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

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 CRISPRdirected 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.

Homology directed correction, a new pathway model for point mutation repair catalyzed by CRISPR-Cas

“…While successful single base mutation correction was observed, unintended on-site changes were also found at an alarmingly high rate. We have also confirmed other laboratories initial observations that unintended genetic changes were prevalent after CRISPR-Cas gene editing in CD34 + cells 4 , 8 , 9 .…”

“…The most prominent challenge centers on the diversity of genetic outcomes that have resulted from sophisticated attempts to correct a single base mutation using CRISPR-based gene editing. Our work 2 – 4 and others 5 – 7 have revealed that while successful point mutation repair can be achieved, it is almost always associated with a significant amount of imprecise editing, particularly at the targeted sites where the CRISPR-Cas complex was designed to act, i.e., on-site mutagenesis .…”

Improvements of nuclease and nickase gene modification techniques for the treatment of genetic diseases

“…This was done for example to knockout PD-1 and CD52 for the different types of cancer by electroporating Cas9 and a sgRNA to edit the cells ex vivo . The results indicated that the clinical applications of CRISPR-Cas9 gene-edited T-cells are generally safe and feasible ( Lu et al, 2020 ; Frangoul et al, 2021 ; Modarai et al, 2021 ). The disruption of erythroid enhancer of the BCL11A gene by CRISPR-Cas9 for the treatment of ß-thalassemia has been observed with serious adverse events.…”

Genome editing tools to modify immune cells

“…Można użyć przenośni, że komórka, w której zachodzi taka edycja, zamiast planowanej dzięki HDR i donorowemu DNA poprawy, wpada niejako z deszczu pod rynnę, po włączeniu się NHEJ. Tego typu wady metody CRISPR prowadzą do tego, że systemy edycji powodują dużo niezamierzonych zmian, pomimo wprowadzenia donorowego DNA [17][18][19]. Technika TALEN również korzysta z HDR oraz NHEJ i jest w związku z tym obciążona podobnymi mankamentami (ryc.…”