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

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.

[1]  S. Chatterjee,et al.  Immune responses to adeno-associated virus and its recombinant vectors , 2003, Gene Therapy.

[2]  Luigi Naldini,et al.  Preclinical modeling highlights the therapeutic potential of hematopoietic stem cell gene editing for correction of SCID-X1 , 2017, Science Translational Medicine.

[3]  B. van Steensel,et al.  Easy quantification of template-directed CRISPR/Cas9 editing , 2017, bioRxiv.

[4]  Alexander Meissner,et al.  Highly efficient reprogramming to pluripotency and directed differentiation of human cells with synthetic modified mRNA. , 2010, Cell stem cell.

[5]  Jiawei Han,et al.  Expression of bbc3, a pro-apoptotic BH3-only gene, is regulated by diverse cell death and survival signals , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[6]  Prashant Mali,et al.  A multifunctional AAV–CRISPR–Cas9 and its host response , 2016, Nature Methods.

[7]  A. Scharenberg,et al.  Efficient modification of CCR5 in primary human hematopoietic cells using a megaTAL nuclease and AAV donor template , 2015, Science Translational Medicine.

[8]  M. van der Burg,et al.  Targeted Genome Editing in Human Repopulating Hematopoietic Stem Cells , 2014, Nature.

[9]  P. Gregory,et al.  Homology-driven genome editing in hematopoietic stem and progenitor cells using zinc finger nuclease mRNA and AAV6 donors , 2015, Nature Biotechnology.

[10]  Jianwei Zhu,et al.  Niclosamide induced cell apoptosis via upregulation of ATF3 and activation of PERK in Hepatocellular carcinoma cells , 2016, BMC Gastroenterology.

[11]  P. Kuppusamy,et al.  Preconditioning mesenchymal stem cells with caspase inhibition and hyperoxia prior to hypoxia exposure increases cell proliferation , 2013, Journal of cellular biochemistry.

[12]  R. Bak,et al.  Priming Human Repopulating Hematopoietic Stem and Progenitor Cells for Cas9/sgRNA Gene Targeting , 2018, Molecular therapy. Nucleic acids.

[13]  Gaelen T. Hess,et al.  Genome-scale measurement of off-target activity using Cas9 toxicity in high-throughput screens , 2017, Nature Communications.

[14]  Y. Yamaguchi-Iwai,et al.  Homologous recombination and non‐homologous end‐joining pathways of DNA double‐strand break repair have overlapping roles in the maintenance of chromosomal integrity in vertebrate cells , 1998, The EMBO journal.

[15]  Gang Bao,et al.  CRISPR/Cas9 systems targeting β-globin and CCR5 genes have substantial off-target activity , 2013, Nucleic acids research.

[16]  Yun Wang,et al.  Tropism and toxicity of adeno-associated viral vector serotypes 1, 2, 5, 6, 7, 8, and 9 in rat neurons and glia in vitro. , 2008, Virology.

[17]  A. Baiker,et al.  Universal real-time PCR for the detection and quantification of adeno-associated virus serotype 2-derived inverted terminal repeat sequences. , 2012, Human gene therapy methods.

[18]  J. Doudna,et al.  A Programmable Dual-RNA–Guided DNA Endonuclease in Adaptive Bacterial Immunity , 2012, Science.

[19]  N. Wong,et al.  Differential Activation of Innate Immune Responses by Adenovirus and Adeno-Associated Virus Vectors , 2002, Journal of Virology.

[20]  B. van Steensel,et al.  Easy quantitative assessment of genome editing by sequence trace decomposition , 2014, Nucleic acids research.

[21]  Junying Yuan,et al.  Selective Inhibition of Eukaryotic Translation Initiation Factor 2α Dephosphorylation Potentiates Fatty Acid-induced Endoplasmic Reticulum Stress and Causes Pancreatic β-Cell Dysfunction and Apoptosis* , 2006, Journal of Biological Chemistry.

[22]  S. Dowdy,et al.  Combining CRISPR/Cas9 and rAAV Templates for Efficient Gene Editing. , 2015, Nucleic acid therapeutics.

[23]  T. Golub,et al.  Genomic Copy Number Dictates a Gene-Independent Cell Response to CRISPR/Cas9 Targeting. , 2016, Cancer discovery.

[24]  Boris Zhivotovsky,et al.  DNA damage-induced apoptosis , 2004, Oncogene.

[25]  W. Merrick,et al.  A new pathway of translational regulation mediated by eukaryotic initiation factor 3 , 2000, The EMBO journal.

[26]  L. Chicaybam,et al.  An Efficient Low Cost Method for Gene Transfer to T Lymphocytes , 2013, PloS one.

[27]  Le Cong,et al.  Multiplex Genome Engineering Using CRISPR/Cas Systems , 2013, Science.

