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. 2019 Jun 27;105(3):598–609. doi: 10.3324/haematol.2019.221804

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Generation of ELANE KO CN iPSC clones. (A) Scheme of the ELANE-specific CRISPR/Cas9-gRNA ribonucleoprotein 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 neutropenia (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.