Genome editing via homologous recombination (HR) (gene targeting) in human hematopoietic stem cells (HSCs) has the power to reveal gene–function relationships and potentially transform curative hematological gene and cell therapies. However, there are no comprehensive and reproducible protocols for targeting HSCs for HR. Herein, we provide a detailed protocol for the production, enrichment, and in vitro and in vivo analyses of HR-targeted HSCs by combining CRISPR/Cas9 technology with the use of rAAV6 and flow cytometry. Using this protocol, researchers can introduce single-nucleotide changes into the genome or longer gene cassettes with the precision of genome editing. Along with our troubleshooting and optimization guidelines, researchers can use this protocol to streamline HSC genome editing at any locus of interest. The in vitro HSC-targeting protocol and analyses can be completed in 3 weeks, and the long-term in vivo HSC engraftment analyses in immunodeficient mice can be achieved in 16 weeks. This protocol enables manipulation of genes for investigation of gene functions during hematopoiesis, as well as for the correction of genetic mutations in HSC transplantation–based therapies for diseases such as sickle cell disease, β-thalassemia, and primary immunodeficiencies.
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R.O.B. was supported through an individual postdoctoral grant (DFF-1333-00106B) and a Sapere Aude, Research Talent grant (DFF-1331-00735B), both from the Danish Council for Independent Research, Medical Sciences. D.P.D. was supported through a Stanford Child Health Research Institute (CHRI) grant and postdoctoral award. M.H.P. gratefully acknowledges the support of the Amon Carter Foundation, the Laurie Kraus Lacob Faculty Scholar Award in Pediatric Translational Research, and NIH grants R01-AI097320 and R01-AI120766. We further thank members of the Porteus and Majeti lab for helpful input, comments, and discussions.
M.H.P. is a consultant and has equity interest in CRISPR Tx, but CRISPR Tx had no input into the design, execution, interpretation, or publication of the methods or results herein.
Integrated supplementary information
Supplementary Figure 1 Gating scheme for identifying and quantifying the number of HSPCs with targeted integration of a GFP reporter gene.
FACS plots show the gating strategy for identifying CD34+ HSPCs targeted with a GFP reporter gene. The figure shows data from cord blood-derived CD34+ HSPCs that were electroporated two days after isolation with Cas9 RNP targeting the HBB locus. Immediately after electroporation, cells were transduced at an MOI of 50,000 using an AAV6 donor vector carrying a UbC-GFP expression cassette. Four days after electroporation and transduction, cells were stained for CD34 (and propidium iodide to identify live cells) and then analyzed on a FACS Aria (BD). Cells are identified in a forward/side scatter plot (FCS-A and SSC-A) and single cells discriminated from doublets using SSC-W/SSC-H and FSC-W/FSC-H plots. Live cells are discriminated based on propidium iodide, and in the final GFP/CD34 plot, the targeted GFP+CD34+ cells are identified. Cell frequencies within gates are shown.
Supplementary Figure 2 Gating strategy to identify stem and progenitor subpopulations within the CD34+ cells.
Representative FACS plots are shown from the analysis of CD34+ cells freshly isolated from cord blood. Cells were stained for human lineage markers (CD2, CD3, CD4, CD8, CD16, CD19, CD20, CD56, CD235a, and CD14), as well as CD34, CD38, CD90, CD45RA, and CD123. The gating strategy identifies cells in a forward/side scatter plot (FCS-A and SSC-A) and then single cells are discriminated from doublets using SSC-W/SSC-H and FSC-W/FSC-H plots. Live cells are discriminated based on propidium iodide. Subpopulations are shown in red gates and identified as follows: HSCs (Lin-/CD34+/CD38−/CD45RA−/CD90+), MPPs (Lin−/CD34+/CD38−/CD45RA−/CD90−), LMPPs (Lin−, CD34+, CD38−, CD90−, CD45RA+), CMPs (Lin−/CD34+/CD38+/CD45RA−/CD123+), GMPs (Lin−/CD34+/CD38+/CD45RA+/CD123+), and MEPs (Lin−/CD34+/CD38+/CD45RA−/CD123−). HSCs: hematopoietic stem cells, MPPs: multipotent progenitors, LMPPs: lymphoid-primed multipotent progenitors, CMPs: common myeloid progenitors, GMPs: granulocyte-macrophage progenitors, MEPs: megakaryocyte-erythrocyte progenitors.
Representative FACS plots show the gating strategy upstream of the FACS plots shown in Figure 4D. Cells are identified in a forward/side scatter plot (FCS-A and SSC-A) and single cells discriminated from doublets using SSC-W/SSC-H and FSC-W/FSC-H plots. Live cells and murine red blood cells (RBCs) are discriminated based on propidium iodide and an anti-murine Ter119 antibody. Out of the live non-RBCs, human cells can be gated based on huCD45 and HLA-ABC expression as depicted in Figure 4D. Adapted with permission from ref. 15, Springer Nature.
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Bak, R., Dever, D. & Porteus, M. CRISPR/Cas9 genome editing in human hematopoietic stem cells. Nat Protoc 13, 358–376 (2018). https://doi.org/10.1038/nprot.2017.143
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