Global Transcriptional Response to CRISPR/Cas9-AAV6-Based Genome Editing in CD34 Hematopoietic Stem and Progenitor Cells

Research output: Contribution to journal/Conference contribution in journal/Contribution to newspaperJournal articleResearchpeer-review

  • M Kyle Cromer, Department of Pediatrics, Stanford University, Stanford, California 94305, USA.
  • ,
  • Sriram Vaidyanathan, Department of Pediatrics, Stanford University, Stanford, California 94305, USA.
  • ,
  • Daniel E Ryan, Agilent Technologies, Santa Clara, CA 95051, USA.
  • ,
  • Bo Curry, Agilent Technologies, Santa Clara, CA 95051, USA.
  • ,
  • Anne Bergstrom Lucas, Agilent Technologies, Santa Clara, CA 95051, USA.
  • ,
  • Joab Camarena, Department of Pediatrics, Stanford University, Stanford, California 94305, USA.
  • ,
  • Milan Kaushik, Department of Pediatrics, Stanford University, Stanford, California 94305, USA.
  • ,
  • Sarah R Hay, Department of Pediatrics, Stanford University, Stanford, California 94305, USA.
  • ,
  • Renata M Martin, Department of Pediatrics, Stanford University, Stanford, California 94305, USA.
  • ,
  • Israel Steinfeld, Agilent Technologies, Santa Clara, CA 95051, USA.
  • ,
  • Rasmus O Bak
  • Daniel P Dever, Department of Pediatrics, Stanford University, Stanford, California 94305, USA.
  • ,
  • Ayal Hendel, Institute for Nanotechnology and Advanced Materials, The Mina and Everard Goodman Faculty of Life Sciences, Bar-Ilan University, Ramat Gan 5290002, Israel. Electronic address: ayal.hendel@biu.ac.il.
  • ,
  • Laurakay Bruhn, Agilent Technologies, Santa Clara, CA 95051, USA.
  • ,
  • Matthew H Porteus, Department of Pediatrics, Stanford University, Stanford, CA 94305, USA. Electronic address: mporteus@stanford.edu.

Genome-editing technologies are currently being translated to the clinic. However, cellular effects of the editing machinery have yet to be fully elucidated. Here, we performed global microarray-based gene expression measurements on human CD34+ hematopoietic stem and progenitor cells that underwent editing. We probed effects of the entire editing process as well as each component individually, including electroporation, Cas9 (mRNA or protein) with chemically modified sgRNA, and AAV6 transduction. We identified differentially expressed genes relative to control treatments, which displayed enrichment for particular biological processes. All editing machinery components elicited immune, stress, and apoptotic responses. Cas9 mRNA invoked the greatest amount of transcriptional change, eliciting a distinct viral response and global transcriptional downregulation, particularly of metabolic and cell cycle processes. Electroporation also induced significant transcriptional change, with notable downregulation of metabolic processes. Surprisingly, AAV6 evoked no detectable viral response. We also found Cas9/sgRNA ribonucleoprotein treatment to be well tolerated, in spite of eliciting a DNA damage signature. Overall, this data establishes a benchmark for cellular tolerance of CRISPR/Cas9-AAV6-based genome editing, ensuring that the clinical protocol is as safe and efficient as possible.

Original languageEnglish
JournalMolecular Therapy
Volume26
Issue10
Pages (from-to)2431-2442
Number of pages12
ISSN1525-0016
DOIs
Publication statusPublished - 2018
Externally publishedYes

    Research areas

  • RNA expression analysis, genome editing, hematopoietic stem cells, APOPTOSIS, DOUBLE-STRAND BREAKS, BETA-GLOBIN, TRANSLATION, ZINC-FINGER NUCLEASES, GENE, MESSENGER-RNA, IMMUNE-RESPONSES, OFF-TARGET ACTIVITY, ADENOASSOCIATED VIRUS VECTORS

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