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.
Metrics
-
- 8,075
- views
-
- 1,684
- downloads
-
- 88
- citations
Views, downloads and citations are aggregated across all versions of this paper published by eLife.
Download links
Downloads (link to download the article as PDF)
Open citations (links to open the citations from this article in various online reference manager services)
Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)
Further reading
-
- Stem Cells and Regenerative Medicine
We developed a 96-well plate assay which allows fast, reproducible, and high-throughput generation of 3D cardiac rings around a deformable optically transparent hydrogel (polyethylene glycol [PEG]) pillar of known stiffness. Human induced pluripotent stem cell-derived cardiomyocytes, mixed with normal human adult dermal fibroblasts in an optimized 3:1 ratio, self-organized to form ring-shaped cardiac constructs. Immunostaining showed that the fibroblasts form a basal layer in contact with the glass, stabilizing the muscular fiber above. Tissues started contracting around the pillar at D1 and their fractional shortening increased until D7, reaching a plateau at 25±1%, that was maintained up to 14 days. The average stress, calculated from the compaction of the central pillar during contractions, was 1.4±0.4 mN/mm2. The cardiac constructs recapitulated expected inotropic responses to calcium and various drugs (isoproterenol, verapamil) as well as the arrhythmogenic effects of dofetilide. This versatile high-throughput assay allows multiple in situ mechanical and structural readouts.