564. The Cytotoxic Effect of RNA-Guided Endonuclease Cas9 on Human Hematopoietic Stem and Progenitor Cells (HSPCs)
Authors: Kyung-Rok Yu, Marcus A.F. Corat, Jean-Yves Metais, Cynthia E. Dunbar
Journal: Molecular Therapy (2016)

The CRISPR (clustered regulatory interspaced short palindromic repeats) - CRISpR-associated protein 9 (Cas9) system (CRISPR/Cas9) has recently emerged as an efficient and powerful approach widely used for targeted genetic engineering. RNA-guided endonuclease Cas9 in combination with a single guide RNA (sgRNA) targets the sequence-specific DNA binding domains and generates double strand breaks (DSBs), which are repaired through non-homologous end joining (NHEJ) or homologous recombination (HR). Although CRISPR/CAS9 system very efficiently disrupts target locations in the genome of multiple model organisms, editing in some primary cell types, especially hematopoietic stem and progenitor cells (HSPCs) has been more challenging, with lower efficiencies particularly in functional engrafting HSPC. However, the exact mechanisms have not been elucidated. To optimize editing via CRISPR/Cas9 in human HSPCs, we generated several different lentiviral vectors (Figure 1); a negative control GFP only non-editing vector (LeGO-GFP), U6 promoter-AAVS1 targeting sgRNA-EF1a promoter-GFP (AAVS1-GFP), U6-EFS promoter-Cas9-EF1a-GFP (Cas9-GFP), U6-AAVS1 targeting sgRNA-EFS-Cas9-EF1a-GFP (AAVS1-Cas9-GFP), U6-EFS-human codon-optimized Cas9-EF1a-GFP (hCas9-GFP), and U6-AAVS1 targeting sgRNA-EFS-human codon-optimized Cas9-EF1a-GFP (AAVS1-hCas9-GFP). These lentiviral vectors simultaneously deliver sgRNA targeting a "safe harbor" genome location (AAVS1 in these experiments), Cas9 and a fluorescent marker, however, we replaced P2A with EF1a promoter between Cas9 and a fluorescent marker, because we found dim or none fluorescent marker expression using P2A co-expression systems. Lentivirus-transduced HSPCs were analyzed by flow cytometry based on their GFP expression in a time-dependent manner (3, 6, 9, 12 days). All vectors resulted in a gradual decrease in the percentage of GFP+ cells over time, however, standard Cas9 containing vectors (Cas9-GFP and AAVS1-Cas9-GFP) showed most significant decreases (Figure 2). Vectors containing the human codon-optimized version of Cas9 (hCas9-GFP, AAVS1-hCas9-GFP) showed intermediate decreases, and vectors without Cas9, whether or not they expressed sgRNA (LeGO, AAVS1-GFP), showed the least decrease in GFP+ cells, with 30% of the cells still expressing GFP at 12 days following transduction. These results led us to investigate the cytotoxicity of Cas9 on HSPCs, using Vivid and Annexin V staining. Both standard Cas9 and human codon-optimized Cas9 expressing HSPCs showed strong Annexin V positivity and a gradual increase in Vivid+ cells, explaining the loss of GFP+ cells with Cas9-expressing vectors. On the other hand, HSPCs transduced with the non-Cas9-expressing vectors remained Vivid and Annexin V low until through 12 days. Our findings suggest that sustained expression of Cas9 in human HSPCs, for instance via an integrating vector, has significant toxicity, and that an alternative Cas9 delivery method for HSPCs is required. We have tried integrase-defective lentivirus (IDLV) and mRNA transfection to deliver Cas9 into HSPCs, and data will be presented. This approach will facilitate the use of CRISPR/Cas9 system to engineer genes of clinical significance in HSPCs with a therapeutically meaningful efficacy.