Multiplexed genetic engineering of human hematopoietic stem and progenitor cells using CRISPR/Cas9 and AAV6

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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.

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Rasmus O Bak

Department of Pediatrics, Stanford University, Stanford, United States

Competing interests
No competing interests declared.

"This ORCID iD identifies the author of this article:" 0000-0002-7383-0297
Daniel P Dever

Department of Pediatrics, Stanford University, Stanford, United States

Competing interests
No competing interests declared.
Andreas Reinisch

Department of Medicine, Division of Hematology, Stanford University, Stanford, United States

Competing interests
No competing interests declared.
David Cruz Hernandez

Department of Medicine, Division of Hematology, Stanford University, Stanford, United States

Competing interests
No competing interests declared.
Ravindra Majeti

Department of Medicine, Division of Hematology, Stanford University, Stanford, United States

For correspondence
rmajeti@stanford.edu

Competing interests
Ravindra Majeti, has equity and consults for Forty Seven Inc..
Matthew H Porteus

Department of Pediatrics, Stanford University, Stanford, United States

For correspondence
mporteus@stanford.edu

Competing interests
Matthew H Porteus, has equity and consults for CRISPR Therapeutics..

"This ORCID iD identifies the author of this article:" 0000-0002-3850-4648

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.

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).

Copyright

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.