Multiplexed genetic engineering of human hematopoietic stem and progenitor cells using CRISPR/Cas9 and AAV6
Abstract
Precise and efficient manipulation of genes is crucial for understanding the molecular mechanisms that govern human hematopoiesis and for developing novel therapies for diseases of the blood and immune system. Current methods do not enable precise engineering of complex genotypes that can be easily tracked in a mixed population of cells. We describe a method to multiplex homologous recombination (HR) in human hematopoietic stem and progenitor cells and primary human T cells by combining rAAV6 donor delivery and the CRISPR/Cas9 system delivered as ribonucleoproteins (RNPs). In addition, the use of reporter genes allows FACS-purification and tracking of cells that have had multiple alleles or loci modified by HR. We believe this method will enable broad applications not only to the study of human hematopoietic gene function and networks, but also to perform sophisticated synthetic biology to develop innovative engineered stem cell-based therapeutics.
Article and author information
Author details
Funding
Danish Council for Independent Research (DFF-1333-00106B)
- Rasmus O Bak
Danish Council for Independent Research (DFF-1331-00735B)
- Rasmus O Bak
National Institutes of Health (R01- AI097320)
- Matthew H Porteus
National Institutes of Health (R01-AI120766)
- Matthew H Porteus
Stanford Child Health Research Institute (Postdoctoral Award)
- Daniel P Dever
Austrian Research Council (Erwin Schroedinger Postdoctoral Fellowship)
- Andreas Reinisch
Amon G. Carter Foundation
- Matthew H Porteus
Laurie Kraus Lacob Faculty Scholar Award in Pediatric Translational Research (Scholar Award)
- Matthew H Porteus
National Institutes of Health (PN2EY018244)
- Matthew H Porteus
Stanford Ludwig Center for Cancer Stem Cell Research
- Ravindra Majeti
National Institutes of Health (R01-CA188055)
- Ravindra Majeti
New York Stem Cell Foundation (Robertson Investigator)
- Ravindra Majeti
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Reviewing Editor
- Ross L Levine, Memorial Sloan Kettering Cancer Center, United States
Ethics
Animal experimentation: Animal experiments were performed in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The experimental protocol was approved by Stanford University's Administrative Panel on Lab Animal Care (IACUC 25065).
Version history
- Received: April 18, 2017
- Accepted: September 26, 2017
- Accepted Manuscript published: September 28, 2017 (version 1)
- Version of Record published: October 25, 2017 (version 2)
- Version of Record updated: November 16, 2018 (version 3)
Copyright
© 2017, Bak et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
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Further reading
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- Cell Biology
- Stem Cells and Regenerative Medicine
Extramedullary erythropoiesis is not expected in healthy adult mice, but erythropoietic gene expression was elevated in lineage-depleted spleen cells from Cd47−/− mice. Expression of several genes associated with early stages of erythropoiesis was elevated in mice lacking CD47 or its signaling ligand thrombospondin-1, consistent with previous evidence that this signaling pathway inhibits expression of multipotent stem cell transcription factors in spleen. In contrast, cells expressing markers of committed erythroid progenitors were more abundant in Cd47−/− spleens but significantly depleted in Thbs1−/− spleens. Single-cell transcriptome and flow cytometry analyses indicated that loss of CD47 is associated with accumulation and increased proliferation in spleen of Ter119−CD34+ progenitors and Ter119+CD34− committed erythroid progenitors with elevated mRNA expression of Kit, Ermap, and Tfrc. Induction of committed erythroid precursors is consistent with the known function of CD47 to limit the phagocytic removal of aged erythrocytes. Conversely, loss of thrombospondin-1 delays the turnover of aged red blood cells, which may account for the suppression of committed erythroid precursors in Thbs1−/− spleens relative to basal levels in wild-type mice. In addition to defining a role for CD47 to limit extramedullary erythropoiesis, these studies reveal a thrombospondin-1-dependent basal level of extramedullary erythropoiesis in adult mouse spleen.