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

Jovan Mircetic,Vanessa Hänchen,Felix Lansing,Maciej Paszkowski-Rogacz,Min Ae Lee-Kirsch,Frank Buchholz,Aksana Schneider,Duran Sürün,Katrin Neumann

doi:10.3390/genes11050511

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.

Highlights

Since the discovery of human induced pluripotent stem cells that can be generated by reprogramming somatic cells, hiPSCs-based disease modeling has been established as a versatile tool [1]. hiPSCs can be propagated indefinitely in vitro under growth conditions that maintain pluripotency and can be differentiated into many different somatic cell types under appropriate culture conditions [2,3,4,5]
The patient iPSCs were nucleofected with Cas9-NLS protein, sgRNA, and a 120-nt-long ssDNA oligonucleotide with 59-nucleotide homology upstream, and 60-nucleotide homology downstream of the mutation, respectively
HiPS cells are critical for studying rare monogenic diseases, because of limited access to affected tissues, such as the central nervous system (CNS) or muscle

Summary

Introduction

Since the discovery of human induced pluripotent stem cells (hiPSC) that can be generated by reprogramming somatic cells, hiPSCs-based disease modeling has been established as a versatile tool [1]. hiPSCs can be propagated indefinitely in vitro under growth conditions that maintain pluripotency and can be differentiated into many different somatic cell types under appropriate culture conditions [2,3,4,5]. Patient-specific hiPSC lines are generated through reprogramming of somatic cells isolated from a donor. These cells can be applied for studying molecular and cellular disease mechanisms, as well as for drug screening [6,7,8] and for the development of possible. A major challenge of using patient-derived hiPSCs for modeling human diseases is their genetic diversity and differences in epigenetic memory [11,12]. This makes it difficult to compare data obtained from mutant iPSCs generated from a patient with other hiPSC lines generated from healthy individuals. Elimination of genetic background effects would be beneficial for loss-of-function and gain-of-function studies or drug screening

Methods

Results

Conclusion