Efficient generation of isogenic primary human myeloid cells using CRISPR-Cas9 ribonucleoproteins

Joseph Hiatt, Devin A. Cavero, Michael J. McGregor, Weihao Zheng, Jonathan M. Budzik, Theodore L. Roth, Kelsey M. Haas, David Wu, Ujjwal Rathore, Anke Meyer-Franke, Mohamed S. Bouzidi, Eric Shifrut, Youjin Lee, Vigneshwari Easwar Kumar, Eric V. Dang, David E. Gordon, Jason A. Wojcechowskyj, Judd F. Hultquist, Krystal A. Fontaine, Satish K. PillaiJeffery S. Cox, Joel D. Ernst, Nevan J. Krogan*, Alexander Marson*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

36 Scopus citations

Abstract

Genome engineering of primary human cells with CRISPR-Cas9 has revolutionized experimental and therapeutic approaches to cell biology, but human myeloid-lineage cells have remained largely genetically intractable. We present a method for the delivery of CRISPR-Cas9 ribonucleoprotein (RNP) complexes by nucleofection directly into CD14+ human monocytes purified from peripheral blood, leading to high rates of precise gene knockout. These cells can be efficiently differentiated into monocyte-derived macrophages or dendritic cells. This process yields genetically edited cells that retain transcript and protein markers of myeloid differentiation and phagocytic function. Genetic ablation of the restriction factor SAMHD1 increased HIV-1 infection >50-fold, demonstrating the power of this system for genotype-phenotype interrogation. This fast, flexible, and scalable platform can be used for genetic studies of human myeloid cells in immune signaling, inflammation, cancer immunology, host-pathogen interactions, and beyond, and could facilitate the development of myeloid cellular therapies.

Original languageEnglish (US)
Article number109105
JournalCell reports
Volume35
Issue number6
DOIs
StatePublished - May 11 2021

