43. CRISPR/Cas9 and rAAV6-Mediated Targeted Integration at the CCR5 Locus in Hematopoietic Stem and Progenitor Cells
Authors: Rasmus O. Bak, Daniel P. Dever, Nivedita Saxena, Joab Camarena, Andreas Reinisch, Alec B. Wilkens, Carmencita E. Nicolas, Ayal Hendel, Ravindra Majeti, Kenneth I. Weinberg, Matthew H. Porteus
Journal: Molecular Therapy (2016)

CRISPR/Cas9-mediated genome editing relies on guide RNAs to direct site-specific DNA cleavage mediated by the Cas9 endonuclease. In this study, we identified a highly potent single guide RNA (sgRNA) targeting exon 3 of CCR5. This sgRNA was chemically synthesized with three modified nucleotides at each terminus with 2′O-Methyl and phosphorothioate modifications, and electroporated into cells either with Cas9 mRNA or complexed with Cas9 protein (RNP). Using Tracking of Indels by DEcomposition (TIDE) to quantify insertions and deletions (INDELs), we observe up to 80% INDELs in CD34+ hematopoietic stem and progenitor cells (HSPCs). To achieve targeted integration by homology-directed repair (HDR), we produced rAAV6 vectors carrying a GFP expression cassette flanked by CCR5 homology arms. Electroporation with Cas9 RNP followed by rAAV6 transduction led to targeted integration in up to 30% of the cells. Interestingly, we observed that cells with targeted integration expressed GFP at fluorescence intensities more than 10-fold higher than from episomal AAV vectors. This allowed us to sort targeted cells as early as four days after nucleofection and transduction. Upon fluorescence-activated cell sorting and culture of this population, >99% of cells remained GFP+ 20 days post sort. In a methylcellulose-based colony-forming unit (CFU) assay, we identified multipotent and lineage-committed progenitor cells in this population, and PCR of gDNA extracted from colonies confirmed targeted integration at CCR5 in at least 98% of cells. Phenotypic characterization of this targeted population confirmed the presence of CD34+ CD38− CD90+ CD45RA− cells, indicating genome editing of hematopoietic stem cells. We transplanted edited cells into immunodeficient NSG mice and analyzed the bone marrow 8 weeks post-transplant. In mice transplanted with cells that were not enriched for targeted integration, we found 0.1-1.9% GFP+ cells among the engrafted human cells. This was a significant decline compared to the 12-13% GFP+ cells in the input cell population following culture, which is a phenomenon consistent with findings reported by other groups using different nuclease platforms. In contrast, when transplanting cells enriched for targeted integration, we found that 75% of the engrafted human cells were GFP+, confirming the presence of cells with long-term engraftment potential in the enriched population. In conclusion, we have found that the combination of CRISPR/Cas9 and rAAV6 is an effective platform for HDR-mediated targeted integration of a transgene into the CCR5 locus. Furthermore, the GFP MFI shift observed when episomal rAAV6 vectors are integrated into the chromosome following HDR enables early isolation of a population highly enriched for targeted integration at this locus. Since CCR5 is considered a 'safe harbor' for targeted insertion of a gene, this approach might find general use in therapeutic genome editing. Additionally, since CCR5 is an important co-receptor during HIV infection, our findings may be used to generate immune cells resistant to HIV infection.