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. 2018 Oct 3;26(10):2431-2442.
doi: 10.1016/j.ymthe.2018.06.002. Epub 2018 Jul 11.

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

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Free PMC article

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

M Kyle Cromer et al. Mol Ther. .
Free PMC article

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.

Keywords: RNA expression analysis; genome editing; hematopoietic stem cells.

Figures

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Figure 1
Figure 1
Unsupervised Clustering Reveals Cas9 mRNA-Induced Expression Signature (A) Indel rates and HR rates per treatment group are plotted as determined by TIDER analysis and TOPO cloning, respectively. Each column represents four HSPC CD34+ donors. All columns are means and error bars depict SEM. (B) At 6 hr and 24 hr post-treatment, two principal components (PC1 and PC2) accounting for majority of variation are plotted for each treatment for each donor, grouped by treatment. (C) At 24 hr post-treatment, two principal components (PC1 and PC2) accounting for majority of variation are plotted for each treatment for each donor, grouped by donor. Signals from all probes on the microarray were used as input for unsupervised clustering by PCA. Each data point represents the average PC1 and PC2 values of two technical replicates for an individual CD34+ donor for each treatment.
Figure 2
Figure 2
Volcano Plots Depict Differential Expression Signatures of All Treatments (A) MLogP value is plotted against log2 of the ratio of the probe expression difference between specific treatment and mock electroporation treatment at 6 hr and 24 hr. (B) At 24 hr post-treatment, electroporation and AAV-only samples were compared to non-electroporated control sample. The horizontal line indicates Bonferroni-corrected significance threshold of MLogP ≥ 7. The total numbers of significantly down- and upregulated probes is shown in the top left and top right of each plot, respectively.
Figure 3
Figure 3
Cas9 mRNA and Electroporation Treatments Elicit Greatest Cellular Response (A) At 24 hr post-treatment, percentage of enriched GO processes (FDR q value ≤ 0.01) normalized by total number in each category across all treatments are plotted for electroporated and AAV treatments compared to non-electroporated control. (B) At 24 hr post-treatment, normalized percentage of enriched GO processes compared to electroporated control. GO processes differentially up- or downregulated are shown in blue and orange, respectively. All differentially expressed genes as well as enriched GO processes are provided in Tables S1 and S2, respectively.
Figure 4
Figure 4
Gene-Level Resolution of the Most Heavily Enriched GO Processes At 24 hr post-treatment, the genes contributing to GO enrichment were compiled and the top 10 most significantly differentially expressed genes in each GO category are shown (based on median MLogP values per GO category across all treatments). All differentially expressed genes as well as enriched GO processes are provided in Tables S1 and S2, respectively.

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