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 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.

Trial registration: ClinicalTrials.gov NCT03745287.

Fig. 1. Overview of experimental design and CRISPR/Cas9 RNPs used for this study.

a Schematic of the cleavage sites, G5 and G10, for the human beta globin gene and seed sequences for the crRNAs. Also shown is the 72-mer ssODN sequence that contains the single base pair exchange to create a mismatch to produce the sickle cell sequence. The oligonucleotide used in these experiments is 72 bases in length, bearing phosphorothioate-modified linkages at the three terminal bases. b This panel represents the overall workflow of how each donor’s CD34+ cells were treated and analyzed for this study. A total of 12 different donor’s CD34+ cells were obtained and analyzed. Of note, the use of the word donor and patient is interchangeable for the samples in this study, as donor 1 is also patient 1 or P1 for short. c This panel represents a more detailed schematic of the workflow done for each sample of CD34+ cells that were analyzed in this study.

Fig. 2. Nucleofection of CD34 + cells using different CRISPR/Cas9 RNPs.

a Cells targeted with 8 µg G5 sgRNA/15 µg Cas9 RNP and 5.4 µg of 72-mer ssODN and analyzed with TIDER software for percent indels and HDR. This concentration of CRISPR/Cas9 and ssODN was found to be the optimal concentrations, in our hands, for targeting the CD34+ cells with significant HDR. The graph represents data from two experiments targeting the same source of CD34+ cells with CRISPR/Cas9 with the G5 sgRNA (#1 and #2 are independent experiments), and two experiments targeting the same CD34+ cells with CRIPSR/Cas9 with the G10 sgRNA (#1 and #2 for each experimental set). b, c Representative TIDER plots of indel and HDR activity catalyzed by the G5 sgRNA and G10 sgRNA CRISPR/Cas9 (experimental set #1 for each CRISPR/Cas9). The majority of the indels are 5 bp deletion or less when using CRISPR/Cas9 close to the mutation site, as can be seen by the black box (b), while there are larger sized indels when using the CRISPR/Cas9 that cuts further away from the HBB mutation site. The data presented in this figure are from patient 8 CD34+ cells that were targeted using both the G5 and G10 CRISPR/Cas9 RNPs and 72-mer ssODN, in duplicate reactions.

Fig. 3. The wild-type sequencing of each patient’s CD34+ cells prior to nucleofection with CRISPR/Cas9 RNPs.

a Schematic of the cleavage sites, G5 and G10, for the beta globin gene and where they each cut in relation to the sickle cell mutation. In addition, the HDR 72-mer oligo sequence is shown for point of reference to where the seed sequences cut on the beta globin gene. b Cells from each patient were collected as a reference for what the individual’s DNA sequence was prior to CRISPR/Cas9 RNP targeting. Every patient had similar nucleotide sequences near the beta globin site, expect for a single nucleotide polymorphism (SNP) found in patients 2, 5, and 10, denoted by a Y, and that could be a cytosine or thymine that location.

Fig. 4. Nucleofection of CD34+ cells using different CRISPR/Cas9 RNPs.

a The table represents the total indel pattern and value output from TIDER software analysis for each of the 12 patient samples targeted with both G5 and G10 CRISPR/Cas9 RNPs. b Cells targeted with 8 µg G5 sgRNA/15 µg Cas9 with 5.4 µg of 72-mer ssODN and analyzed with TIDER for percent indels and HDR. The pie charts represent the total indel pattern that resulted from each patient’s experimental targeting. The 0 bp percentages represent the combined value of the total amount of sequence that did not equal any insertions or deletions (gray area of pie charts), as well as any HDR reactions (yellow area of pie charts). c Cells targeted with 8 µg G10 sgRNA/15 µg Cas9 with 5.4 µg of 72-mer ssODN and analyzed with TIDER for percent indels and HDR. The pie charts represent the total indel pattern that resulted from each patient’s experimental targeting. The 0 bp percentages represent the combined value of the total amount of sequence that did not equal any insertions or deletions (gray area of pie charts), as well as any HDR reactions (yellow area of pie charts). All values shown in the pie chart are indels that were scored 1% or higher on TIDER software analysis. The total percentages of indels are shown fully in panel A. All nucleofection reactions were performed with the same number of CD34+ cells, same nucleofection parameters and same time of analysis. For each panel, an N = 12 (12 different individuals) was analyzed. All pie charts with red asterisks means a significant HDR reaction as occurred, and a colored key is present in each figure panel to correspond to the indel sizes.

Fig. 5. p53 expression in CRISPR-targeted CD34+ cells.

Approximately 100,000 CD34+ cells that were targeted with either the G5 CRISPR/Cas 9 RNP + 72-mer ssODN, or G10 CRISPR/Cas9 RNP+ 72-mer ssODN were used for p53 analysis. All cells were collected 72 h post nucleofection and analyzed for p53 expression. The samples were fixed and stained for p53 and the gating of the histograms for all samples were set against an appropriate IgG antibody control. a The gray shaded histograms represent the mock control samples which are the cells treated with the appropriate CRISPR RNP and ssODN, but no nucleofection. It is important to note that mock nucleofector samples were also run as another control and there was no difference seen between any of the controls. The red lined histograms represent the targeted cells with the appropriate CRISPR RNP and ssODN. The samples were analyzed on the FITC channel on the BD LSRFortessa. b The graph represents the average percent positive p53 expression from eight of the patient samples that were analyzed in this study. The remaining four that were not analyzed, either did not tolerate the CRISPR/Cas9 RNP targeting or not enough cells survived post-nucleofection to performs both sequencing analysis and p53 expression. The graph shows that the average values, of all eight samples, between the control cells and the targeted cells. There seems to be no significant change in p53 expression when the CRISPR/Cas9 RNP targeting was employed. The error bars represent the S.E.M.