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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

Yudovich, David LU ; Bäckström, Alexandra LU ; Schmiderer, Ludwig LU ; Žemaitis, Kristijonas LU ; Subramaniam, Agatheeswaran LU and Larsson, Jonas LU (2020) In Scientific Reports 10(1).
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... (More)

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.

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author
; ; ; ; and
organization
publishing date
type
Contribution to journal
publication status
published
subject
in
Scientific Reports
volume
10
issue
1
article number
22393
publisher
Nature Publishing Group
external identifiers
  • pmid:33372184
  • scopus:85098254128
ISSN
2045-2322
DOI
10.1038/s41598-020-79724-x
language
English
LU publication?
yes
id
16001eb9-30a0-4c0b-b26c-48c568ca8983
date added to LUP
2021-01-07 09:52:11
date last changed
2024-05-16 00:52:05
@article{16001eb9-30a0-4c0b-b26c-48c568ca8983,
  abstract     = {{<p>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<sup>+</sup> 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.</p>}},
  author       = {{Yudovich, David and Bäckström, Alexandra and Schmiderer, Ludwig and Žemaitis, Kristijonas and Subramaniam, Agatheeswaran and Larsson, Jonas}},
  issn         = {{2045-2322}},
  language     = {{eng}},
  number       = {{1}},
  publisher    = {{Nature Publishing Group}},
  series       = {{Scientific Reports}},
  title        = {{Combined lentiviral- and RNA-mediated CRISPR/Cas9 delivery for efficient and traceable gene editing in human hematopoietic stem and progenitor cells}},
  url          = {{http://dx.doi.org/10.1038/s41598-020-79724-x}},
  doi          = {{10.1038/s41598-020-79724-x}},
  volume       = {{10}},
  year         = {{2020}},
}