Base Editing Mediated Generation of Point Mutations Into Human Pluripotent Stem Cells for Modeling Disease

Human pluripotent stem cells (hPSC) are known to acquire chromosomal abnormalities, which range from point mutations to large copy number changes, including full chromosome aneuploidy. These aberrations have a wide-ranging influence on the state of cells, in both the undifferentiated and differentiated state. Currently, very little is known on how these abnormalities will impact the clinical translation of hPSC, and particularly their potential to prime cells for oncogenic transformation. A further complication is that many of these abnormalities exist in a mosaic state in culture, which complicates their detection with conventional karyotyping methods. In this review we discuss current knowledge on how these aberrations influence the cell state and how this may impact the future of research and the cells’ clinical potential.

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Base Editing of Human Pluripotent Stem Cells for modeling Long QT syndrome

10.21203/rs.3.rs-954667/v1 ◽

2021 ◽

Author(s):

Fujian Wu ◽

Tianwei Guo ◽

Lixiang Sun ◽

Furong Li ◽

Xiaofei Yang

Keyword(s):

Stem Cells ◽

Long Qt Syndrome ◽

Pluripotent Stem Cells ◽

Disease Modeling ◽

Human Pluripotent Stem Cells ◽

Electrode Arrays ◽

Long Qt ◽

Base Editing ◽

Qt Syndrome ◽

Disease Specific

Abstract Human pluripotent stem cells (hPSCs) have great potential for disease modeling, drug discovery, and regenerative medicine as they can differentiate into many different functional cell types via directed differentiation. However, the application of disease modeling is limited due to a time-consuming and labor-intensive process of introducing known pathogenic mutations into hPSCs. Base editing is a newly developed technology that enables the facile introduction of point mutations into specific loci within the genome of living cells without unwanted genome injured. We describe an optimized stepwise protocol to introduce disease-specific mutations of long QT syndrome (LQTs) into hPSCs. We highlight technical issues, especially those associated with introducing a point mutation to obtain isogenic hPSCs without inserting any resistance cassette and reproducible cardiomyocyte differentiation. Based on the protocol, we succeeded in getting hPSCs carrying LQTs pathogenic mutation with excellent efficiency (21.7 % of heterozygous clones, 9.1 % of homozygous clones) in less than 20 days. In addition, we also provide protocols to analyze electrophysiological of hPSC-derived cardiomyocytes using multi-electrode arrays. This protocol is also applicable to introduce other disease-specific mutations into hPSCs.

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Base Editing of Human Pluripotent Stem Cells for Modeling Long QT Syndrome

Stem Cell Reviews and Reports ◽

10.1007/s12015-021-10324-6 ◽

2022 ◽

Author(s):

Fujian Wu ◽

Tianwei Guo ◽

Lixiang Sun ◽

Furong Li ◽

Xiaofei Yang

Keyword(s):

Stem Cells ◽

Long Qt Syndrome ◽

Pluripotent Stem Cells ◽

Disease Modeling ◽

Human Pluripotent Stem Cells ◽

Electrode Arrays ◽

Long Qt ◽

Base Editing ◽

Qt Syndrome ◽

Disease Specific

AbstractHuman pluripotent stem cells (hPSCs) have great potential for disease modeling, drug discovery, and regenerative medicine as they can differentiate into many different functional cell types via directed differentiation. However, the application of disease modeling is limited due to a time-consuming and labor-intensive process of introducing known pathogenic mutations into hPSCs. Base editing is a newly developed technology that enables the facile introduction of point mutations into specific loci within the genome of living cells without unwanted genome injured. We describe an optimized stepwise protocol to introduce disease-specific mutations of long QT syndrome (LQTs) into hPSCs. We highlight technical issues, especially those associated with introducing a point mutation to obtain isogenic hPSCs without inserting any resistance cassette and reproducible cardiomyocyte differentiation. Based on the protocol, we succeeded in getting hPSCs carrying LQTs pathogenic mutation with excellent efficiency (31.7% of heterozygous clones, 9.1% of homozygous clones) in less than 20 days. In addition, we also provide protocols to analyze electrophysiological of hPSC-derived cardiomyocytes using multi-electrode arrays. This protocol is also applicable to introduce other disease-specific mutations into hPSCs. Graphical abstract

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Efficient GNE myopathy disease modeling with mutation specific phenotypes in human pluripotent stem cells by base editors

10.1101/2020.11.25.397711 ◽

2020 ◽

Author(s):

Ju-Chan Park ◽

Jumee Kim ◽

Hyun-Ki Jang ◽

Seung-Yeon Lee ◽

Keun-Tae Kim ◽

...

