell and cover nearly every mouse gene (~18,000). By looking at CRISPR-edited neutrophils in mice with acute lung injury from bacterial pneumonia, we will learn which genes control an important protein called interferon gamma (IFN gamma) that neutrophils make to fight infection and that can cause lung damage. This is drastically more efficient than making 18,000 mouse strains with one inactivated gene each. This proposal aims to solve a major problem in neutrophil and inflammation research: the inability to rapidly and efficiently introduce genetic changes to neutrophils. The method we propose is generally applicable and can easily be used in other injury models (such as trauma or blast injuries) and other illnesses (such as influenza or inflammatory joint diseases). Because of the rapid ongoing developments in CRISPR technology, we will also be able to pair this method with other CRISPR enzymes that activate genes instead of inactivating them, making it a generally useful method for neutrophil biology. This project will identify targets for patient-directed therapies in lung injuries and infections with broad impact across multiple disease and injury areas.