Combined lentiviral- and RNA-mediated CRISPR/Cas9 delivery for efficient and traceable gene editing in human hematopoietic stem and progenitor cells

Abstract The CRISPR/Cas9 system is a versatile tool for functional genomics and forward genetic screens in mammalian cells. However, it has been challenging to deliver the CRISPR components to sensitive cell types, such as primary human hematopoietic stem and progenitor cells (HSPCs), partly due to...

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Autores principales:	David Yudovich, Alexandra Bäckström, Ludwig Schmiderer, Kristijonas Žemaitis, Agatheeswaran Subramaniam, Jonas Larsson
Formato:	article
Lenguaje:	EN
Publicado:	Nature Portfolio 2020
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/37fcffdd00f84de08d81d45c0b3fdfd6
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spelling	oai:doaj.org-article:37fcffdd00f84de08d81d45c0b3fdfd62021-12-02T14:01:28ZCombined lentiviral- and RNA-mediated CRISPR/Cas9 delivery for efficient and traceable gene editing in human hematopoietic stem and progenitor cells10.1038/s41598-020-79724-x2045-2322https://doaj.org/article/37fcffdd00f84de08d81d45c0b3fdfd62020-12-01T00:00:00Zhttps://doi.org/10.1038/s41598-020-79724-xhttps://doaj.org/toc/2045-2322Abstract The CRISPR/Cas9 system is a versatile tool for functional genomics and forward genetic screens in mammalian cells. However, it has been challenging to deliver the CRISPR components to sensitive cell types, such as primary human hematopoietic stem and progenitor cells (HSPCs), partly due to lentiviral transduction of Cas9 being extremely inefficient in these cells. Here, to overcome these hurdles, we developed a combinatorial system using stable lentiviral delivery of single guide RNA (sgRNA) followed by transient transfection of Cas9 mRNA by electroporation in human cord blood-derived CD34+ HSPCs. We further applied an optimized sgRNA structure, that significantly improved editing efficiency in this context, and we obtained knockout levels reaching 90% for the cell surface proteins CD45 and CD44 in sgRNA transduced HSPCs. Our combinatorial CRISPR/Cas9 delivery approach had no negative influence on CD34 expression or colony forming capacity in vitro compared to non-treated HSPCs. Furthermore, gene edited HSPCs showed intact in vivo reconstitution capacity following transplantation to immunodeficient mice. Taken together, we developed a paradigm for combinatorial CRISPR/Cas9 delivery that enables efficient and traceable gene editing in primary human HSPCs, and is compatible with high functionality both in vitro and in vivo.David YudovichAlexandra BäckströmLudwig SchmidererKristijonas ŽemaitisAgatheeswaran SubramaniamJonas LarssonNature PortfolioarticleMedicineRScienceQENScientific Reports, Vol 10, Iss 1, Pp 1-11 (2020)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q David Yudovich Alexandra Bäckström Ludwig Schmiderer Kristijonas Žemaitis Agatheeswaran Subramaniam Jonas Larsson Combined lentiviral- and RNA-mediated CRISPR/Cas9 delivery for efficient and traceable gene editing in human hematopoietic stem and progenitor cells
description	Abstract The CRISPR/Cas9 system is a versatile tool for functional genomics and forward genetic screens in mammalian cells. However, it has been challenging to deliver the CRISPR components to sensitive cell types, such as primary human hematopoietic stem and progenitor cells (HSPCs), partly due to lentiviral transduction of Cas9 being extremely inefficient in these cells. Here, to overcome these hurdles, we developed a combinatorial system using stable lentiviral delivery of single guide RNA (sgRNA) followed by transient transfection of Cas9 mRNA by electroporation in human cord blood-derived CD34+ HSPCs. We further applied an optimized sgRNA structure, that significantly improved editing efficiency in this context, and we obtained knockout levels reaching 90% for the cell surface proteins CD45 and CD44 in sgRNA transduced HSPCs. Our combinatorial CRISPR/Cas9 delivery approach had no negative influence on CD34 expression or colony forming capacity in vitro compared to non-treated HSPCs. Furthermore, gene edited HSPCs showed intact in vivo reconstitution capacity following transplantation to immunodeficient mice. Taken together, we developed a paradigm for combinatorial CRISPR/Cas9 delivery that enables efficient and traceable gene editing in primary human HSPCs, and is compatible with high functionality both in vitro and in vivo.
format	article
author	David Yudovich Alexandra Bäckström Ludwig Schmiderer Kristijonas Žemaitis Agatheeswaran Subramaniam Jonas Larsson
author_facet	David Yudovich Alexandra Bäckström Ludwig Schmiderer Kristijonas Žemaitis Agatheeswaran Subramaniam Jonas Larsson
author_sort	David Yudovich
title	Combined lentiviral- and RNA-mediated CRISPR/Cas9 delivery for efficient and traceable gene editing in human hematopoietic stem and progenitor cells
title_short	Combined lentiviral- and RNA-mediated CRISPR/Cas9 delivery for efficient and traceable gene editing in human hematopoietic stem and progenitor cells
title_full	Combined lentiviral- and RNA-mediated CRISPR/Cas9 delivery for efficient and traceable gene editing in human hematopoietic stem and progenitor cells
title_fullStr	Combined lentiviral- and RNA-mediated CRISPR/Cas9 delivery for efficient and traceable gene editing in human hematopoietic stem and progenitor cells
title_full_unstemmed	Combined lentiviral- and RNA-mediated CRISPR/Cas9 delivery for efficient and traceable gene editing in human hematopoietic stem and progenitor cells
title_sort	combined lentiviral- and rna-mediated crispr/cas9 delivery for efficient and traceable gene editing in human hematopoietic stem and progenitor cells
publisher	Nature Portfolio
publishDate	2020
url	https://doaj.org/article/37fcffdd00f84de08d81d45c0b3fdfd6
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Combined lentiviral- and RNA-mediated CRISPR/Cas9 delivery for efficient and traceable gene editing in human hematopoietic stem and progenitor cells

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