Genome Editing Using Cas9-gRNA Ribonucleoprotein in Human Pluripotent Stem Cells for Disease Modeling

2021 ◽  
Author(s):  
Noelia Benetó ◽  
Daniel Grinberg ◽  
Lluïsa Vilageliu ◽  
Isaac Canals
Keyword(s):  
Stem Cells ◽  
Genome Editing ◽  
Pluripotent Stem Cells ◽  
Disease Modeling ◽  
Human Pluripotent Stem Cells

scholarly journals Selection-free, high frequency genome editing by homologous recombination of human pluripotent stem cells using Cas9 RNP and AAV6

2018 ◽  
Author(s):  
Renata M. Martin ◽  
Kazuya Ikeda ◽  
Nobuko Uchida ◽  
Kyle Cromer ◽  
Toshi Nishimura ◽  
...  
Keyword(s):  
Stem Cells ◽  
Genome Editing ◽  
Pluripotent Stem Cells ◽  
Disease Modeling ◽  
Human Pluripotent Stem Cells ◽  
Guide Rna ◽  
Large Gene ◽  
Marker Selection ◽  
Cas9 Protein

AbstractCombination of genome editing and human pluripotent stem cells (hPSCs) offers a platform for in vitro disease modeling, drug discovery and personalized stem cell therapeutics. However, incorporation of large modifications using CRISPR/Cas9-based genome editing in hPSCs typically requires the use of selection markers due to low editing efficiencies. Here we report a novel editing technology in hPSCs using Cas9 protein complexed with chemically modified single guide RNA (sgRNA) and recombinant AAV6 (rAAV6) vectors for donor delivery without marker selection. With these components, we demonstrate targeted integration of a 2.2 kb DNA expression cassette in hPSCs at frequencies up to 94% and 67% at the HBB and MYD88 loci, respectively. We used this protocol to correct the homozygous sickle cell disease (SCD) mutation in an iPSC line derived from a SCD patient with a frequency of 63%. This Cas9/AAV6 system allows for both the integration of large gene cassettes and the creation of single nucleotide changes in hPSCs at high frequencies, eliminating the need for multiple editing steps and marker selection, thus increasing the potential of editing human pluripotent cells for both research and translational applications.

scholarly journals Selection-free non-viral method revealed highly efficient CRISPR-Cas9 genome editing of human pluripotent stem cells guided by cellular autophagy

2021 ◽  
Author(s):  
Michelle Surma ◽  
Kavitha Anbarasu ◽  
Arupratan Das
Keyword(s):  
Stem Cells ◽  
Genome Editing ◽  
Pluripotent Stem Cells ◽  
Disease Modeling ◽  
Human Pluripotent Stem Cells ◽  
Cell Replacement Therapy ◽  
Gene Expressions ◽  
Guide Rna ◽  
Transfection Method ◽  
Cellular Autophagy

CRISPR-Cas9 mediated genome editing of human pluripotent stem cells (hPSCs) provides strong avenues for human disease modeling, drug discovery and cell replacement therapy. Genome editing of hPSCs is an extremely inefficient process and requires complex gene delivery and selection methods to improve edit efficiency which are not ideal for clinical applications. Here, we have shown a selection free simple lipofectamine based transfection method where a single plasmid encoding guide RNA (gRNA) and Cas9 selectively transfected hPSCs at the colony edges. Upon dissection and sequencing, the edge cells showed more than 30% edit frequency compared to the reported 3% rate under no selections. Increased cellular health of the edge cells as revealed by reduced autophagy gene-expressions is critical for such transfection pattern. Edge specific transfection was inhibited by blocking lysosomal activity which is essential for autophagy. Hence, our method provides robust scarless genome-editing of hPSCs which is ideal for translational research.

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.

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

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.

scholarly journals Scarless Genome Editing of Human Pluripotent Stem Cells via Transient Puromycin Selection

2018 ◽  
Vol 10 (2) ◽  
pp. 642-654 ◽  
Author(s):  
Benjamin Steyer ◽  
Qian Bu ◽  
Evan Cory ◽  
Keer Jiang ◽  
Stella Duong ◽  
...  
Keyword(s):  
Stem Cells ◽  
Genome Editing ◽  
Pluripotent Stem Cells ◽  
Human Pluripotent Stem Cells ◽  
Puromycin Selection

scholarly journals CRISPR/Cas9-based Targeted Genome Editing for the Development of Monogenic Diseases Models with Human Pluripotent Stem Cells

2018 ◽  
Vol 45 (1) ◽  
pp. e50 ◽  
Author(s):  
Navin Gupta ◽  
Koichiro Susa ◽  
Yoko Yoda ◽  
Joseph V. Bonventre ◽  
M. Todd Valerius ◽  
...  
Keyword(s):  
Stem Cells ◽  
Genome Editing ◽  
Pluripotent Stem Cells ◽  
Human Pluripotent Stem Cells ◽  
Monogenic Diseases
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