Abstract The goal of this proposed research is to optimize the CRISPR-Cas9 delivery system for efficient in vivo gene editing of T cells. CRISPR-Cas9 has emerged as a powerful tool for genome engineering in diverse organisms, as well as for developing therapeutics for genetic and infectious diseases. T cells are known to play critical role in orchestrating cell-mediated immunity and humoral immunity; thus, T cell genome engineering offers promise to treat HIV infection, cancer, as well as autoimmune diseases. However, in vivo targeting delivery of the CRISPR-Cas9 machinery to T cells to efficiently modify their genome remains a major difficulty. CD7 is a pan-T cell molecule that is specifically expressed on T cells. This receptor is rapidly internalized after antibody (Ab) binding, and it has been used for Ab-mediated in vivo delivery of toxins to treat T cell lymphomas and leukemias in preclinical studies and clinical trials. We hypothesize that anti-human CD7 monoclonal antibody (?-hCD7 mAb)-mediated delivery of Cas9 protein to T cells via Cas9 ribonucleoprotein (RNP) and Cas9 protein prepackaged lentivirus-like particles (Cas9P LV) will allow efficient gene editing in vivo. In preliminary studies, we found that protein A ZZ domain-fused fluorescence protein, once conjugated with ?-hCD7 mAb, could be effectively delivered into CD4+ T cells; we also found that ?-hCD7 mAb-conjugated Cas9-ZZ fusion protein could be efficiently delivered and internalized into CD4+ T cells. Additionally, to overcome the potential immunogenicity problem by direct delivery of Cas9 protein, we developed a novel lentiviral (LV) particle-based Cas9 protein delivery strategy to shield Cas9 protein in the LV particles. Once this Cas9P LV was pseudotyped with a Sindbis/ZZ domain envelope, it could be conjugated to ?-hCD7 mAb via Fc portion of the mAb and delivered into CD4+ T cells. Finally, we found that once a CCR5 single guide RNA (sgRNA) had been co-packaged into a Cas9P LV linked to an ?-hCD7 mAb, we could efficiently perform gene editing in T cells. These proof-of-concept results demonstrate the potential of ?-hCD7 mAb-mediated delivery of CRISPR-Cas9 into T cells for in vivo gene editing. Therefore, in this proposed research, we will use ?-hCD7 mAb-mediated Cas9 RNP and Cas9P LV to deliver the CRISPR-Cas9 machinery to target the CCR5 and PD-1 genes. These genes have been shown to be important in HIV infection and cancer. We will test and compare the in vivo gene editing efficiency using ?-hCD7 mAb-mediated Cas9 RNP and Cas9P LV delivery to target the CCR5 and PD-1 genes. We also seek to validate functional changes after efficient disruption of these genes in humanized mouse models. Finally, we will validate the disruption of rhesus monkey (RM) ccr5 caused by in vivo gene editing and functional changes in a RM model of simian-human immunodeficiency virus (SHIV) infection. Our research has been designed to open a new avenue toward efficient in vivo gene editing of T cells for clinical applications.