Modeling Human Hematopoiesis Using the CRISPR/Cas9 System
Författare
Summary, in English
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.
Avdelning/ar
Publiceringsår
2023
Språk
Engelska
Publikation/Tidskrift/Serie
Lund University, Faculty of Medicine Doctoral Dissertation Series
Issue
2023:44
Dokumenttyp
Doktorsavhandling
Förlag
Lund University, Faculty of Medicine
Ämne
- Cell and Molecular Biology
Nyckelord
- Hematopoiesis
- Hematopoietic stem and progenitor cells
- HSC regulation
- CRISPR/Cas9
- Gene editing
Status
Published
Handledare
ISBN/ISSN/Övrigt
- ISSN: 1652-8220
- ISBN: 978-91-8021-383-7
Försvarsdatum
30 mars 2023
Försvarstid
09:00
Försvarsplats
Segerfalksalen, BMC A10, Sölvegatan 17 i Lund
Opponent
- Rasmus O. Bak (Associate Professor, PhD)