My lab has developed a rapid and efficacious Cas9-based approach to introduce programmed sequence changes to human cells with ease. This work takes advantage of our discovery that Cas9 is extremely long-lived on its target DNA, yet releases a flap of single stranded DNA after cleavage. By using Cas9 ribonucleoprotein complexes (RNPs) and programming single stranded oligonucleotide donors (ssODNs) to anneal to the released flap, we have been able to achieve up to 60% allele replacement in a wide variety of cell types. Building upon this initial discovery, we have further demonstrated highly effective replacement of SNPs in human hematopoietic stem cells (HSCs). Targeting the causative mutation of sickle cell anemia in HSCs, we can swap sequences at up to ~40% of alleles, with concordant benefit for the production of wild type hemoglobin over sickle hemoglobin.

Our approach could be promising for the treatment of other hematopoietic disorders, including Fanconi Anemia. However, recent work unpublished work in my lab has surprisingly revealed that interstrand crosslink repair may underlie Cas9-mediated sequence replacement using ssODNs. In fact, our data indicates that knocking down one of several FA complement groups completely abolishes editing when using ssODNs. We hypothesize that this is due to how cells recognize and process ssODN templates during Cas9-induced HDR and has major implications for the potential treatment of FA by genome editing, since it could suggest that certain editing modalities will be far more efficacious than others.

We propose to define editing modalities that will be efficacious in the cells of FA patients. We will determine whether FA mutations are truly inimical to ssODN-mediated HDR and determine alternate editing strategies in cell lines. We apply these alternate strategies in immortalized human progenitor cells as well as adult mobilized hematopoietic stem cells.