PROJECT SUMMARY/ABSTRACT: Since its emergence, the Human Immunodeficiency Virus (HIV) has infected 84 million people worldwide and led to the deaths of 40 million people1. Despite the development of over 20 different treatment options, there is still no cure available. As a result, many individuals infected with HIV experience long-term side effects from antiretroviral therapy and have more comorbidities earlier in their lives than those who are not infected2,3. Therefore, there is a need to further research alternative ways to reduce or prevent HIV infection. The two coreceptors for HIV infection, CCR5 and CXCR4, which play an essential role in HIV infecting CD4 cells, are promising opportunities to limit HIV. While elimination of CCR5 in hematopoietic cells is being pursued, it is clear that CCR5-null hematopoietic cells can still be infected by CXCR4-tropic forms of HIV. Therefore, I seek to develop HIV resistant hematopoietic stem cells by targeting both of the CCR5 and CXCR4 co-receptors with base editing to prevent HIV entry while not impairing central functions of these proteins in hematopoiesis. My first AIM will use a base editor screen to identify CCR5 mutants that knock out CCR5 expression but also decrease the cell surface expression of CXCR4, which has been seen with other CCR5 mutants. The results of the screen will be analyzed with fluorescence-activated cell sorting (FACS) and single cell DNA sequencing. My second AIM will also use a base editor screen to identify CXCR4 mutants that prevent the binding of the HIV surface proteins, but allow for the binding of the natural ligand. These CXCR4 mutants will be tested with HIV pseudoviruses to identify mutations that prevent viral infection of the cells and a transwell migration assay to identify mutants that maintain CXCR4 responsiveness. Single cell DNA sequencing will be run on the cells that pass both of these tests to identify mutation candidates that meet the goals of this AIM. The Sankaran lab has extensive expertise in base editor screens and hematopoiesis. To complement this skill set I have contacted other faculty in the area, such as Dr. Alajandro Balazs, to assist with any HIV related questions I have and assist with the production of HIV pseudoviruses to test the hits from my base editor screens. My training plan focuses on gaining further expertise in hematopoiesis, HIV biology, DNA sequencing, processing of sequencing data, written and oral scientific communication, and scientific mentoring. With access to the Broad Institute of MIT and Harvard, Boston Children’s Hospital, and Harvard University I have all the tools, resources, and access to expertise necessary to excel in my research.