A Cas9 Ribonucleoprotein Platform for Functional Genetic Studies of HIV-Host Interactions in Primary Human T Cells.

Hultquist JF, Schumann K, Woo JM, Manganaro L, McGregor MJ, Doudna J, Simon V, Krogan NJ, Marson A.
Source: Cell Rep
Publication Date: (2016)
Issue: 17(5): 1438-1452
Research Area:
Immunotherapy / Hematology
Gene Expression
Cells used in publication:
T cell, human stim.
Species: human
Tissue Origin: blood
CD4+, human
Species: human
Tissue Origin: blood
Culture Media:
4D-Nucleofector® 96-well Systems
4D-Nucleofector® X-Unit

primary isolated CD4+ T cells (by ficoll separation and negative Easy sep enrichment kit) were stimulated with CD3/CD28 for 48h in the presence of IL-2. Used Cas9 protein with two NLS signals to transfect in solution P3 and program EH-115 Details: Cas9 RNPs were prepared fresh for each experiment. crRNA and tracrRNA were first mixed 1:1 and incubated 30 min at 37°C to generate 40 µM crRNA: tracrRNA duplexes. An equal volume of 40 µM Cas9-NLS was slowly added to the crRNA:tracrRNA and incubated for 15 min at 37°C to generate 20 µM Cas9 RNPs. For each reaction, around 300,000 stimulated T cells were pelleted and resuspended in 20 µL P3 buffer (Lonza). 3 µL of 20 µM Cas9 RNP mix was added directly to these cells and the entire volume transferred to the 96-well reaction cuvette. For double-editing reactions, 3 µL of each Cas9 RNP was added to 20 µL cells. Cells were electroporated using program EH-115 on the Amaxa 4D-Nucleofector (Lonza). 80µL pre-warmed, complete RPMI was added to each well, and the cells were allowed to recover for 30 min at 37°C. Cells were then re-stimulated on plates coated overnight with 10 µg/mL anti-CD3 (UCHT1, Tonbo Biosciences) and 10 µg/mL anti-CD28 (CD28.2, Tonbo Biosciences) for 24 hr prior to infection.


New genetic tools are needed to understand the functional interactions between HIV and human host factors in primary cells. We recently developed a method to edit the genome of primary CD4+ T cells by electroporation of CRISPR/Cas9 ribonucleoproteins (RNPs). Here, we adapted this methodology to a high-throughput platform for the efficient, arrayed editing of candidate host factors. CXCR4 or CCR5 knockout cells generated with this method are resistant to HIV infection in a tropism-dependent manner, whereas knockout of LEDGF or TNPO3 results in a tropism-independent reduction in infection. CRISPR/Cas9 RNPs can furthermore edit multiple genes simultaneously, enabling studies of interactions among multiple host and viral factors. Finally, in an arrayed screen of 45 genes associated with HIV integrase, we identified several candidate dependency/restriction factors, demonstrating the power of this approach as a discovery platform. This technology should accelerate target validation for pharmaceutical and cell-based therapies to cure HIV infection.