Gene Therapy as the New Frontier for Sickle Cell Disease.

Sickle Cell Disease (SCD) is one of the most common monogenic disorders caused by a point mutation in the ß-globin gene. This mutation results in polymerization of hemoglobin (Hb) under reduced oxygenation conditions, causing rigid sickle-shaped RBCs and hemolytic anemia. This clearly defined fundamental molecular mechanism makes SCD a prototypical target for precision therapy. Both the mutant ß-globin protein and its downstream pathophysiology are pharmacological targets of intensive research. SCD also is a disease well-suited for biological interventions like gene therapy. Recent advances in hematopoietic stem cell (HSC) transplantation and gene therapy platforms, like Lentiviral vectors and gene editing strategies, expand the potentially curative options for patients with SCD. This review discusses the recent advances in precision therapy for SCD and the preclinical and clinical advances in autologous HSC gene therapy for SCD.

Rational Selection of CRISPR-Cas9 Guide RNAs for Homology-Directed Genome Editing.

Homology-directed repair (HDR) of a DNA break allows copying of genetic material from an exogenous DNA template and is frequently exploited in CRISPR-Cas9 genome editing. However, HDR is in competition with other DNA repair pathways, including non-homologous end joining (NHEJ) and microhomology-mediated end joining (MMEJ), and the efficiency of HDR outcomes is not predictable. Consequently, to optimize HDR editing, panels of CRISPR-Cas9 guide RNAs (gRNAs) and matched homology templates must be evaluated. We report here that CRISPR-Cas9 indel signatures can instead be used to identify gRNAs that maximize HDR outcomes. Specifically, we show that the frequency of deletions resulting from MMEJ repair, characterized as deletions greater than or equal to 3 bp, better predicts HDR frequency than consideration of total indel frequency. We further demonstrate that tools that predict gRNA indel signatures can be repurposed to identify gRNAs to promote HDR. Finally, by comparing indels generated by S. aureus and S. pyogenes Cas9 targeted to the same site, we add to the growing body of data that the targeted DNA sequence is a major factor governing genome editing outcomes.