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. 2024 Aug 13;8(15):3941-3945.
doi: 10.1182/bloodadvances.2023012279.

CRISPR/Cas9-mediated gene disruption determines the roles of MITF and CITED2 in human mast cell differentiation

Jiezhen Mo  1 , Fredrik Wermeling  1 , Gunnar Nilsson  1   2 , Joakim S Dahlin  1
Affiliations

CRISPR/Cas9-mediated gene disruption determines the roles of MITF and CITED2 in human mast cell differentiation

Jiezhen Mo et al. Blood Adv. .
No abstract available

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Conflict of interest statement

Conflict-of-interest disclosure: The authors declare no competing financial interests.

Figures

Figure 1.
Figure 1.
MITF is required for human mast cell differentiation. (A) Overview of the CRISPR/Cas9 RNP knockout strategy. (B) Representative flow cytometry plots showing the ablation of CD45 5 days after electroporation. Cells gated on live singlets. (C) The percentage of CD45-deficient cells with increasing doses of Cas9 RNP. Two independent experiments are shown. Analysis of cells by flow cytometry 5 days after electroporation. (D) Representative flow cytometry plots showing the frequency of CD45– and CD45+ mast cells after genetic knockout. (E) The frequency of CD45-deficient mast cells assessed by flow cytometry. Analysis was conducted on day 11 or day 12 after electroporation, with Cas9 RNP dosages of 780 to 960 pmol. Each dot in panel E represents 1 independent experiment. (F) Single-cell RNA-sequencing data visualized using the UMAP embedding. The gene expression level of MITF is plotted in the hematopoietic progenitor landscape. MPPs, neutrophil (Neu) progenitors, erythoid (Ery) progenitors, and MCPs are annotated. (G) The left panel shows a representative flow cytometry analysis plot of in vitro–differentiated CRISPR/Cas9-modified CD34+ progenitors 5 days after electroporation with MITF sgRNA. The right panels show representative flow cytometry analysis plots of the cells 12 days after electroporation. Cells gated on live singlets. (H) Example sequencing results of the sorted live cells after electroporation with MITF sgRNA1. Data generated by the Inference of CRISPR Edits (ICE) software based on the Sanger sequencing results of the polymerase chain reaction amplicon. (I) Gene editing efficiency shown as percentage of insertions-deletions (InDels) of MITF sgRNA1 and sgRNA2 in live cells sorted 5 days after electroporation. (J) Fraction of c-Kithi FcεRI+ mast cells normalized to NC (negative control) sgRNA in live singlets 12 days after electroporation. Two-tailed 1 sample t test, hypothetical value is 1. The NC sgRNA condition refers to nontargeting sgRNA. The cells were cultured without interleukin-3 (IL-3) during the second week, apart from in 1 of the independent experiments in which IL-3 was present. (K) Editing efficiency calculated by percentage of InDel (Ki) and percentage of frameshift (Kii) in the mast cell population and the non-mast cell population within individual samples. Cells were analyzed 12 days after electroporation. The editing efficiency was analyzed in 2 independent experiments using MITF sgRNA1 and 3 independent experiments using MITF sgRNA2. (L) Flow cytometry plots showing the 2 outputs of sorted FcεRI– progenitors and MCPs 12 days after electroporation with MITF sgRNA2, followed by differentiation into mast cells. Cells gated on live singlets. DAPI, 4′,6-diamidino-2-phenylindole; FSC, forward scatter; MACS, magnetic-activated cell sorting; MC, mast cell; NC, negative control; PAM, protospacer adjacent motif.
Figure 2.
Figure 2.
CITED2 is dispensable for human mast cell differentiation, survival, and proliferation. (A) Single-cell RNA-sequencing data visualized using the UMAP embedding. The gene expression level of CITED2 is plotted in the hematopoietic progenitor landscape. (B) Editing efficiency shown as percentage of InDels and percentage of frameshift mutations for CITED2 sgRNA1 of live cells sorted 5 days after electroporation. Five independent experiments were performed. (C) Flow cytometry analysis of mast cells 12 days after electroporation with CITED2 sgRNA1 in CD34+ cells. Representative of 5 independent experiments. Live single cells are shown. (D) The fraction of mast cells 11 to 12 days after electroporation. The results show the frequency of c-Kithi FcεRI+ mast cells (among live singlets) in the CITED2 sgRNA1 sample normalized to the corresponding NC sgRNA sample. The cell culture medium was supplemented with IL-3 during the second week in 2 of the 5 experiments. Two-tailed 1 sample t test, hypothetical value is 1. The experiments highlighted with red dots were analyzed with regards to editing efficiency in panel E. (E) Editing efficiency of percentage of InDels and percentage of frameshift for CITED2 sgRNA1 of c-Kithi FcεRI+ mast cells and non-mast cells within individual samples sorted 12 days after electroporation. Two independent experiments were performed. (F) Representative flow cytometry plots showing the frequency of mast cells derived from sorted FcεRI– progenitors and MCPs 12 days after electroporation with CITED2 sgRNA1. (G) The fraction of mast cells after electroporation and culture of sorted FcεRI– progenitors and MCPs. The results show the frequency of c-Kithi FcεRI+ mast cells (among live singlets) in the CITED2 sgRNA1 sample normalized to the corresponding NC sgRNA sample. The plots show 3 independent experiments in which CITED2 was knocked out in sorted FcεRI– progenitors and 2 independent experiments in which CITED2 was knocked out in sorted MCPs. Two-tailed 1 sample t test, hypothetical value is 1. (H) Editing efficiency shown as percentage of InDel and percentage of frameshift of CITED2 sgRNA1 in c-Kithi FcεRI+ mast cells derived from FcεRI- progenitors (upper panel) and MCPs (lower panel). (I) Flow cytometry plots and histograms showing the survival and the proliferation of LAD2 and HMC-1 electroporated with NC sgRNA and CITED2 sgRNA1, respectively. Representative of 2 independent experiments. Control (gray) refers to all live cells analyzed after staining with CellTrace Far Red and incubating the cells in the fridge. Live single cells are shown. (J) Editing efficiency shown as percentage of InDel and percentage of frameshift in LAD2 and HMC-1 in 2 independent experiments. The NC sgRNA condition refers to nontargeting sgRNA. ns, nonsignificant.
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References

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