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. Pillai; Jeffery S. Cox; Joel D. Ernst; Nevan J. Krogan; Alexander Marson

doi:10.1016/j.celrep.2021.109105

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 journal › Article › peer-review

30 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 language	English
Article number	109105
Journal	Cell Reports
Volume	35
Issue number	6
DOIs	https://doi.org/10.1016/j.celrep.2021.109105
State	Published - 11 May 2021
Externally published	Yes

Funding

Funders	Funder number
Arsenal Biosciences
UCSF MSTP	T32GM007618
Vir Biotechnology
National Science Foundation	T32AI00733429, 1650113
National Institutes of Health	1S10OD010786-01
National Institute of Allergy and Infectious Diseases	P01AI063302
Burroughs Wellcome Fund
Cancer Research Institute
GlaxoSmithKline
Gilead Sciences
F. Hoffmann-La Roche
University of California, Davis
University of California, San Diego
Gladstone Institutes
James B. Pendleton Charitable Trust	P01 AI063302, P50 AI150476, R01 AI150449, U19 AI135990, R01 AI124471
Parker Institute for Cancer Immunotherapy

Keywords

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

UN SDGs

This output contributes to the following UN Sustainable Development Goals (SDGs)

Access to Document

10.1016/j.celrep.2021.109105

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Hiatt, J., Cavero, D. A., McGregor, M. J., Zheng, W., Budzik, J. M., Roth, T. L., Haas, K. M., Wu, D., Rathore, U., Meyer-Franke, A., Bouzidi, M. S., Shifrut, E., Lee, Y., Kumar, V. E., Dang, E. V., Gordon, D. E., Wojcechowskyj, J. A., Hultquist, J. F., Fontaine, K. A., ... Marson, A. (2021). Efficient generation of isogenic primary human myeloid cells using CRISPR-Cas9 ribonucleoproteins. Cell Reports, 35(6), Article 109105. https://doi.org/10.1016/j.celrep.2021.109105

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title = "Efficient generation of isogenic primary human myeloid cells using CRISPR-Cas9 ribonucleoproteins",

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.",

keywords = "CRISPR, Cas9, dendritic cells, electroporation, host-pathogen interactions, knockout, macrophages, monocytes, myeloid cells, ribonculeoproteins (RNPs)",

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

note = "Publisher Copyright: {\textcopyright} 2021 The Authors",

year = "2021",

month = may,

day = "11",

doi = "10.1016/j.celrep.2021.109105",

language = "אנגלית",

volume = "35",

journal = "Cell Reports",

issn = "2211-1247",

publisher = "Cell Press",

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Hiatt, J, Cavero, DA, McGregor, MJ, Zheng, W, Budzik, JM, Roth, TL, Haas, KM, Wu, D, Rathore, U, Meyer-Franke, A, Bouzidi, MS, Shifrut, E, Lee, Y, Kumar, VE, Dang, EV, Gordon, DE, Wojcechowskyj, JA, Hultquist, JF, Fontaine, KA, Pillai, SK, Cox, JS, Ernst, JD, Krogan, NJ & Marson, A 2021, 'Efficient generation of isogenic primary human myeloid cells using CRISPR-Cas9 ribonucleoproteins', Cell Reports, vol. 35, no. 6, 109105. https://doi.org/10.1016/j.celrep.2021.109105

TY - JOUR

T1 - Efficient generation of isogenic primary human myeloid cells using CRISPR-Cas9 ribonucleoproteins

AU - Hiatt, Joseph

AU - Cavero, Devin A.

AU - McGregor, Michael J.

AU - Zheng, Weihao

AU - Budzik, Jonathan M.

AU - Roth, Theodore L.

AU - Haas, Kelsey M.

AU - Wu, David

AU - Rathore, Ujjwal

AU - Meyer-Franke, Anke

AU - Bouzidi, Mohamed S.

AU - Shifrut, Eric

AU - Lee, Youjin

AU - Kumar, Vigneshwari Easwar

AU - Dang, Eric V.

AU - Gordon, David E.

AU - Wojcechowskyj, Jason A.

AU - Hultquist, Judd F.

AU - Fontaine, Krystal A.

AU - Pillai, Satish K.

AU - Cox, Jeffery S.

AU - Ernst, Joel D.

AU - Krogan, Nevan J.

AU - Marson, Alexander

PY - 2021/5/11

Y1 - 2021/5/11

N2 - 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.

AB - 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.

KW - CRISPR

KW - Cas9

KW - dendritic cells

KW - electroporation

KW - host-pathogen interactions

KW - knockout

KW - macrophages

KW - monocytes

KW - myeloid cells

KW - ribonculeoproteins (RNPs)

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U2 - 10.1016/j.celrep.2021.109105

DO - 10.1016/j.celrep.2021.109105

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C2 - 33979618

AN - SCOPUS:85105548119

SN - 2211-1247

VL - 35

JO - Cell Reports

JF - Cell Reports

IS - 6

M1 - 109105

ER -

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

Abstract

Funding

Keywords

UN SDGs

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