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Efficient gene knockout in primary human and murine myeloid cells by non-viral delivery of CRISPR-Cas9

Myeloid cells play critical and diverse roles in mammalian physiology, including tissue development and repair, innate defense against pathogens, and generation of adaptive immunity. As cells that show prolonged recruitment to sites of injury or pathology, myeloid cells represent therapeutic targets...

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Autores principales: Freund, Emily C., Lock, Jaclyn Y., Oh, Jaehak, Maculins, Timurs, Delamarre, Lelia, Bohlen, Christopher J., Haley, Benjamin, Murthy, Aditya
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Rockefeller University Press 2020
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7336301/
https://www.ncbi.nlm.nih.gov/pubmed/32357367
http://dx.doi.org/10.1084/jem.20191692
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author Freund, Emily C.
Lock, Jaclyn Y.
Oh, Jaehak
Maculins, Timurs
Delamarre, Lelia
Bohlen, Christopher J.
Haley, Benjamin
Murthy, Aditya
author_facet Freund, Emily C.
Lock, Jaclyn Y.
Oh, Jaehak
Maculins, Timurs
Delamarre, Lelia
Bohlen, Christopher J.
Haley, Benjamin
Murthy, Aditya
author_sort Freund, Emily C.
collection PubMed
description Myeloid cells play critical and diverse roles in mammalian physiology, including tissue development and repair, innate defense against pathogens, and generation of adaptive immunity. As cells that show prolonged recruitment to sites of injury or pathology, myeloid cells represent therapeutic targets for a broad range of diseases. However, few approaches have been developed for gene editing of these cell types, likely owing to their sensitivity to foreign genetic material or virus-based manipulation. Here we describe optimized strategies for gene disruption in primary myeloid cells of human and murine origin. Using nucleofection-based delivery of Cas9-ribonuclear proteins (RNPs), we achieved near population-level genetic knockout of single and multiple targets in a range of cell types without selection or enrichment. Importantly, we show that cellular fitness and response to immunological stimuli is not significantly impacted by the gene editing process. This provides a significant advance in the study of myeloid cell biology, thus enabling pathway discovery and drug target validation across species in the field of innate immunity.
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spelling pubmed-73363012020-07-16 Efficient gene knockout in primary human and murine myeloid cells by non-viral delivery of CRISPR-Cas9 Freund, Emily C. Lock, Jaclyn Y. Oh, Jaehak Maculins, Timurs Delamarre, Lelia Bohlen, Christopher J. Haley, Benjamin Murthy, Aditya J Exp Med Technical Advances and Resources Myeloid cells play critical and diverse roles in mammalian physiology, including tissue development and repair, innate defense against pathogens, and generation of adaptive immunity. As cells that show prolonged recruitment to sites of injury or pathology, myeloid cells represent therapeutic targets for a broad range of diseases. However, few approaches have been developed for gene editing of these cell types, likely owing to their sensitivity to foreign genetic material or virus-based manipulation. Here we describe optimized strategies for gene disruption in primary myeloid cells of human and murine origin. Using nucleofection-based delivery of Cas9-ribonuclear proteins (RNPs), we achieved near population-level genetic knockout of single and multiple targets in a range of cell types without selection or enrichment. Importantly, we show that cellular fitness and response to immunological stimuli is not significantly impacted by the gene editing process. This provides a significant advance in the study of myeloid cell biology, thus enabling pathway discovery and drug target validation across species in the field of innate immunity. Rockefeller University Press 2020-05-01 /pmc/articles/PMC7336301/ /pubmed/32357367 http://dx.doi.org/10.1084/jem.20191692 Text en © 2020 Freund et al. https://creativecommons.org/licenses/by/4.0/This article is available under a Creative Commons License (Attribution 4.0 International, as described at https://creativecommons.org/licenses/by/4.0/).
spellingShingle Technical Advances and Resources
Freund, Emily C.
Lock, Jaclyn Y.
Oh, Jaehak
Maculins, Timurs
Delamarre, Lelia
Bohlen, Christopher J.
Haley, Benjamin
Murthy, Aditya
Efficient gene knockout in primary human and murine myeloid cells by non-viral delivery of CRISPR-Cas9
title Efficient gene knockout in primary human and murine myeloid cells by non-viral delivery of CRISPR-Cas9
title_full Efficient gene knockout in primary human and murine myeloid cells by non-viral delivery of CRISPR-Cas9
title_fullStr Efficient gene knockout in primary human and murine myeloid cells by non-viral delivery of CRISPR-Cas9
title_full_unstemmed Efficient gene knockout in primary human and murine myeloid cells by non-viral delivery of CRISPR-Cas9
title_short Efficient gene knockout in primary human and murine myeloid cells by non-viral delivery of CRISPR-Cas9
title_sort efficient gene knockout in primary human and murine myeloid cells by non-viral delivery of crispr-cas9
topic Technical Advances and Resources
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7336301/
https://www.ncbi.nlm.nih.gov/pubmed/32357367
http://dx.doi.org/10.1084/jem.20191692
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