@article{52c4e683f55846f584001cfe190e2f62,
title = "Efficient generation and correction of mutations in human iPS cells utilizing mRNAs of CRISPR base editors and prime editors",
abstract = "In contrast to CRISPR/Cas9 nucleases, CRISPR base editors (BE) and prime editors (PE) enable predefined nucleotide exchanges in genomic sequences without generating DNA double strand breaks. Here, we employed BE and PE mRNAs in conjunction with chemically synthesized sgRNAs and pegRNAs for efficient editing of human induced pluripotent stem cells (iPSC). Whereas we were unable to correct a diseasecausing mutation in patient derived iPSCs using a CRISPR/Cas9 nuclease approach, we corrected the mutation back to wild type with high efficiency utilizing an adenine BE. We also used adenine and cytosine BEs to introduce nine different cancer associated TP53 mutations into human iPSCs with up to 90% efficiency, generating a panel of cell lines to investigate the biology of these mutations in an isogenic background. Finally, we pioneered the use of prime editing in human iPSCs, opening this important cell type for the precise modification of nucleotides not addressable by BEs and to multiple nucleotide exchanges. These approaches eliminate the necessity of deriving disease specific iPSCs from human donors and allows the comparison of different diseasecausing mutations in isogenic genetic backgrounds.",
keywords = "Base editors, CRISPR/Cas9, Human induced pluripotent stem cells, MRNA, Prime editors",
author = "Duran S{\"u}r{\"u}n and Aksana Schneider and Jovan Mircetic and Katrin Neumann and Felix Lansing and Maciej Paszkowskirogacz and Vanessa H{\"a}nchen and Leekirsch, {Min Ae} and Frank Buchholz",
note = "Funding Information: Funding: This research was funded by: F‐B, European Union (ERC 742133, H2020 UPGRADE 825825); BMBF GO‐Bio (031B0633); J‐M, by the Deutsche Krebshilfe/MSNZ P2 Dresden; ML‐K, by the Deutsche Forschungsgemeinschaft (German Research Foundation), grant 369799452/404459235; and V‐H, measurements by the European Social Fund and is co‐financed by taxes based on the budget decided by members of the Saxon State Parliament. Funding Information: This research was funded by: FB, European Union (ERC 742133, H2020 UPGRADE 825825); BMBF GOBio (031B0633); JM, by the Deutsche Krebshilfe/MSNZ P2 Dresden; MLK, by the Deutsche Forschungsgemeinschaft (German Research Foundation), grant 369799452/404459235; and VH, measurements by the European Social Fund and is cofinanced by taxes based on the budget decided by members of the Saxon State Parliament. We thank Sebastian Diecke from the Core Facility Stem Cells at the Berlin Institute of Health (BIH) for providing the AAVS1PuroCAGeGFP targeted hiPS cells (hPSCreg name: BIHi001A2) and the Institute of Human Genetics, University Clinics Jena, Germany for analyzing metaphase spreads. Furthermore, we thank the DRESDENconcept Genome Center at the CMCB, TU Dresden for performing the NGS experiments and the Flow Cytometry Core Facility and Stem Cell Engineering Core Facility of the CMCB Technology Platform at TU Dresden for their excellent support. Publisher Copyright: {\textcopyright} 2020 by the authors. Licensee MDPI, Basel, Switzerland.",
year = "2020",
month = may,
doi = "10.3390/genes11050511",
language = "English",
volume = "11",
journal = "Genes",
issn = "2073-4425",
publisher = "Multidisciplinary Digital Publishing Institute (MDPI)",
number = "5",
}