Functional profiling of single CRISPR/Cas9-edited human long-term hematopoietic stem cells

Elvin Wagenblast,Eric R Lechman,Leonard D Shultz,Joana Araújo,Olga I Gan,Maria Azkanaz,John E Dick,Jessica L Mcleod,Sabrina A Smith,Lorien Shakib,Gabriela Krivdova

doi:10.1038/s41467-019-12726-0

Abstract

In the human hematopoietic system, rare self-renewing multipotent long-term hematopoietic stem cells (LT-HSCs) are responsible for the lifelong production of mature blood cells and are the rational target for clinical regenerative therapies. However, the heterogeneity in the hematopoietic stem cell compartment and variable outcomes of CRISPR/Cas9 editing make functional interrogation of rare LT-HSCs challenging. Here, we report high efficiency LT-HSC editing at single-cell resolution using electroporation of modified synthetic gRNAs and Cas9 protein. Targeted short isoform expression of the GATA1 transcription factor elicit distinct differentiation and proliferation effects in single highly purified LT-HSC when analyzed with functional in vitro differentiation and long-term repopulation xenotransplantation assays. Our method represents a blueprint for systematic genetic analysis of complex tissue hierarchies at single-cell resolution.

Highlights

In the human hematopoietic system, rare self-renewing multipotent long-term hematopoietic stem cells (LT-HSCs) are responsible for the lifelong production of mature blood cells and are the rational target for clinical regenerative therapies
In order to simplify LTHSC targeting within heterogeneous CD34+ hematopoietic stem and progenitor cell (HSPC), we explored the possibility of CRISPR/Cas[9] editing in highly purified LTHSCs
Acquired and inherited GATA1 mutations contribute to hematological disorders such as Down syndrome acute megakaryoblastic leukemia (AMKL), DiamondBlackfan anemia, transient myeloproliferative disorder and congenital dyserythropoietic anemias with thrombocytopenia[22,23,24,25,26,27]

Summary

Introduction

In the human hematopoietic system, rare self-renewing multipotent long-term hematopoietic stem cells (LT-HSCs) are responsible for the lifelong production of mature blood cells and are the rational target for clinical regenerative therapies. Previous studies have reported long-term engraftment of up to 16 weeks following xenotransplantation of CRISPR/Cas[9] edited human CD34+ HSPCs10,16,18, suggesting that rare LT-HSCs within the CD34+ population can be gene edited. These studies utilized considerable numbers of CD34+ HSPCs and lacked the resolution to functionally interrogate the differentiation and proliferation properties of individual LT-HSCs. In order to simplify LTHSC targeting within heterogeneous CD34+ HSPCs, we explored the possibility of CRISPR/Cas[9] editing in highly purified LTHSCs. In order to simplify LTHSC targeting within heterogeneous CD34+ HSPCs, we explored the possibility of CRISPR/Cas[9] editing in highly purified LTHSCs This approach would enable direct functional characterization of LT-HSCs, rather than bulk populations. We show successful editing of highly purified LT-HSCs via CRISPR/Cas9mediated NHEJ or HDR and their subsequent functional investigation using single cell in vitro differentiation and near-clonal xenotransplantation assays

Methods

Results

Conclusion