Transcriptional Response to Genome Editing Machinery in CD34+ HSPCs

Genome editing technology is now being translated to the clinic. However, the cellular effects of genome editing machinery have yet to be fully elucidated. Here, we performed global gene expression microarrays on human CD34+ HSPCs that underwent editing. In addition to probing effects of the entire editing process, we sought to parse out contributions of each component individually, including electroporation, Cas9 (mRNA or protein) with gRNA, and AAV6. Of these, treatment with Cas9 mRNA resulted in the greatest number of differentially-expressed genes and gene ontology (GO) processes. Cas9 mRNA evoked dramatic immune and viral responses as well as global transcriptional down-regulation, particularly of metabolic and cell cycle GOs. Electroporation also induced a large degree of transcriptional change, particularly in immune and apoptotic processes. However, AAV6 evoked no detectable viral or immune response. Among the relatively small number of enriched GOs in Cas9/gRNA ribonucleoprotein (RNP)- and AAV6-treated HSPCs, we observed DNA damage and apoptotic responses. Therefore, we sought to determine if inhibition of apoptosis could reduce loss of viability upon editing. We found that treating cells with irreversible caspase inhibitor Z-VAD-FMK significantly improved cell viability during the editing process in HSPCs.