Funding

The authors acknowledge their partners and families for supporting us during this work. We thank all of the members of the Marson lab and the Krogan lab for helpful conversations and advice. We are grateful for discussions with B.R. Conklin, M. Ott, D.G. Russel, M. Jost, V. Ramani, G. Alberts, S. Pyle, D. Sainz, and G. Ehle. We are grateful for the generosity of our blood donors and the help of Y. Dayter and the research support team at Vitalant. R. Gummuluru and S. Stanley graciously shared the HIV-1 plasmid and the Mtb-GFP bacteria, respectively. Parts of Figure 1A were created with BioRender.com . The RNA sequencing was carried out by V. Rashbrook, S. Ashtari, and C. Goldman at the DNA Technologies and Expression Analysis Core at the University of California, Davis (UCSD) Genome Center, supported by NIH Shared Instrumentation Grant 1S10OD010786-01 . J.H. was supported by the UCSF MSTP ( T32GM007618 ). K.M.H. is supported by the National Science Foundation ( 1650113 ). Y.L. was supported by the Ruth L. Kirschstein Fellowship ( T32AI00733429 ). The Marson lab has received gifts from J. Aronov , G. Hoskin , K. Jordan , B. Bakar , and the Caufield family , and has received funds from the Gladstone Institutes , the Innovative Genomics Institute (IGI), and the Parker Institute for Cancer Immunotherapy (PICI). A.M. holds a Career Award for Medical Scientists from the Burroughs Wellcome Fund , the Lloyd J. Old STAR award from the Cancer Research Institute (CRI), and is an investigator at the Chan Zuckerberg Biohub. The Krogan Laboratory has received research support from Vir Biotechnology and F. Hoffmann-La Roche . The Marson, Krogan, and Cox labs have received funding from the BioFulcrum Viral and Infectious Disease Research Program . This work was supported by funding from the James B. Pendleton Charitable Trust and by NIH grants P50 AI150476 (A.M. and N.J.K.), U19 AI135990 (A.M., N.J.K., and J.S.C.), P01 AI063302 (N.J.K. and J.S.C), R01 AI150449 (S.K.P.), and R01 AI124471 (J.D.E). The authors acknowledge their partners and families for supporting us during this work. We thank all of the members of the Marson lab and the Krogan lab for helpful conversations and advice. We are grateful for discussions with B.R. Conklin, M. Ott, D.G. Russel, M. Jost, V. Ramani, G. Alberts, S. Pyle, D. Sainz, and G. Ehle. We are grateful for the generosity of our blood donors and the help of Y. Dayter and the research support team at Vitalant. R. Gummuluru and S. Stanley graciously shared the HIV-1 plasmid and the Mtb-GFP bacteria, respectively. Parts of Figure 1A were created with BioRender.com. The RNA sequencing was carried out by V. Rashbrook, S. Ashtari, and C. Goldman at the DNA Technologies and Expression Analysis Core at the University of California, Davis (UCSD) Genome Center, supported by NIH Shared Instrumentation Grant 1S10OD010786-01. J.H. was supported by the UCSF MSTP (T32GM007618). K.M.H. is supported by the National Science Foundation (1650113). Y.L. was supported by the Ruth L. Kirschstein Fellowship (T32AI00733429). The Marson lab has received gifts from J. Aronov, G. Hoskin, K. Jordan, B. Bakar, and the Caufield family, and has received funds from the Gladstone Institutes, the Innovative Genomics Institute (IGI), and the Parker Institute for Cancer Immunotherapy (PICI). A.M. holds a Career Award for Medical Scientists from the Burroughs Wellcome Fund, the Lloyd J. Old STAR award from the Cancer Research Institute (CRI), and is an investigator at the Chan Zuckerberg Biohub. The Krogan Laboratory has received research support from Vir Biotechnology and F. Hoffmann-La Roche. The Marson, Krogan, and Cox labs have received funding from the BioFulcrum Viral and Infectious Disease Research Program. This work was supported by funding from the James B. Pendleton Charitable Trust and by NIH grants P50 AI150476 (A.M. and N.J.K.), U19 AI135990 (A.M. N.J.K. and J.S.C.), P01 AI063302 (N.J.K. and J.S.C), R01 AI150449 (S.K.P.), and R01 AI124471 (J.D.E). Conceptualization, J.H. D.A.C. M.J.M. T.L.R. M.S.B. E.V.D. K.A.F. S.K.P. N.J.K. and A.M. Investigation, J.H. D.A.C. M.J.M. V.E.K. D.E.G. W.Z. J.M.B. K.M.H. U.R. A.M.-F. and J.A.W. Resources, A.M.-F. K.A.F. J.S.C. J.D.E. N.J.K. and A.M. Formal analysis, J.H. D.A.C. M.J.M. D.W. and E.S. Supervision, J.H. K.A.F. J.S.C. J.D.E. N.J.K. and A.M. Funding acquisition, K.A.F. J.S.C. J.D.E. N.J.K. and A.M. Writing ? original draft, J.H. D.A.C. M.J.M. and A.M. Writing ? review & editing, J.H. D.A.C. M.J.M. W.Z. T.L.R. K.M.H. Y.L. M.S.B. J.F.H. J.A.W. K.A.F. J.D.E. N.J.K. and A.M. The authors declare competing interests: T.L.R. is a co-founder of Arsenal Biosciences. A.M. is a compensated co-founder, member of the boards of directors, and a member of the scientific advisory boards of Spotlight Therapeutics and Arsenal Biosciences. A.M. was a compensated member of the scientific advisory board at PACT Pharma and was a compensated advisor to Juno Therapeutics and Trizell. A.M. owns stock in Arsenal Biosciences, Spotlight Therapeutics, and PACT Pharma. A.M. has received honoraria from Merck and Vertex, a consulting fee from AlphaSights, and is an investor in and informal advisor to Offline Ventures. The Marson lab has received research support from Juno Therapeutics, Epinomics, Sanofi, GlaxoSmithKline, Gilead, and Anthem. A.M. T.L.R. and E.S. are holders of patents pertaining to, but not resulting from, this work. The Krogan laboratory has received research support from Vir Biotechnology and F. Hoffmann-La Roche.

Keywords

  • CRISPR
  • Cas9
  • dendritic cells
  • electroporation
  • host-pathogen interactions
  • knockout
  • macrophages
  • monocytes
  • myeloid cells
  • ribonculeoproteins (RNPs)

ASJC Scopus subject areas

  • General Biochemistry, Genetics and Molecular Biology

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