Page 88 - Haematologica March 2020

M. Nasri et al.
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Figure 3. Generation of ELANE KO CN iPSC clones. (A) Scheme of the ELANE-specific CRISPR/Cas9-gRNA ribonu- cleoprotein electroporation of induced pluripotent stem cells (iPSC) and generation of ELANE KO iPSC clones. Generation of single ELANE knockout iPSC clones was made by seeding single iPSC, subsequent picking of each clone and transferring them into 96 well plates. Screening of each iPSC clone was done by Cas9 in vitro digestion and Sanger sequencing. (B) Scheme of CRISPR/Cas9 introduced modifications in the ELANE gene in iPSC clones of congenital neu- tropenia (CN) patients. Red inserts show positions of indels in ELANE mRNA (NM_001972.2) and numbers refer to bp position after ATG. (C) Scheme of the EB-based hematopoietic/neutrophilic differentiation of iPSC.
tions: 274 bp del/ins in CN p.C151Y ELANE KO iPSC, and 17 bp del in CN p.A57V ELANE KO iPSC (Figure 3B). The editing efficiency of healthy ctrl iPSC was 97 % (Online Supplementary Figure S4A), therefore, we used the total population of gene-edited healthy control (ctrl) iPSC for further analysis. We did not detect any off-target activity of the gRNA for the selected cDNA sites in all studied iPSC, as assessed using Sanger sequencing (Online Supplementary Figure S4B and Tables S1, S2).
Applying a slightly modified in vitro embryoid body (EB)-based iPSC differentiation method that allows gener- ation of hematopoietic cells and mature myeloid cells for approximately 30 days,22,23 we found an increase in the percentage of CD15+CD16+CD45+ granulocytes in ELANE KO CN-iPSC cell culture, as compared to CN-iPSC. The generation of granulocytes from ELANE KO CN-iPSC was comparable to iPSC generated from a healthy donor (Figure 3C, 4A, and Online Supplementary Figure S5A). Generation of immature hematopoietic cells (CD34+KDR+, CD34+CD43+, CD45–CD235+CD41a+ and CD45+CD34+ cells) and CD45+CD33+ myeloid progenitor cells in ELANE KO CN- and CN-iPSC lines were similar or increased, in comparison to corresponding MOCK treated iPSC lines (Online Supplementary Figure S6A).
A CFU assay was performed with ELANE-/- iPSC-derived CD34+ cells from CN patients and showed elevated levels of CFU-G but reduced CFU-M colony numbers, as com- pared to CD34+ cells derived from MOCK treated CN iPSC clones (Figure 4C). These data suggest that ELANE knockout restores granulocytic differentiation in CN.
We did not observe any significant defects in in vitro granulocytic differentiation of ELANE KO iPSC generated from a healthy donor, as compared to MOCK treated cells (Figure 4A-C, Online Supplementary Figures S5 and S6).
THP-1, which has high basal expression levels of ELANE and NE. The efficiency of ELANE knockout in the total population of edited THP-1 cells was 77 %, as assessed by Sanger sequencing and tracking of indels by decomposi- tion (TIDE) analysis (data not shown). The pure ELANE KO THP-1 cell clone has compound heterozygosity of 14 and 17 bp deletions on each allele (Figure 2B-C). The NE expression was completely absent in the pure ELANE KO THP-1 cell clone, as determined by Western blotting (WB) using anti-NE antibody against the C-terminus of NE pro- tein (Figure 2D, Online Supplementary Figure S2A-B). These data suggest that sgRNA targeting ELANE that we designed led to a complete loss of NE protein.
Restoration of the in vitro granulocytic differentiation in ELANE-CN iPSC clones after ELANE knockout
We generated iPSC from peripheral blood mononuclear cells (PB MNC) of two ELANE-CN patients, harboring ELANE mutations p.C151Y or p.A57V (CN p.C151Y iPSC and CN p.A57V iPSC, respectively). Additionally, iPSC of one healthy control (healthy ctrl iPSC) were evaluated. All three iPSC lines expressed elevated mRNA and protein levels of pluripotent stem cell-specific factors, displayed alkaline phosphatase activity and expression of pluripo- tent embryonic stem cell surface markers (Online Supplementary Figure 3A-C).
Next, we used electroporation of iPSC clones with ELANE-specific CRISPR/Cas9-sgRNA RNP to generate pure ELANE KO CN iPSC clones. For this, electroporated iPSC were seeded on a geltrex coated culture dish and sin- gle-cell derived iPSC clones were isolated transferred to geltrex coated 96 well-plates for the subsequent selection of ELANE knockout clones (Figure 3A). Confirmed ELANE knockout iPSC clones have followed ELANE modifica-
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