Abstract
Heterozygous variants in POLR2A, encoding the largest subunit of RNA polymerase II, cause severe neurodevelopmental and multisystem abnormalities in humans. Using CRISPR/Cas9 we generated the human iPSC line KICRi002A-5 with a heterozygous truncating 4 bp insertion in exon 5 of the POLR2A gene. Analysis using qRT-PCR confirmed reduced POLR2A mRNA in KICRi002A-5 vs. the isogenic WT iPSC line. The edited iPSC line expressed pluripotency markers and exhibited differentiation capacity into the three germ layers. Assessment of genomic integrity revealed a normal karyotype and OFF-target editing was excluded. The iPSC line KICRi002A-5 provides a useful resource to study mechanisms underlying developmental defects caused by RBP1 insufficiency.
Highlights
Heterozygous variants in POLR2A, encoding the largest subunit of RNA polymerase II, cause severe neurodevelopmental and multisystem abnormalities in humans
Resource utility The generated iPSC line UUIGPi012A-5 provides a useful resource to model and study mechanisms underlying the various human develop mental defects caused by heterozygous variants in the POLR2A gene
We further evaluated the differentiation capacity of the KICRi002A25 iPSC line by embryoid body formation and subsequent non-directed differentiation
Summary
Evidence of the reprogramming transgene loss (including genomic copy if applicable). Feeder-free maintenance on LN521 coated dishes in Essential®8-medium Targeted gene knock-down using Cas Neurodevelopmental disorder with dysmorphisms, intellectual disability, seizures and behavioral abnormalities (OMIM #618603) RNA-polymerase II, subunit A (POLR2A)/ chr17: 7,484,365–7,514,615 CRISPR-Cas. Name of transgene Eukaryotic selective agent resistance (including inducible/gene expressing cell-specific) Inducible/constitutive system details Date archived/stock date Cell line repository/bank. Eu/user/cellline/edit/KICRi002-A-5 The study was approved by the Regional Ethics Committee Uppsala 2016 (Registration: Dnr 2016/209). December 2020 https://hpscreg. eu/user/cellline/edit/KICRi002-A-5 The study was approved by the Regional Ethics Committee Uppsala 2016 (Registration: Dnr 2016/209). pSpCas (BB)-2A-GFP (PX458), Addgene plasmid #48138
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