Functional screening in human HSPCs identifies optimized protein-based enhancers of Homology Directed Repair.

Homology Directed Repair (HDR) enables precise genome editing, but the implementation of HDR-based therapies is hindered by limited efficiency in comparison to methods that exploit alternative DNA repair routes, such as Non-Homologous End Joining (NHEJ). In this study, we develop a functional, pooled screening platform to identify protein-based reagents that improve HDR in human hematopoietic stem and progenitor cells (HSPCs). We leverage this screening platform to explore sequence diversity at the binding interface of the NHEJ inhibitor i53 and its target, 53BP1, identifying optimized variants that enable new intermolecular bonds and robustly increase HDR.

Molecular dynamics of genome editing with CRISPR-Cas9 and rAAV6 virus in human HSPCs to treat sickle cell disease.

gene correction with CRISPR-Cas9 and a recombinant adeno-associated virus serotype 6 (rAAV6) in autologous hematopoietic stem/progenitor cells (HSPCs) to treat sickle cell disease (SCD) has now entered early-phase clinical investigation. To facilitate the progress of CRISPR-Cas9/rAAV6 genome editing technology, we analyzed the molecular changes in key reagents and cellular responses during and after the genome editing procedure in human HSPCs. We demonstrated the high stability of rAAV6 to serve as the donor DNA template.

Development of β-globin gene correction in human hematopoietic stem cells as a potential durable treatment for sickle cell disease.

Sickle cell disease (SCD) is the most common serious monogenic disease with 300,000 births annually worldwide. SCD is an autosomal recessive disease resulting from a single point mutation in codon six of the β-globin gene (). Ex vivo β-globin gene correction in autologous patient-derived hematopoietic stem and progenitor cells (HSPCs) may potentially provide a curative treatment for SCD.