4.6 Article

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

期刊

GENES
卷 11, 期 5, 页码 -

出版社

MDPI
DOI: 10.3390/genes11050511

关键词

CRISPR; Cas9; base editors; prime editors; human induced pluripotent stem cells; mRNA

资金

  1. European Union [ERC 742133, H2020 UPGRADE 825825]
  2. BMBF GO-Bio [031B0633]
  3. Deutsche Krebshilfe/MSNZ P2 Dresden
  4. Deutsche Forschungsgemeinschaft (German Research Foundation) [369799452/404459235]
  5. European Social Fund

向作者/读者索取更多资源

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.

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