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

M. Kyle Cromer; Sriram Vaidyanathan; Daniel E. Ryan; Bo Curry; Anne Bergstrom Lucas; Joab Camarena; Milan Kaushik; Sarah R. Hay; Renata M. Martin; Israel Steinfeld; Rasmus O. Bak; Daniel P. Dever; Ayal Hendel; Laurakay Bruhn; Matthew H. Porteus

doi:10.1016/j.ymthe.2018.06.002

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

M. Kyle Cromer, Sriram Vaidyanathan, Daniel E. Ryan, Bo Curry, Anne Bergstrom Lucas, Joab Camarena, Milan Kaushik, Sarah R. Hay, Renata M. Martin, Israel Steinfeld, Rasmus O. Bak, Daniel P. Dever, Ayal Hendel, Laurakay Bruhn, Matthew H. Porteus

The Mina and Everard Goodman Faculty of Life Sciences

Research output: Contribution to journal › Article › peer-review

77 Scopus citations

Abstract

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. In this issue of Molecular Therapy, Cromer et al. (2018) describe the global transcriptional response to individual components of Cas9/AAV6-mediated genome editing in hematopoietic stem and progenitor cells. They found that Cas9 in the form of mRNA elicits a dramatic viral/interferon response, while AAV6 causes little transcriptional disturbance.

Original language	English
Pages (from-to)	2431-2442
Number of pages	12
Journal	Molecular Therapy
Volume	26
Issue number	10
DOIs	https://doi.org/10.1016/j.ymthe.2018.06.002
State	Published - 3 Oct 2018

Bibliographical note

Funding Information:
We would like to thank D. Russell for the pDGM6 plasmid and the Binns Program for Cord Blood Research at Stanford University for cord blood-derived CD34+ HSPCs. We also would like to give thanks to the members of the Porteus laboratory for input, comments, and discussion. M.H.P. gratefully acknowledges the support of the Amon Carter Foundation, the Laurie Kraus Lacob Faculty Scholar Award in Pediatric Translational Research, the Sutardja Foundation, and NIH grant support (R01-AI097320 and R01-AI120766). M.K.C. was supported by an NIH T32 Institutional Training Grant in Hematology (5T32HL120824-04). D.P.D. was supported by the Stanford Child Health Research Institute (CHRI) grant and the Bass Cancer Center Program Endowment. R.O.B. was supported through an Individual Postdoctoral grant (DFF-1333-00106B) and a Sapere Aude Research Talent grant (DFF-1331-00735B), both from the Danish Council for Independent Research, Medical Sciences.

Funding Information:
We would like to thank D. Russell for the pDGM6 plasmid and the Binns Program for Cord Blood Research at Stanford University for cord blood-derived CD34 + HSPCs. We also would like to give thanks to the members of the Porteus laboratory for input, comments, and discussion. M.H.P. gratefully acknowledges the support of the Amon Carter Foundation , the Laurie Kraus Lacob Faculty Scholar Award in Pediatric Translational Research , the Sutardja Foundation , and NIH grant support ( R01-AI097320 and R01-AI120766 ). M.K.C. was supported by an NIH T32 Institutional Training Grant in Hematology ( 5T32HL120824-04 ). D.P.D. was supported by the Stanford Child Health Research Institute (CHRI) grant and the Bass Cancer Center Program Endowment . R.O.B. was supported through an Individual Postdoctoral grant ( DFF-1333-00106B ) and a Sapere Aude Research Talent grant ( DFF-1331-00735B ), both from the Danish Council for Independent Research, Medical Sciences .

Publisher Copyright:
© 2018 The American Society of Gene and Cell Therapy

Keywords

RNA expression analysis
genome editing
hematopoietic stem cells

Access to Document

10.1016/j.ymthe.2018.06.002

Cite this

Cromer, M. K., Vaidyanathan, S., Ryan, D. E., Curry, B., Lucas, A. B., Camarena, J., Kaushik, M., Hay, S. R., Martin, R. M., Steinfeld, I., Bak, R. O., Dever, D. P., Hendel, A., Bruhn, L., & Porteus, M. H. (2018). Global Transcriptional Response to CRISPR/Cas9-AAV6-Based Genome Editing in CD34⁺ Hematopoietic Stem and Progenitor Cells. Molecular Therapy, 26(10), 2431-2442. https://doi.org/10.1016/j.ymthe.2018.06.002

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abstract = "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. In this issue of Molecular Therapy, Cromer et al. (2018) describe the global transcriptional response to individual components of Cas9/AAV6-mediated genome editing in hematopoietic stem and progenitor cells. They found that Cas9 in the form of mRNA elicits a dramatic viral/interferon response, while AAV6 causes little transcriptional disturbance.",

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Cromer, MK, Vaidyanathan, S, Ryan, DE, Curry, B, Lucas, AB, Camarena, J, Kaushik, M, Hay, SR, Martin, RM, Steinfeld, I, Bak, RO, Dever, DP, Hendel, A, Bruhn, L & Porteus, MH 2018, 'Global Transcriptional Response to CRISPR/Cas9-AAV6-Based Genome Editing in CD34⁺ Hematopoietic Stem and Progenitor Cells', Molecular Therapy, vol. 26, no. 10, pp. 2431-2442. https://doi.org/10.1016/j.ymthe.2018.06.002

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AU - Cromer, M. Kyle

AU - Vaidyanathan, Sriram

AU - Ryan, Daniel E.

AU - Curry, Bo

AU - Lucas, Anne Bergstrom

AU - Camarena, Joab

AU - Kaushik, Milan

AU - Hay, Sarah R.

AU - Martin, Renata M.

AU - Steinfeld, Israel

AU - Bak, Rasmus O.

AU - Dever, Daniel P.

AU - Hendel, Ayal

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