Cargando…
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...
Autores principales: | , , , , , , , |
---|---|
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 |
_version_ | 1783554291449987072 |
---|---|
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. |
format | Online Article Text |
id | pubmed-7336301 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2020 |
publisher | Rockefeller University Press |
record_format | MEDLINE/PubMed |
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 |
work_keys_str_mv | AT freundemilyc efficientgeneknockoutinprimaryhumanandmurinemyeloidcellsbynonviraldeliveryofcrisprcas9 AT lockjaclyny efficientgeneknockoutinprimaryhumanandmurinemyeloidcellsbynonviraldeliveryofcrisprcas9 AT ohjaehak efficientgeneknockoutinprimaryhumanandmurinemyeloidcellsbynonviraldeliveryofcrisprcas9 AT maculinstimurs efficientgeneknockoutinprimaryhumanandmurinemyeloidcellsbynonviraldeliveryofcrisprcas9 AT delamarrelelia efficientgeneknockoutinprimaryhumanandmurinemyeloidcellsbynonviraldeliveryofcrisprcas9 AT bohlenchristopherj efficientgeneknockoutinprimaryhumanandmurinemyeloidcellsbynonviraldeliveryofcrisprcas9 AT haleybenjamin efficientgeneknockoutinprimaryhumanandmurinemyeloidcellsbynonviraldeliveryofcrisprcas9 AT murthyaditya efficientgeneknockoutinprimaryhumanandmurinemyeloidcellsbynonviraldeliveryofcrisprcas9 |