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Article

Efficient Generation and Correction of Mutations in Human iPS Cells Utilizing mRNAs of CRISPR Base Editors and Prime Editors

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Medical Systems Biology, Medical Faculty and University Hospital Carl Gustav Carus, TU Dresden, 01307 Dresden, Germany
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Mildred Scheel Early Career Center, National Center for Tumor Diseases Dresden (NCT/UCC), Medical Faculty and University Hospital Carl Gustav Carus, TU Dresden, 01307 Dresden, Germany
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Stem Cell Engineering Facility, Center for Molecular and Cellular Bioengineering (CMCB), TU Dresden, 01307 Dresden, Germany
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Department of Pediatrics, Medical Faculty and University Hospital Carl Gustav Carus, TU Dresden, 01307 Dresden, Germany
*
Author to whom correspondence should be addressed.
Genes 2020, 11(5), 511; https://doi.org/10.3390/genes11050511
Received: 10 April 2020 / Revised: 30 April 2020 / Accepted: 4 May 2020 / Published: 6 May 2020
(This article belongs to the Special Issue Genes at Ten)
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 disease-causing 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 disease-causing mutations in isogenic genetic backgrounds. View Full-Text
Keywords: CRISPR/Cas9; base editors; prime editors; human induced pluripotent stem cells; mRNA CRISPR/Cas9; base editors; prime editors; human induced pluripotent stem cells; mRNA
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MDPI and ACS Style

Sürün, D.; Schneider, A.; Mircetic, J.; Neumann, K.; Lansing, F.; Paszkowski-Rogacz, M.; Hänchen, V.; Lee-Kirsch, M.A.; Buchholz, F. Efficient Generation and Correction of Mutations in Human iPS Cells Utilizing mRNAs of CRISPR Base Editors and Prime Editors. Genes 2020, 11, 511. https://doi.org/10.3390/genes11050511

AMA Style

Sürün D, Schneider A, Mircetic J, Neumann K, Lansing F, Paszkowski-Rogacz M, Hänchen V, Lee-Kirsch MA, Buchholz F. Efficient Generation and Correction of Mutations in Human iPS Cells Utilizing mRNAs of CRISPR Base Editors and Prime Editors. Genes. 2020; 11(5):511. https://doi.org/10.3390/genes11050511

Chicago/Turabian Style

Sürün, Duran, Aksana Schneider, Jovan Mircetic, Katrin Neumann, Felix Lansing, Maciej Paszkowski-Rogacz, Vanessa Hänchen, Min Ae Lee-Kirsch, and Frank Buchholz. 2020. "Efficient Generation and Correction of Mutations in Human iPS Cells Utilizing mRNAs of CRISPR Base Editors and Prime Editors" Genes 11, no. 5: 511. https://doi.org/10.3390/genes11050511

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