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Efficient and stable CRISPR/Cas9-mediated genome-editing of human type 2 innate lymphoid cells

Innate lymphoid cells (ILCs) are a family of innate lymphocytes with important roles in immune response coordination and maintenance of tissue homeostasis. The ILC family includes group 1 (ILC1s), group 2 (ILC2s) and group 3 (ILC3s) ‘helper’ ILCs, as well as cytotoxic Natural Killer (NK) cells. Stud...

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Autores principales: Audouze-Chaud, Johanne, Mathews, Jessica A., Crome, Sarah Q.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Frontiers Media S.A. 2023
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10585162/
https://www.ncbi.nlm.nih.gov/pubmed/37868976
http://dx.doi.org/10.3389/fimmu.2023.1275413
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author Audouze-Chaud, Johanne
Mathews, Jessica A.
Crome, Sarah Q.
author_facet Audouze-Chaud, Johanne
Mathews, Jessica A.
Crome, Sarah Q.
author_sort Audouze-Chaud, Johanne
collection PubMed
description Innate lymphoid cells (ILCs) are a family of innate lymphocytes with important roles in immune response coordination and maintenance of tissue homeostasis. The ILC family includes group 1 (ILC1s), group 2 (ILC2s) and group 3 (ILC3s) ‘helper’ ILCs, as well as cytotoxic Natural Killer (NK) cells. Study of helper ILCs in humans presents several challenges, including their low proportions in peripheral blood or needing access to rare samples to study tissue resident ILC populations. In addition, the lack of established protocols harnessing genetic manipulation platforms has limited the ability to explore molecular mechanism regulating human helper ILC biology. CRISPR/Cas9 is an efficient genome editing tool that enables the knockout of genes of interest, and is commonly used to study molecular regulation of many immune cell types. Here, we developed methods to efficiently knockout genes of interest in human ILC2s. We discuss challenges and lessons learned from our CRISPR/Cas9 gene editing optimizations using a nucleofection transfection approach and test a range of conditions and nucleofection settings to obtain a protocol that achieves effective and stable gene knockout while maintaining optimal cell viability. Using IL-4 as a representative target, we compare different ribonucleoprotein configurations, as well as assess effects of length of time in culture and other parameters that impact CRISPR/Cas9 transfection efficiency. Collectively, we detail a CRISPR/Cas9 protocol for efficient genetic knockout to aid in studying molecular mechanism regulating human ILC2s.
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spelling pubmed-105851622023-10-20 Efficient and stable CRISPR/Cas9-mediated genome-editing of human type 2 innate lymphoid cells Audouze-Chaud, Johanne Mathews, Jessica A. Crome, Sarah Q. Front Immunol Immunology Innate lymphoid cells (ILCs) are a family of innate lymphocytes with important roles in immune response coordination and maintenance of tissue homeostasis. The ILC family includes group 1 (ILC1s), group 2 (ILC2s) and group 3 (ILC3s) ‘helper’ ILCs, as well as cytotoxic Natural Killer (NK) cells. Study of helper ILCs in humans presents several challenges, including their low proportions in peripheral blood or needing access to rare samples to study tissue resident ILC populations. In addition, the lack of established protocols harnessing genetic manipulation platforms has limited the ability to explore molecular mechanism regulating human helper ILC biology. CRISPR/Cas9 is an efficient genome editing tool that enables the knockout of genes of interest, and is commonly used to study molecular regulation of many immune cell types. Here, we developed methods to efficiently knockout genes of interest in human ILC2s. We discuss challenges and lessons learned from our CRISPR/Cas9 gene editing optimizations using a nucleofection transfection approach and test a range of conditions and nucleofection settings to obtain a protocol that achieves effective and stable gene knockout while maintaining optimal cell viability. Using IL-4 as a representative target, we compare different ribonucleoprotein configurations, as well as assess effects of length of time in culture and other parameters that impact CRISPR/Cas9 transfection efficiency. Collectively, we detail a CRISPR/Cas9 protocol for efficient genetic knockout to aid in studying molecular mechanism regulating human ILC2s. Frontiers Media S.A. 2023-10-05 /pmc/articles/PMC10585162/ /pubmed/37868976 http://dx.doi.org/10.3389/fimmu.2023.1275413 Text en Copyright © 2023 Audouze-Chaud, Mathews and Crome https://creativecommons.org/licenses/by/4.0/This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
spellingShingle Immunology
Audouze-Chaud, Johanne
Mathews, Jessica A.
Crome, Sarah Q.
Efficient and stable CRISPR/Cas9-mediated genome-editing of human type 2 innate lymphoid cells
title Efficient and stable CRISPR/Cas9-mediated genome-editing of human type 2 innate lymphoid cells
title_full Efficient and stable CRISPR/Cas9-mediated genome-editing of human type 2 innate lymphoid cells
title_fullStr Efficient and stable CRISPR/Cas9-mediated genome-editing of human type 2 innate lymphoid cells
title_full_unstemmed Efficient and stable CRISPR/Cas9-mediated genome-editing of human type 2 innate lymphoid cells
title_short Efficient and stable CRISPR/Cas9-mediated genome-editing of human type 2 innate lymphoid cells
title_sort efficient and stable crispr/cas9-mediated genome-editing of human type 2 innate lymphoid cells
topic Immunology
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10585162/
https://www.ncbi.nlm.nih.gov/pubmed/37868976
http://dx.doi.org/10.3389/fimmu.2023.1275413
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