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Combinatorial gene targeting in primary human hematopoietic stem and progenitor cells

Bäckström, Alexandra LU ; Yudovich, David LU ; Žemaitis, Kristijonas LU ; Nilsén Falck, Ludvig LU ; Subramaniam, Agatheeswaran LU and Larsson, Jonas LU (2022) In Scientific Reports 12(1).
Abstract

The CRISPR/Cas9 system offers enormous versatility for functional genomics but many applications have proven to be challenging in primary human cells compared to cell lines or mouse cells. Here, to establish a paradigm for multiplexed gene editing in primary human cord blood-derived hematopoietic stem and progenitor cells (HSPCs), we used co-delivery of lentiviral sgRNA vectors expressing either Enhanced Green Fluorescent Protein (EGFP) or Kusabira Orange (KuO), together with Cas9 mRNA, to simultaneously edit two genetic loci. The fluorescent markers allow for tracking of either single- or double-edited cells, and we could achieve robust double knockout of the cell surface molecules CD45 and CD44 with an efficiency of ~ 70%. As a... (More)

The CRISPR/Cas9 system offers enormous versatility for functional genomics but many applications have proven to be challenging in primary human cells compared to cell lines or mouse cells. Here, to establish a paradigm for multiplexed gene editing in primary human cord blood-derived hematopoietic stem and progenitor cells (HSPCs), we used co-delivery of lentiviral sgRNA vectors expressing either Enhanced Green Fluorescent Protein (EGFP) or Kusabira Orange (KuO), together with Cas9 mRNA, to simultaneously edit two genetic loci. The fluorescent markers allow for tracking of either single- or double-edited cells, and we could achieve robust double knockout of the cell surface molecules CD45 and CD44 with an efficiency of ~ 70%. As a functional proof of concept, we demonstrate that this system can be used to model gene dependencies for cell survival, by simultaneously targeting the cohesin genes STAG1 and STAG2. Moreover, we show combinatorial effects with potential synergy for HSPC expansion by targeting the Aryl Hydrocarbon Receptor (AHR) in conjunction with members of the CoREST complex. Taken together, our traceable multiplexed CRISPR/Cas9 system enables studies of genetic dependencies and cooperation in primary HSPCs, and has important implications for modelling polygenic diseases, as well as investigation of the underlying mechanisms of gene interactions.

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author
; ; ; ; and
organization
publishing date
type
Contribution to journal
publication status
published
subject
in
Scientific Reports
volume
12
issue
1
article number
18169
publisher
Nature Publishing Group
external identifiers
  • pmid:36307542
  • scopus:85140901312
ISSN
2045-2322
DOI
10.1038/s41598-022-23118-8
language
English
LU publication?
yes
id
1ab1c4e6-e620-499b-bd69-1ebec356d94e
date added to LUP
2022-12-06 12:15:38
date last changed
2024-06-13 23:01:57
@article{1ab1c4e6-e620-499b-bd69-1ebec356d94e,
  abstract     = {{<p>The CRISPR/Cas9 system offers enormous versatility for functional genomics but many applications have proven to be challenging in primary human cells compared to cell lines or mouse cells. Here, to establish a paradigm for multiplexed gene editing in primary human cord blood-derived hematopoietic stem and progenitor cells (HSPCs), we used co-delivery of lentiviral sgRNA vectors expressing either Enhanced Green Fluorescent Protein (EGFP) or Kusabira Orange (KuO), together with Cas9 mRNA, to simultaneously edit two genetic loci. The fluorescent markers allow for tracking of either single- or double-edited cells, and we could achieve robust double knockout of the cell surface molecules CD45 and CD44 with an efficiency of ~ 70%. As a functional proof of concept, we demonstrate that this system can be used to model gene dependencies for cell survival, by simultaneously targeting the cohesin genes STAG1 and STAG2. Moreover, we show combinatorial effects with potential synergy for HSPC expansion by targeting the Aryl Hydrocarbon Receptor (AHR) in conjunction with members of the CoREST complex. Taken together, our traceable multiplexed CRISPR/Cas9 system enables studies of genetic dependencies and cooperation in primary HSPCs, and has important implications for modelling polygenic diseases, as well as investigation of the underlying mechanisms of gene interactions.</p>}},
  author       = {{Bäckström, Alexandra and Yudovich, David and Žemaitis, Kristijonas and Nilsén Falck, Ludvig and Subramaniam, Agatheeswaran and Larsson, Jonas}},
  issn         = {{2045-2322}},
  language     = {{eng}},
  number       = {{1}},
  publisher    = {{Nature Publishing Group}},
  series       = {{Scientific Reports}},
  title        = {{Combinatorial gene targeting in primary human hematopoietic stem and progenitor cells}},
  url          = {{http://dx.doi.org/10.1038/s41598-022-23118-8}},
  doi          = {{10.1038/s41598-022-23118-8}},
  volume       = {{12}},
  year         = {{2022}},
}