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Modeling Human Hematopoiesis Using the CRISPR/Cas9 System

Bäckström, Alexandra LU (2023) In Lund University, Faculty of Medicine Doctoral Dissertation Series
Abstract
Hematopoietic stem cells (HSCs) have the ability to self-renew and to give rise to all blood cells of the different lineages, and are thereby responsible for the replenishment of blood cells throughout life. These cells are tightly regulated by extrinsic and intrinsic regulators, such as signals from the bone marrow microenvironment, complex transcription factor networks and epigenetic regulators. To understand this complex regulatory process has been a long-standing goal, and the emergence of the CRISPR/Cas9 genome engineering system has posed yet another tool that could contribute to the understandings of HSC biology.

To deliver the CRISPR/Cas9 system by lentiviral transduction to sensitive primary cells such as human... (More)
Hematopoietic stem cells (HSCs) have the ability to self-renew and to give rise to all blood cells of the different lineages, and are thereby responsible for the replenishment of blood cells throughout life. These cells are tightly regulated by extrinsic and intrinsic regulators, such as signals from the bone marrow microenvironment, complex transcription factor networks and epigenetic regulators. To understand this complex regulatory process has been a long-standing goal, and the emergence of the CRISPR/Cas9 genome engineering system has posed yet another tool that could contribute to the understandings of HSC biology.

To deliver the CRISPR/Cas9 system by lentiviral transduction to sensitive primary cells such as human hematopoietic stem and progenitor cells (HSPCs) has proven challenging due to the inefficient transduction of Cas9. In Paper I, we developed a split CRISPR/Cas9 system, which combines lentiviral single guide RNA (sgRNA) transduction with transient delivery of Cas9 mRNA by electroporation, to overcome this challenge. This system allows for traceable and efficient gene editing in primary human cord blood-derived HSPCs.

Our subsequent work focused on applications of this system to model human hematopoiesis. In Paper II, we extended the use of the split delivery system to multiplexed perturbations in primary CD34 HSPCs. By using two separate sgRNA vectors containing different traceable fluorescent markers, we achieved a robust double knockout of two genomic loci in double-transduced cells. Furthermore, as a proof of concept, we demonstrate that this system can be used to study gene interactions and gene dependencies.

In Paper III, we further explored the use of our split delivery system in a targeted CRISPR/Cas9 screen to find regulators of HSC self-renewal and differentiation. By screening for sgRNAs that promote expansion or maintenance of the immature CD34 HSPC phenotype, we found several potential gene targets, which can be studied further to elucidate their role in HSC regulation. Amongst the prominent candidates, we identified the putative RNA helicase DDX6 as a critical regulator of HSC self-renewal and differentiation.

In Paper IV, we further demonstrate the usefulness of this CRISPR/Cas9 delivery system for individual gene targeting. The small molecule UM171 has been shown to promote ex vivo expansion of cord blood-derived CD34 HSPCs. While being evaluated in clinical trials, the mechanistic basis of UM171-mediated expansion remained unknown at the time. As part of delineating this mechanism, we targeted REST Corepressor 1 (RCOR1), a member of the CoREST complex that we found to be the primary target of UM171.

Taken together, we have developed a split CRISPR/Cas9 delivery system for traceable and efficient gene editing in primary human HSPCs that enables further studies of the genetic regulation of these clinically relevant cells through combinatorial perturbations and high-throughput screens. (Less)
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author
supervisor
opponent
  • Associate Professor, PhD Bak, Rasmus O., Aarhus University
organization
publishing date
type
Thesis
publication status
published
subject
keywords
Hematopoiesis, Hematopoietic stem and progenitor cells, HSC regulation, CRISPR/Cas9, Gene editing
in
Lund University, Faculty of Medicine Doctoral Dissertation Series
issue
2023:44
pages
83 pages
publisher
Lund University, Faculty of Medicine
defense location
Segerfalksalen, BMC A10, Sölvegatan 17 i Lund
defense date
2023-03-30 09:00:00
ISSN
1652-8220
ISBN
978-91-8021-383-7
language
English
LU publication?
yes
id
949d2cba-f785-46fd-903d-ee5bab951c83
date added to LUP
2023-03-08 13:42:55
date last changed
2023-03-10 14:46:23
@phdthesis{949d2cba-f785-46fd-903d-ee5bab951c83,
  abstract     = {{Hematopoietic stem cells (HSCs) have the ability to self-renew and to give rise to all blood cells of the different lineages, and are thereby responsible for the replenishment of blood cells throughout life. These cells are tightly regulated by extrinsic and intrinsic regulators, such as signals from the bone marrow microenvironment, complex transcription factor networks and epigenetic regulators. To understand this complex regulatory process has been a long-standing goal, and the emergence of the CRISPR/Cas9 genome engineering system has posed yet another tool that could contribute to the understandings of HSC biology. <br/><br/>To deliver the CRISPR/Cas9 system by lentiviral transduction to sensitive primary cells such as human hematopoietic stem and progenitor cells (HSPCs) has proven challenging due to the inefficient transduction of Cas9. In Paper I, we developed a split CRISPR/Cas9 system, which combines lentiviral single guide RNA (sgRNA) transduction with transient delivery of Cas9 mRNA by electroporation, to overcome this challenge. This system allows for traceable and efficient gene editing in primary human cord blood-derived HSPCs. <br/><br/>Our subsequent work focused on applications of this system to model human hematopoiesis. In Paper II, we extended the use of the split delivery system to multiplexed perturbations in primary CD34 HSPCs. By using two separate sgRNA vectors containing different traceable fluorescent markers, we achieved a robust double knockout of two genomic loci in double-transduced cells. Furthermore, as a proof of concept, we demonstrate that this system can be used to study gene interactions and gene dependencies. <br/><br/>In Paper III, we further explored the use of our split delivery system in a targeted CRISPR/Cas9 screen to find regulators of HSC self-renewal and differentiation. By screening for sgRNAs that promote expansion or maintenance of the immature CD34 HSPC phenotype, we found several potential gene targets, which can be studied further to elucidate their role in HSC regulation. Amongst the prominent candidates, we identified the putative RNA helicase DDX6 as a critical regulator of HSC self-renewal and differentiation. <br/><br/>In Paper IV, we further demonstrate the usefulness of this CRISPR/Cas9 delivery system for individual gene targeting. The small molecule UM171 has been shown to promote ex vivo expansion of cord blood-derived CD34 HSPCs. While being evaluated in clinical trials, the mechanistic basis of UM171-mediated expansion remained unknown at the time. As part of delineating this mechanism, we targeted REST Corepressor 1 (RCOR1), a member of the CoREST complex that we found to be the primary target of UM171. <br/><br/>Taken together, we have developed a split CRISPR/Cas9 delivery system for traceable and efficient gene editing in primary human HSPCs that enables further studies of the genetic regulation of these clinically relevant cells through combinatorial perturbations and high-throughput screens.}},
  author       = {{Bäckström, Alexandra}},
  isbn         = {{978-91-8021-383-7}},
  issn         = {{1652-8220}},
  keywords     = {{Hematopoiesis; Hematopoietic stem and progenitor cells; HSC regulation; CRISPR/Cas9; Gene editing}},
  language     = {{eng}},
  number       = {{2023:44}},
  publisher    = {{Lund University, Faculty of Medicine}},
  school       = {{Lund University}},
  series       = {{Lund University, Faculty of Medicine Doctoral Dissertation Series}},
  title        = {{Modeling Human Hematopoiesis Using the CRISPR/Cas9 System}},
  year         = {{2023}},
}