Precise and error-prone CRISPR-directed gene editing activity in human CD34+ cells varies widely among patient samples

Shirin R Modarai , Sambee Kanda , Kevin Bloh , Lynn M Opdenaker , Eric B Kmiec 
Source: Gene Ther
Publication Date: (2020)
Issue: :
Research Area:
Immunotherapy / Hematology
Regenerative medicine
Cells used in publication:
CD34+ cell, human
Species: human
Tissue Origin: blood
4D-Nucleofector™ X-Unit

We varied the sgRNA concentrations from 4–8 µg, Cas9 concentrations from 7.5–15 µg, and ssODNs from 1.35–8.1 µg in 2.0 × 105 CD34+ cells and used program ER-100 on the Lonza 4D nucleofector. The Lonza P3 Primary cell 4DNucleofector X-Kit was used for these experiments. The optimized RNP condition and ssODN used in all the experiments in the manuscript was 8 µg sgRNA to 15 µg Cas9, which is a 2:1 pmol ratio of sgRNA to Cas9. In addition, a 5.4 µg concentration of the 72-mer ssODN was the optimal concentration. Preliminary work showed optimization of nucleofection kit on a variety of CD34+ cell sources (i.e., donors) based on the pmax GFP plasmid uptake analyzed by flow cytometry. From the optimized parameters of the nucleofector and Lonza kit, the optimal transfection efficiency was chosen, and the same protocol was used for subsequent CD34+ targeted samples.


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.