Keyword(s):

Stem Cells ◽

Pluripotent Stem Cells ◽

Disease Modeling ◽

Point Mutations ◽

Human Pluripotent Stem Cells ◽

Drug Candidate ◽

Disease Models ◽

Target Cells ◽

Gne Myopathy ◽

Low Efficiency

AbstractIsogenic pairs of cell lines derived from human pluripotent stem cells (hPSCs) enable the precise assessment of mutation-specific phenotypes through differentiation to target cells, as this method of disease modeling excludes the bias of genetic variation. However, the extremely low efficiency of precise gene editing based on homology-directed repair (HDR) with Cas9 in hPSCs remains a technical hurdle for this approach. Herein, we took advantage of currently available base editors (BEs) in hPSCs to epitomize the isogenic disease model from hPSCs with a pathophysiological indicator. Using this method, we established 14 hPSCs that harbor point mutations on the GNE gene, including four different mutations found in GNE myopathy patients. Because BEs activated p53 to a lesser degree than Cas9, we observed a higher editing efficiency with BEs. Four different mutations in the epimerase or kinase domains of GNE revealed mutation-specific hyposialylation, which was closely correlated to pathological clinical phenotypes. These mutation-specific hyposialylation patterns were evident in GNE protein structure modeling. Furthermore, treatment with a drug candidate currently under clinical trials showed a mutation-specific drug response in GNE myopathy disease models. These data suggest that isogenic disease models from hPSCs using BEs could serve as a useful tool for mimicking the pathophysiology of GNE myopathy and for predicting drug responses.

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Cytosine and adenosine base editing in human pluripotent stem cells using transient reporters for editing enrichment

Nature Protocols ◽

10.1038/s41596-021-00552-y ◽

2021 ◽

Author(s):

Stefan J. Tekel ◽

Nicholas Brookhouser ◽

Kylie Standage-Beier ◽

Xiao Wang ◽

David A. Brafman

Keyword(s):

Stem Cells ◽

Pluripotent Stem Cells ◽

Human Pluripotent Stem Cells ◽

Base Editing

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Pipeline for the Generation and Characterization of Transgenic Human Pluripotent Stem Cells Using the CRISPR/Cas9 Technology

Cells ◽

10.3390/cells9051312 ◽

2020 ◽

Vol 9 (5) ◽

pp. 1312 ◽

Cited By ~ 2

Author(s):

Joffrey Mianné ◽

Chloé Bourguignon ◽

Chloé Nguyen Van ◽

Mathieu Fieldès ◽

Amel Nasri ◽

...

Keyword(s):

Stem Cells ◽

Cell Lines ◽

Pluripotent Stem Cells ◽

Genome Engineering ◽

Disease Modeling ◽

Mutant Cell ◽

Point Mutations ◽

Human Pluripotent Stem Cells ◽

Wide Range

Recent advances in genome engineering based on the CRISPR/Cas9 technology have revolutionized our ability to manipulate genomic DNA. Its use in human pluripotent stem cells (hPSCs) has allowed a wide range of mutant cell lines to be obtained at an unprecedented rate. The combination of these two groundbreaking technologies has tremendous potential, from disease modeling to stem cell-based therapies. However, the generation, screening and molecular characterization of these cell lines remain a cumbersome and multi-step endeavor. Here, we propose a pipeline of strategies to efficiently generate, sub-clone, and characterize CRISPR/Cas9-edited hPSC lines in the function of the introduced mutation (indels, point mutations, insertion of large constructs, deletions).

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