Abstract
Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and their associated CRISPR-associated nucleases (Cas) are among the most promising technologies for the treatment of hemoglobinopathies including Sickle Cell Disease (SCD). We are only beginning to identify the molecular variables that influence the specificity and the efficiency of CRISPR- directed gene editing, including the position of the cleavage site and the inherent variability among patient samples selected for CRISPR-directed gene editing. Here, we target the beta globin gene in human CD34+ cells to assess the impact of these two variables and find that both contribute to the global diversity of genetic outcomes. Our study demonstrates a unique genetic profile of indels that is generated based on where along the beta globin gene attempts are made to correct the SCD single base mutation. Interestingly, even within the same patient sample, the location of where along the beta globin gene the DNA is cut, HDR activity varies widely. Our data establish a framework upon which realistic protocols inform strategies for gene editing for SCD overcoming the practical hurdles that often impede clinical success.
Highlights
Sickle Cell Disease (SCD) is a severe hereditary form of anemia caused by a single nucleotide mutation altering the sixth codon of the beta globin gene (HBB) [1]
Schematic of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) ribonucleoprotein and overall workflow of this study Recent reports suggest that the RNP catalyzes high levels of gene editing activity at the beta globin locus [7, 8, 18] and
Our results indicate that the genomic target in most of the cells has been altered as demonstrated by the presence of insertions and/or deletions (Fig. 2a)
Summary
Sickle Cell Disease (SCD) is a severe hereditary form of anemia caused by a single nucleotide mutation altering the sixth codon of the beta globin gene (HBB) [1]. There is renewed optimism for the development of gene therapy because novel genome editing technologies have emerged [2]. CRISPR-directed gene editing is being used to either disable suppressive elements upstream from the start of the fetal globin gene or to correct the well-known mutation in the defective beta globin gene [1]. A Phase 1/2 clinical trial for sickle cell disease examining safety and efficacy of autologous CRISPR-Cas edited CD34+ cells (ClinicalTrials.gov; NCT03745287) is in the recruitment
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