A CRISPR-Cas9 Genome Engineering Platform in Primary CD4+ T Cells for the Interrogation of HIV Host Factors
CRISPR-Cas9 gene editing strategies have revolutionized our ability to engineer the human genome for robust functional interrogation of complex biological processes. We have recently adapted this technology to primary human T cells to generate a high-throughput platform for analyzing the role of host factors in pathogen infection and lifecycle. Here, we describe applications of this system to investigate HIV pathogenesis in CD4+ T cells. Briefly, CRISPR-Cas9 ribonucleoproteins (crRNPs) are synthesized in vitro and delivered to activated primary human CD4+ T cells by nucleofection. These edited cells are then validated and expanded for use in downstream cellular, genetic, or protein-based assays. Our platform supports the arrayed generation of several gene manipulations in only a few hours time and is widely adaptable across culture conditions, infection protocols, and downstream applications. We present detailed protocols for crRNP synthesis, primary T cell culture, 96-well nucleofection, molecular validation, and HIV infection with additional considerations for guide and screen design as well as crRNP multiplexing.