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

被引:97
作者
Sueruen, Duran [1 ,2 ]
Schneider, Aksana [1 ,2 ]
Mircetic, Jovan [1 ,2 ,3 ]
Neumann, Katrin [4 ]
Lansing, Felix [1 ,2 ]
Paszkowski-Rogacz, Maciej [1 ,2 ]
Haenchen, Vanessa [2 ,5 ]
Lee-Kirsch, Min Ae [2 ,5 ]
Buchholz, Frank [1 ,2 ]
机构
[1] Tech Univ Dresden, Med Fac, Med Syst Biol, D-01307 Dresden, Germany
[2] Tech Univ Dresden, Univ Hosp Carl Gustav Carus, D-01307 Dresden, Germany
[3] Tech Univ Dresden, Mildred Scheel Early Career Ctr, Natl Ctr Tumor Dis Dresden NCT UCC, Med Fac, D-01307 Dresden, Germany
[4] Tech Univ Dresden, Ctr Mol & Cellular Bioengn CMCB, Stem Cell Engn Facil, D-01307 Dresden, Germany
[5] Tech Univ Dresden, Dept Pediat, Med Fac, D-01307 Dresden, Germany
基金
欧盟地平线“2020”;
关键词
CRISPR; Cas9; base editors; prime editors; human induced pluripotent stem cells; mRNA; GENOMIC DNA; DIFFERENTIATION; REPORTER; LOCUS;
D O I
10.3390/genes11050511
中图分类号
Q3 [遗传学];
学科分类号
071007 ; 090102 ;
摘要
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.
引用
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页数:19
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