[28]  M. Gale,et al.  Immune signaling by RIG-I-like receptors. , 2011, Immunity.

[29]  D. Russell,et al.  AAV-mediated gene targeting methods for human cells , 2011, Nature Protocols.

[30]  Gang Bao,et al.  Quantifying genome-editing outcomes at endogenous loci with SMRT sequencing. , 2014, Cell reports.

[31]  A. Scharenberg,et al.  In Vivo Outcome of Homology-Directed Repair at the HBB Gene in HSC Using Alternative Donor Template Delivery Methods , 2019, Molecular therapy. Nucleic acids.

[32]  John C. Rose,et al.  Rapidly inducible Cas9 and DSB-ddPCR to probe editing kinetics , 2017, Nature Methods.

[33]  Daniel G. Miller,et al.  Human Gene Targeting by Adeno-Associated Virus Vectors Is Enhanced by DNA Double-Strand Breaks , 2003, Molecular and Cellular Biology.

[34]  Jacob E Corn,et al.  Synthetically modified guide RNA and donor DNA are a versatile platform for CRISPR-Cas9 engineering , 2017, eLife.

[35]  Shondra M Pruett-Miller,et al.  High-frequency genome editing using ssDNA oligonucleotides with zinc-finger nucleases , 2011, Nature Methods.

[36]  Sruthi Mantri,et al.  CRISPR/Cas9 β-globin gene targeting in human haematopoietic stem cells , 2016, Nature.

[37]  Israel Steinfeld,et al.  BMC Bioinformatics BioMed Central , 2008 .

[38]  Luigi Naldini,et al.  Gene editing in human stem cells using zinc finger nucleases and integrase-defective lentiviral vector delivery , 2007, Nature Biotechnology.

[39]  A. Srivastava,et al.  High-Efficiency Transduction of Primary Human Hematopoietic Stem/Progenitor Cells by AAV6 Vectors: Strategies for Overcoming Donor-Variation and Implications in Genome Editing , 2016, Scientific Reports.

[40]  G. Stamatoyannopoulos.  The molecular basis of hemoglobin disease. , 1972, Annual review of genetics.

[41]  Milos D. Radovic,et al.  Real-time monitoring of cytotoxic effects of electroporation on breast and colon cancer cell lines. , 2017, Bioelectrochemistry.

[42]  M. Weitzman,et al.  Efficient Gene Targeting Mediated by Adeno-Associated Virus and DNA Double-Strand Breaks , 2003, Molecular and Cellular Biology.

[43]  E. Lander,et al.  Development and Applications of CRISPR-Cas9 for Genome Engineering , 2014, Cell.

[44]  P. Cohen,et al.  Use of the Pharmacological Inhibitor BX795 to Study the Regulation and Physiological Roles of TBK1 and IκB Kinase ϵ , 2009, Journal of Biological Chemistry.

[45]  C. Van Waes,et al.  Therapeutic Small Molecules Target Inhibitor of Apoptosis Proteins in Cancers with Deregulation of Extrinsic and Intrinsic Cell Death Pathways , 2016, Clinical Cancer Research.

[46]  C. Bennett,et al.  Synthetic CRISPR RNA-Cas9–guided genome editing in human cells , 2015, Proceedings of the National Academy of Sciences.

[47]  J. Doudna,et al.  The new frontier of genome engineering with CRISPR-Cas9 , 2014, Science.

[48]  L. Ivashkiv,et al.  Regulation of type I interferon responses , 2013, Nature Reviews Immunology.

[49]  S. Orkin,et al.  Genetic treatment of a molecular disorder: gene therapy approaches to sickle cell disease. , 2016, Blood.

[50]  L. Platanias,et al.  Role of the Akt pathway in mRNA translation of interferon-stimulated genes , 2008, Proceedings of the National Academy of Sciences.

[51]  Bryan R. G. Williams,et al.  Interferon-inducible antiviral effectors , 2008, Nature Reviews Immunology.

[52]  Israel Steinfeld,et al.  Chemically modified guide RNAs enhance CRISPR-Cas genome editing in human primary cells , 2015, Nature Biotechnology.

[53]  E. Neumann,et al.  Gene transfer into mouse lyoma cells by electroporation in high electric fields. , 1982, The EMBO journal.

[54]  Alterations in the host transcriptome in vitro following Rift Valley fever virus infection , 2017, Scientific Reports.

[55]  B. Byrne,et al.  Recombinant adeno-associated virus purification using novel methods improves infectious titer and yield , 1999, Gene Therapy.

[56]  Castle Raley,et al.  Targeted Gene Addition to a Safe Harbor locus in human CD34+ Hematopoietic Stem Cells for Correction of X-linked Chronic Granulomatous Disease , 2016, Nature Biotechnology.