Project Summary Tuberculosis (TB), caused by the intracellular bacterial pathogen Mycobacterium tuberculosis (M.tb), remains one of the most deadly infectious diseases in the world. According to the WHO annual report, one third of the population has been infected by M.tb across the world, leading to 10 million active TB cases and 1.4 million deaths in 2019. About 5-10% of infected people develop active TB disease during their lifetime. The mechanism of M.tb-host interactions remains to be defined. Host long noncoding RNAs (lncRNAs) are noncoding RNAs with greater than 200 nucleotides. Our preliminary data and previous publications demonstrated the engagement of host lncRNAs in host defense against mycobacterial infections in macrophages. However, there are limited studies addressing the functions of host lncRNAs in the context of M.tb infection. The long-term goal of this proposed study is to identify host lncRNAs that are beneficial to host defense against M.tb infection in macrophages, and further understand the mechanism how host lncRNAs serve as cis- or trans-regulatory elements in mediating the host immunity in macrophages in response to M.tb infection. In the current proposal, we hypothesize that lncRNAs are essential components of antimycobacterial response within M.tb-infected macrophages. To test the hypothesis, we propose a genome-wide screening for host lncRNAs that regulate host defense against mycobacterial infection in human macrophages using a CRISPR-Cas9 transcription activation system that targets over 10,000 host lncRNAs. Aim 1 will screen host intergenic lncRNAs that improve the survival of THP-1 cells during M.tb infection. Aim 2 will validate candidate host lncRNAs in genetically engineered THP-1 cells during the course of M.tb infection. Nucleic acid- based therapy is becoming an emerging new class of therapeutics for treating unmet medical conditions. Our study will shed light on the development of nucleic acid as a novel host-directed therapy for the treatment of TB, especially drug-resistant TB.