1. Stem Cells and Regenerative Medicine
  2. Genetics and Genomics
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Multiplexed genetic engineering of human hematopoietic stem and progenitor cells using CRISPR/Cas9 and AAV6

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Cite this article as: eLife 2017;6:e27873 doi: 10.7554/eLife.27873

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

  1. Rasmus O Bak

    Department of Pediatrics, Stanford University, Stanford, United States
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-7383-0297
  2. Daniel P Dever

    Department of Pediatrics, Stanford University, Stanford, United States
    Competing interests
    No competing interests declared.
  3. Andreas Reinisch

    Department of Medicine, Division of Hematology, Stanford University, Stanford, United States
    Competing interests
    No competing interests declared.
  4. David Cruz Hernandez

    Department of Medicine, Division of Hematology, Stanford University, Stanford, United States
    Competing interests
    No competing interests declared.
  5. 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..
  6. 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..
    ORCID icon "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).

Reviewing Editor

  1. Ross L Levine, Memorial Sloan Kettering Cancer Center, United States

Publication history

  1. Received: April 18, 2017
  2. Accepted: September 26, 2017
  3. Accepted Manuscript published: September 28, 2017 (version 1)
  4. Version of Record published: October 25, 2017 (version 2)
  5. 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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    2. Stem Cells and Regenerative Medicine
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    The gut is the primary interface between an animal and food, but how it adapts to qualitative dietary variation is poorly defined. We find that the Drosophila midgut plastically resizes following changes in dietary composition. A panel of nutrients collectively promote gut growth, which sugar opposes. Diet influences absolute and relative levels of enterocyte loss and stem cell proliferation, which together determine cell numbers. Diet also influences enterocyte size. A high sugar diet inhibits translation and uncouples ISC proliferation from expression of niche-derived signals but, surprisingly, rescuing these effects genetically was not sufficient to modify diet's impact on midgut size. However, when stem cell proliferation was deficient, diet's impact on enterocyte size was enhanced, and reducing enterocyte-autonomous TOR signaling was sufficient to attenuate diet-dependent midgut resizing. These data clarify the complex relationships between nutrition, epithelial dynamics, and cell size, and reveal a new mode of plastic, diet-dependent organ resizing.