Stem Cells, Genome Editing, and the Path to Translational Medicine

A mechanistic understanding of the genetic basis of complex diseases such as diabetes mellitus remain elusive due in large part to the activity of genetic disease modifiers that impact the penetrance and/or presentation of disease phenotypes. In the face of such complexity, rare forms of diabetes that result from single-gene mutations (monogenic diabetes) can be used to model the contribution of individual genetic factors to pancreatic β-cell dysfunction and the breakdown of glucose homeostasis. Here we review the contribution of protein coding and non-protein coding genetic disease modifiers to the pathogenesis of diabetes subtypes, as well as how recent technological advances in the generation, differentiation, and genome editing of human pluripotent stem cells (hPSC) enable the development of cell-based disease models. Finally, we describe a disease modifier discovery platform that utilizes these technologies to identify novel genetic modifiers using induced pluripotent stem cells (iPSC) derived from patients with monogenic diabetes caused by heterozygous mutations.

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Systematic gene tagging using CRISPR/Cas9 in human stem cells to illuminate cell organization

10.1101/123042 ◽

2017 ◽

Cited By ~ 1

Author(s):

Brock Roberts ◽

Amanda Haupt ◽

Andrew Tucker ◽

Tanya Grancharova ◽

Joy Arakaki ◽

...

Keyword(s):

Stem Cells ◽

Quality Control ◽

Genome Editing ◽

Cell Biology ◽

Cellular Structures ◽

Human Stem Cells ◽

Alpha Tubulin ◽

Junction Protein ◽

Endogenous Proteins ◽

Clonal Lines

AbstractWe present a CRISPR/Cas9 genome editing strategy to systematically tag endogenous proteins with fluorescent tags in human inducible pluripotent stem cells. To date we have generated multiple human iPSC lines with GFP tags for 10 proteins representing key cellular structures. The tagged proteins include alpha tubulin, beta actin, desmoplakin, fibrillarin, lamin B1, non-muscle myosin heavy chain IIB, paxillin, Sec61 beta, tight junction protein ZO1, and Tom20. Our genome editing methodology using Cas9 ribonuclear protein electroporation and fluorescence-based enrichment of edited cells resulted in <0.1-24% HDR across all experiments. Clones were generated from each edited population and screened for precise editing. ∼25% of the clones contained precise mono-allelic edits at the targeted locus. Furthermore, 92% (36/39) of expanded clonal lines satisfied key quality control criteria including genomic stability, appropriate expression and localization of the tagged protein, and pluripotency. Final clonal lines corresponding to each of the 10 cellular structures are now available to the research community. The data described here, including our editing protocol, genetic screening, quality control assays, and imaging observations, can serve as an initial resource for genome editing in cell biology and stem cell research.

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Induced pluripotent stem cells and genome editing technology as therapeutic strategies for Duchenne muscular dystrophy

Journal of thee Medical Sciences (Berkala Ilmu Kedokteran) ◽

10.19106/jmedsci005301202109 ◽

2021 ◽

Vol 53 (1) ◽

Author(s):

Irwan Saputra Batubara

Keyword(s):

Stem Cells ◽

Duchenne Muscular Dystrophy ◽

Muscular Dystrophy ◽

Induced Pluripotent Stem Cells ◽

Genome Editing ◽

Pluripotent Stem Cells ◽

Therapeutic Strategies ◽

Induced Pluripotent

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Efficient genome editing in primary cells and in vivo using viral-derived “Nanoblades” loaded with Cas9/sgRNA ribonucleoproteins

10.1101/202010 ◽

2017 ◽

Cited By ~ 1

Author(s):

Philippe E. Mangeot ◽

Valérie Risson ◽

Floriane Fusil ◽

Aline Marnef ◽

Emilie Laurent ◽

...

Keyword(s):

Stem Cells ◽

Genome Editing ◽

Target Genes ◽

Bone Marrow Cells ◽

Leukemia Virus ◽

Target Cells ◽

Mouse Bone Marrow ◽

Primary Cells ◽

Hematopoietic Stem

AbstractProgrammable nucleases have enabled rapid and accessible genome engineering in eukaryotic cells and living organisms. However, their delivery into target cells can be technically challenging when working with primary cells or in vivo. Using engineered murine leukemia virus-like particles loaded with Cas9/sgRNA ribonucleoproteins (“Nanoblades”), we were able to induce efficient genome-editing in cell lines and primary cells including human induced pluripotent stem cells, human hematopoietic stem cells and mouse bone-marrow cells. Transgene-free Nanoblades were also capable of in vivo genome-editing in mouse embryos and in the liver of injected mice. Nanoblades can be complexed with donor DNA for “all-in-one” homology-directed repair or programmed with modified Cas9 variants to mediate transcriptional up-regulation of target genes. Nanoblades preparation process is simple, relatively inexpensive and can be easily implemented in any laboratory equipped for cellular biology.

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Targeting repair pathways with small molecules increases precise genome editing in pluripotent stem cells

Nature Communications ◽

10.1038/s41467-018-04609-7 ◽

2018 ◽

Vol 9 (1) ◽

Cited By ~ 48

Author(s):

Stephan Riesenberg ◽

Tomislav Maricic

Keyword(s):

Stem Cells ◽

Small Molecules ◽

Genome Editing ◽

Pluripotent Stem Cells ◽

Repair Pathways

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Stem Cells Applications in Therapeutics and Site-Specific Genome Editing Through CRISPR Cas9 System

Journal of Stem Cell Research and Transplantation ◽

10.26420/jstemcellrestransplant.2021.1033 ◽

2021 ◽

Vol 8 (1) ◽

Author(s):

Javed M ◽

◽

Khan A ◽

Mukheed M ◽

◽

...

Keyword(s):

Stem Cells ◽

Genome Editing ◽

Pluripotent Stem Cells ◽

Disease Modeling ◽

Cell Types ◽

Multipotent Stem Cells ◽

Site Specific ◽

Regenerative Medicines ◽

Induced Pluripotent ◽

Adult Cells

Stem cells ae immature cells that have ability to differentiate into all specific and mature cells in body. The two main characteristics of stem cells are selfrenewable and ability to differentiate into all mature, functional and adult cells types. There are the two major classes a) pluripotent stem cells which have potential to differentiate in all adult cell and b) multipotent stem cells which have capacity to differentiate into many adult cells but not in all cell types. Due to the self-renewable ability stem cells are use in therapeutics, tissue regeneration, disease modeling and regenerative medicines and to treat cardiovascular diseases, neural disorders such as Parkinson’s disease and most importantly to treat carcinomas. The human induced pluripotent stem cells provide a great platform to study and treatment of human diseases because these are able to differentiate into many functional and specialized adult cells of body. The genome editing tools such as CRISPR Cas9 system and TALENs are used to generate multiple DNA variants in hPSCs by inducing site specific mutations, frame shift mutation and deletion. In present days CRISPR Cas9 is more efficient and frequent method for genome editing which is derived from bacterial cell.

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