Rapid, efficient and activation-neutral gene editing of polyclonal primary human resting CD4 + T cells allows complex functional analyses

Manuel Albanese, Adrian Ruhle, Jennifer Mittermaier, Ernesto Mejías-Pérez, Madeleine Gapp, Andreas Linder, Niklas A Schmacke, Katharina Hofmann, Alexandru A Hennrich, David N Levy, Andreas Humpe, Karl-Klaus Conzelmann, Veit Hornung, Oliver T Fackler, Oliver T Keppler
Source: Nat Methods
Publication Date: (2021)
Issue: 1: 1-27
Research Area:
Immunotherapy / Hematology
Gene Expression
Cells used in publication:
T cell, human peripheral blood unstim.
Species: human
Tissue Origin: blood
CD4+, human
Species: human
Tissue Origin: blood
4D-Nucleofector® X-Unit

Knockout generation in resting human CD4+ T cells. Freshly isolated CD4+ T cells (2 × 106) were washed twice with PBS and resuspended in 20 µl buffer P3 (Lonza; V4XP-3032). In parallel, synthetic sgRNAs (Synthego) were incubated together with recombinant NLS-Cas9 (IDT; 1081059) for 10 min at room temperature, at a ratio of 1:2.5 (40 pmol Cas9 protein per 100 pmol gRNA) to form the CRISPR–CAS9–gRNA RNP complex. The CAS9–gRNA mix was diluted with sterile filtered (0.22 µm) PBS to reach a final concentration of 20 µM RNPs. For single gRNA editing, 5 µl of the 20 µM RNPs were then mixed with the cell suspension and transferred into a 16-well reaction cuvette of the 4D-Nucleofector System (Lonza). For efficient KO of individual targets, a mix of two specific, pre-validated gRNAs was used, if not indicated differently. Here, only 2 µl of RNP complexes for each gRNAs were used. For co-editing of up to six genes, only 0.5 µl of each RNP complex were used. Cells were nucleofected using program EH-100 on the 4D-Nucleofector system12. Then, 100 µl of pre-warmed RPMI (without supplements) was added to each well and cells were transferred to 48-well plates and allowed to recover for 10 min at 37 °C. Subsequently, complete culture medium
supplemented with IL-7 (Peprotech; 200-07) and IL-15 (Peprotech; 200-15) (2 ng ml-1 each) was added.

Knock in of resting CD4+ T cells. For KIs, the same nucleofection conditions as for the KO generation were used (P3 buffer and program EH-100; Lonza). In addition to the RNP, the donor DNA template was added to the P3 cell suspension at 1 µg, unless stated otherwise. Additional information on the overall strategies to generate KIs into different loci in resting CD4+ T cells, including DNA templates, gRNAs and primers is included in Extended Data Table 1.


CD4+ T cells are central mediators of adaptive and innate immune responses and constitute a major reservoir for human immunodeficiency virus (HIV) in vivo. Detailed investigations of resting human CD4+ T cells have been precluded by the absence of efficient approaches for genetic manipulation limiting our understanding of HIV replication and restricting efforts to find a cure. Here we report a method for rapid, efficient, activation-neutral gene editing of resting, polyclonal human CD4+ T cells using optimized cell cultivation and nucleofection conditions of Cas9-guide RNA ribonucleoprotein complexes. Up to six genes, including HIV dependency and restriction factors, were knocked out individually or simultaneously and functionally characterized. Moreover, we demonstrate the knock in of double-stranded DNA donor templates into different endogenous loci, enabling the study of the physiological interplay of cellular and viral components at single-cell resolution. Together, this technique allows improved molecular and functional characterizations of HIV biology and general immune functions in resting CD4+ T cells.