scholarly journals “Ancestralization” of human pluripotent stem cells by multiplexed precise genome editing

2018 ◽  
Author(s):  
Stephan Riesenberg ◽  
Tomislav Maricic ◽  
Svante Pääbo
Keyword(s):  
Stem Cells ◽  
Genome Editing ◽  
Pluripotent Stem Cells ◽  
Nucleotide Substitution ◽  
Human Stem Cells ◽  
Animal Cells ◽  
Isolated Cells ◽  
Dependent Protein Kinase ◽  
Drastic Increase ◽  
Dependent Protein

We show that inactivation of the DNA-dependent protein kinase catalytic subunit (DNA-PKcs) results in a drastic increase in efficiency of precise genome editing with CRISPR enzymes in human stem cells, allowing up to 79% of chromosomes to carry an intended nucleotide substitution when a single genomic site is targeted. When three different genes are simultaneously targeted, 12% of the isolated cells carry the targeted amino acid-changing substitutions in homozygous forms. These substitutions represent the first step towards resurrecting the proteome ancestral to Neandertals and modern humans. DNA-PKcs inactivation will greatly facilitate multiplexed precise genome editing in animal cells.

scholarly journals Telomere length set point regulation in human pluripotent stem cells critically depends on the shelterin protein TPP1

2020 ◽  
Vol 31 (23) ◽  
pp. 2583-2596
Author(s):  
John M. Boyle ◽  
Kelsey M. Hennick ◽  
Samuel G. Regalado ◽  
Jacob M. Vogan ◽  
Xiaozhu Zhang ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Telomere Length ◽  
Pluripotent Stem Cells ◽  
Human Stem Cells ◽  
Set Point ◽  
Hypomorphic Allele ◽  
Point Control ◽  
Set Point Control ◽  
Stem Cell Lines

To better understand telomere length set point control in human stem cells, we generated knockout stem cell lines for TPP1 and contrasted their phenotypes with those of homozygous TPP1 L104A mutant stem cells. This comparison reveals that TPP1 L104A is not a hypomorphic allele but formally establishes TPP1 L104 as a dissociation of function mutant.

scholarly journals An iCRISPR Platform for Rapid, Multiplexable, and Inducible Genome Editing in Human Pluripotent Stem Cells

2014 ◽  
Vol 15 (2) ◽  
pp. 215-226 ◽  
Author(s):  
Federico González ◽  
Zengrong Zhu ◽  
Zhong-Dong Shi ◽  
Katherine Lelli ◽  
Nipun Verma ◽  
...  
Keyword(s):  
Stem Cells ◽  
Genome Editing ◽  
Pluripotent Stem Cells ◽  
Human Pluripotent Stem Cells

scholarly journals Genome Editing Human Pluripotent Stem Cells to Model β-Cell Disease and Unmask Novel Genetic Modifiers

2021 ◽  
Vol 12 ◽  
Author(s):  
Matthew N. George ◽  
Karla F. Leavens ◽  
Paul Gadue
Keyword(s):  
Stem Cells ◽  
Genome Editing ◽  
Pluripotent Stem Cells ◽  
Genetic Disease ◽  
Single Gene ◽  
Monogenic Diabetes ◽  
Human Pluripotent Stem Cells ◽  
Genetic Modifiers ◽  
Β Cell ◽  
Protein Coding

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.

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

2017 ◽  
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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