Efficient Generation and Correction of Mutations in Human iPS Cells Utilizing mRNAs of CRISPR Base Editors and Prime Editors.
| Title: | Efficient Generation and Correction of Mutations in Human iPS Cells Utilizing mRNAs of CRISPR Base Editors and Prime Editors. |
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| Authors: | Sürün D; Medical Systems Biology, Medical Faculty and University Hospital Carl Gustav Carus, TU Dresden, Germany.; Schneider A; Medical Systems Biology, Medical Faculty and University Hospital Carl Gustav Carus, TU Dresden, Germany.; Mircetic J; Medical Systems Biology, Medical Faculty and University Hospital Carl Gustav Carus, TU Dresden, Germany.; Mildred Scheel Early Career Center, National Center for Tumor Diseases Dresden (NCT/UCC), Medical Faculty and University Hospital Carl Gustav Carus, TU Dresden, Germany.; Neumann K; Stem Cell Engineering Facility, Center for Molecular and Cellular Bioengineering (CMCB), TU Dresden, Germany.; Lansing F; Medical Systems Biology, Medical Faculty and University Hospital Carl Gustav Carus, TU Dresden, Germany.; Paszkowski-Rogacz M; Medical Systems Biology, Medical Faculty and University Hospital Carl Gustav Carus, TU Dresden, Germany.; Hänchen V; Department of Pediatrics, Medical Faculty and University Hospital Carl Gustav Carus, TU Dresden, Germany.; Lee-Kirsch MA; Department of Pediatrics, Medical Faculty and University Hospital Carl Gustav Carus, TU Dresden, Germany.; Buchholz F; Medical Systems Biology, Medical Faculty and University Hospital Carl Gustav Carus, TU Dresden, Germany. |
| Source: | Genes [Genes (Basel)] 2020 May 06; Vol. 11 (5). Date of Electronic Publication: 2020 May 06. |
| Publication Type: | Journal Article; Research Support, Non-U.S. Gov't |
| Language: | English |
| Journal Info: | Publisher: MDPI Country of Publication: Switzerland NLM ID: 101551097 Publication Model: Electronic Cited Medium: Internet ISSN: 2073-4425 (Electronic) Linking ISSN: 20734425 NLM ISO Abbreviation: Genes (Basel) Subsets: MEDLINE |
| Imprint Name(s): | Original Publication: Basel : MDPI |
| MeSH Terms: | CRISPR-Cas Systems* ; Mutagenesis* ; Mutation*; Adenine/*chemistry ; Cytosine/*chemistry ; Gene Editing/*methods ; Induced Pluripotent Stem Cells/*metabolism ; RNA, Messenger/*genetics; Autoimmune Diseases of the Nervous System/genetics ; Imidazoles/pharmacology ; Nervous System Malformations/genetics ; Piperazines/pharmacology ; RNA, Guide, CRISPR-Cas Systems/genetics ; RNA, Messenger/metabolism ; SAM Domain and HD Domain-Containing Protein 1/genetics ; Aminohydrolases ; Base Sequence ; CRISPR-Associated Protein 9 ; Cell Line ; Cellular Reprogramming Techniques ; Embryoid Bodies ; Genes, p53 ; HEK293 Cells ; Humans ; Transfection |
| 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 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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| Contributed Indexing: | Keywords: CRISPR/Cas9; base editors; human induced pluripotent stem cells; mRNA; prime editors |
| Substance Nomenclature: | 0 (Imidazoles); 0 (Piperazines); 0 (RNA, Guide, CRISPR-Cas Systems); 0 (RNA, Messenger); 53IA0V845C (nutlin 3); 8J337D1HZY (Cytosine); EC 3.1.- (CRISPR-Associated Protein 9); EC 3.1.5.- (SAM Domain and HD Domain-Containing Protein 1); EC 3.1.5.- (SAMHD1 protein, human); EC 3.5.4.- (Aminohydrolases); EC 3.5.4.2 (adenine deaminase); JAC85A2161 (Adenine) |
| SCR Disease Name: | Aicardi-Goutieres syndrome |
| Entry Date(s): | Date Created: 20200510 Date Completed: 20210326 Latest Revision: 20240104 |
| Update Code: | 20260130 |
| PubMed Central ID: | PMC7288465 |
| DOI: | 10.3390/genes11050511 |
| PMID: | 32384610 |
| Database: | MEDLINE |
Journal Article; Research Support, Non-U.S. Gov't