利用CRISPR碱基编辑器和引导编辑器的mRNA在人诱导多能干细胞中高效产生和纠正突变
Efficient Generation and Correction of Mutations in Human iPS Cells Utilizing mRNAs of CRISPR Base Editors and Prime Editors.
作者信息
Sürün Duran, Schneider Aksana, Mircetic Jovan, Neumann Katrin, Lansing Felix, Paszkowski-Rogacz Maciej, Hänchen Vanessa, Lee-Kirsch Min Ae, Buchholz Frank
机构信息
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.
出版信息
Genes (Basel). 2020 May 6;11(5):511. doi: 10.3390/genes11050511.
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 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.
与CRISPR/Cas9核酸酶不同,CRISPR碱基编辑器(BE)和引导编辑器(PE)能够在基因组序列中进行预定义的核苷酸交换,而不会产生DNA双链断裂。在此,我们将BE和PE mRNA与化学合成的sgRNA和pegRNA结合使用,以高效编辑人类诱导多能干细胞(iPSC)。虽然我们无法使用CRISPR/Cas9核酸酶方法纠正患者来源的iPSC中的致病突变,但我们利用腺嘌呤BE将该突变高效地纠正回野生型。我们还使用腺嘌呤和胞嘧啶BE将九种不同的癌症相关突变引入人类iPSC,效率高达90%,生成了一组细胞系,以在同基因背景下研究这些突变的生物学特性。最后,我们率先在人类iPSC中使用引导编辑,为这种重要的细胞类型开启了对BE无法处理的核苷酸进行精确修饰以及进行多个核苷酸交换的可能性。这些方法消除了从人类供体获取疾病特异性iPSC的必要性,并允许在同基因遗传背景下比较不同的致病突变。