TY - JOUR
T1 - Rapid, efficient and activation-neutral gene editing of polyclonal primary human resting CD4+ T cells allows complex functional analyses
AU - Albanese, Manuel
AU - Ruhle, Adrian
AU - Mittermaier, Jennifer
AU - Mejías-Pérez, Ernesto
AU - Gapp, Madeleine
AU - Linder, Andreas
AU - Schmacke, Niklas A.
AU - Hofmann, Katharina
AU - Hennrich, Alexandru A.
AU - Levy, David N.
AU - Humpe, Andreas
AU - Conzelmann, Karl Klaus
AU - Hornung, Veit
AU - Fackler, Oliver T.
AU - Keppler, Oliver T.
N1 - Funding Information:
This study is dedicated to the memory of Professor Valerie Bosch. We thank L. Falk and X. Sewald for sharing their expertise on in vitro digestion of RNPs and E. Akidil for helping with Miseq analysis. We are grateful to F. Pinci, L. Gregor and J. Doering for technical support and the FACS core facility (FlowCyt) of the BMC and of the BioSysM for cell sorting. This work was funded in part by the Deutsche Forschungsgemeinschaft (grant no. FA378/11-2 to O.T.F. and grant no. KE742/4-2 to O.T.K.), a grant as part of SPP-1923 (to V.H. and O.T.K.), project grant ID 452881907-TRR338 (to V.H.), project grant ID 369799452-TRR237 A12 (to K.K.C.), the Deutsche Zentrum für Infektionsforschung (project TTU 04.820 to O.T.K.), National Institutes of Health grant R01-AI145753 and NSF/NIGMS 1662096 (to D.N.L.), grants of LMUexcellent and the Friedrich-Baur-Stiftung (to M.A.) and funding by the FöFoLe program of the LMU Medical Faculty (to A.R.).
Publisher Copyright:
© 2021, The Author(s).
PY - 2022/1
Y1 - 2022/1
N2 - 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.
AB - 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.
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U2 - 10.1038/s41592-021-01328-8
DO - 10.1038/s41592-021-01328-8
M3 - Article
C2 - 34949807
AN - SCOPUS:85121593610
VL - 19
SP - 81
EP - 89
JO - Nature Methods
JF - Nature Methods
SN - 1548-7091
IS - 1
ER -