Page 87 - Haematologica March 2020

ELANE knockout restores granulopoiesis in congenital neutropenia
mutant HL60 clones revealed a typical impairment of granulocytic differentiation capacities in both mutant cell lines, as assessed by the significantly lower proportion of cells expressing CD11b granulocytic differentiation mark- er in p.P139L and p.C151Y mutant cell lines compared to the wild-type (P<0.0001 and P=0.00043, respectively) on day 5 of differentiation (Figure 1A-B and Online Supplementary Figure S1A-C). These findings are consistent with ELANE associated neutropenia patients phenotype.
As a proof-of-principle experiment, we have used RNA interference (RNAi) technology to knock down the expression of the ELANE gene in these cell lines. Thereby, we investigated the biological effects of inhibition of mutant NE on the granulocytic differentiation. Indeed, transfection of commercially available siRNA against the exon 4 of ELANE, completely knocked down the expres- sion of NE in all cell lines. Production of CD11b positive cells was significantly restored in both mutant cell lines (P=0.00041 for p.P139L and P=0.00048 for p.C151Y), but
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not in wild-type cells (Figure 1A-B and Online Supplementary Figure S1A-C).
Design and validation of sgRNA targeting ELANE
We further generated guide RNA (gRNA) specifically targeting exon 2 of ELANE by annealing CRISPR-RNA (crRNA) with trans-activating crRNA (tracrRNA). gRNA was incubated with recombinant Cas9 protein to generate CRISPR/Cas9-gRNA RNP complexes. The gRNA targeting exon 2 of ELANE (cut site: chr19[+852.969:-852.969], Figure 2A) was selected to introduce stop-codon muta- tions and to induce nonsense-mediated mRNA decay (NMD) of ELANE mRNA, which is caused by stop-codon or frameshift mutations at the beginning of ELANE mRNA. Based on our experimental analysis, the selected gRNA has high on-target activity with low off-target score (data not shown). To evaluate inhibition of NE expression by CRISPR/Cas9 RNP mediated targeting of exon 2 of ELANE, we generated an ELANE KO myeloid cell line
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Figure 2. Establishment of CRISPR/Cas9 RNP-mediated ELANE knockout in THP-1 cells. (A) ELANE was targeted using single guide ribonucleoprotein (RNP)-mediated (highlighted in pink), which creates a double-strand break at NM_001972.2 exon 2, 161 bp after ATG; NP_001963.1, p.F54. Schematic presentation of the cut site by sgRNA. (B-D) Myeloid cell line THP-1 was electroporated with ELANE-specific CRISPR/Cas9 RNP. Single cell clones of ELANE KO THP-1 cells were generated, as described in the Material and Methods. Representative Sanger sequencing image (B), the bar chart of the TIDE assay (C) and representative Western blotting (WB) images of neutrophil elastase (NE) and α-tubulin protein expression (D) in single cell-derived ELANE KO THP-1 cells are depicted.
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