Toward a CRISPR Picture: Use of CRISPR/Cas9 to Model Diseases in Human Stem Cells In Vitro

Freiermuth, Jamie L.; Powell‐Castilla, Ian J.; Gallicano, G. Ian

doi:10.1002/jcb.26162

Cited by 13 publications

(10 citation statements)

References 31 publications

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“…In most of the cases, pluripotent stem cells have been used for CRISPR/Cas9 genome editing because of higher efficiency of HDR . The model presented here is, to our knowledge, the first designed by genome editing on LSCs for ocular disease in general and of ARK in particular.…”

Section: Resultsmentioning

confidence: 99%

Modeling of Aniridia-Related Keratopathy by CRISPR/Cas9 Genome Editing of Human Limbal Epithelial Cells and Rescue by Recombinant PAX6 Protein

et al. 2018

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Heterozygous PAX6 gene mutations leading to haploinsufficiency are the main cause of congenital aniridia, a rare and progressive panocular disease characterized by reduced visual acuity. Up to 90% of patients suffer from aniridia-related keratopathy (ARK), caused by a combination of factors including limbal epithelial stem cell (LSC) deficiency, impaired healing response and abnormal differentiation of the corneal epithelium. It usually begins in the first decade of life, resulting in recurrent corneal erosions, sub-epithelial fibrosis, and corneal opacification. Unfortunately, there are currently no efficient treatments available for these patients and no in vitro model for this pathology. We used CRISPR/Cas9 technology to introduce into the PAX6 gene of LSCs a heterozygous nonsense mutation found in ARK patients. Nine clones carrying a p.E109X mutation on one allele were obtained with no off-target mutations. Compared with the parental LSCs, heterozygous mutant LSCs displayed reduced expression of PAX6 and marked slow-down of cell proliferation, migration and detachment. Moreover, addition to the culture medium of recombinant PAX6 protein fused to a cell penetrating peptide was able to activate the endogenous PAX6 gene and to rescue phenotypic defects of mutant LSCs, suggesting that administration of such recombinant PAX6 protein could be a promising therapeutic approach for aniridia-related keratopathy. More generally, our results demonstrate that introduction of disease mutations into LSCs by CRISPR/Cas9 genome editing allows the creation of relevant cellular models of ocular disease that should greatly facilitate screening of novel therapeutic approaches. Stem Cells 2018;36:1421-1429.

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Section: Resultsmentioning

confidence: 99%

Modeling of Aniridia-Related Keratopathy by CRISPR/Cas9 Genome Editing of Human Limbal Epithelial Cells and Rescue by Recombinant PAX6 Protein

et al. 2018

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“…[48] Regenerative medicine and expanded stem cell research shows the potential for the development of in vitro applications to resolve problems encountered in the treatment of many diseases such as Parkinson's disease, Alzheimer's disease, spinal cord injuries, diabetes, cancer with expanded stem cell research with technological advances in nanotechnology and materials science. [49] As it can be understood from this stem cell therapy based treatments of genetic based diseases are possible in laboratory conditions or in animal experiments, but the use of stem cell therapy is yet to be verified in humans. However, successful treatments that can be applied to people are being approached step by step fashion.…”

Section: Recent Studies On Stem Cellsmentioning

confidence: 99%

Stem Cells and Regenerative Medicine

Hayretdag¹,

Çoşkunpınar²

2019

Turk J Immunol

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Stem cells are self-renewing, non-differentiated cells that can be transforming into many different cell types and can form all the tissues and organs in our body when proper conditions are provided. Stem cells can be divided into three groups: adult stem cells, stem cells derived from cord blood, and embryonic stem cells. These cells, which have not yet differentiated, have the capacity of self-renewal, unlimited division and can transform into organs and tissues. Adult stem cells are found in many tissues and organs in the body and when damage occurs they multiply at that position to repair the damaged area. Discovery of stem cell differentiation also opened the doors for stem cell therapy in different fields such as; cancer, paralysis, Alzheimer's, parkinsonism, spinal cord injuries, heart diseases and many genetic diseases. ÖzKök hücreler, çok farklı hücre tiplerine dönüşebilen ve uygun koşullar sağlandığında vücudumuzdaki tüm doku ve organları oluşturabilen, kendini yenileyebilen, farklılaşmamış hücrelerdir. Kök hücreler üç gruba ayrılabilir: yetişkin kök hücreler, kord kanından elde edilen kök hücreler ve embriyonik kök hücreler. Henüz farklılaşmamış bu hücreler, kendini yenileme, sınırsız bölünme kapasitesine sahiptir ve organlara ve dokulara dönüşebilirler. Yetişkin kök hücreler, vücuttaki birçok doku ve organda bulunur ve hasar meydana geldiğinde hasarlı alanı onarmak için bu pozisyonda çoğalırlar. Kök hücre farklılaşmasının keşfi, kanser, felç, Alzheimer, parkinsonizm, omurilik yaralanmaları, kalp hastalıkları ve birçok genetik hastalık gibi farklı alanlarda kök hücre tedavisi için kapılar açmıştır.Anahtar Kelimeler: Kök hücre, rejeneratif tıp, kişiselleştirilmiş tıp Stem cellStem cells are cells that have the potential to replace the cells lost in the life of the organism in the embryonic and adulthood with the effect of environmental signals and transform into different cell types. [1,2]

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“…CRISPR-Cas9 genome editing technology is widely used for the establishment of disease models [1,2]. A D10A mutant nickase version of Cas9 (Cas9n) is only able to cut a single DNA strand [3].…”

Section: Introductionmentioning

confidence: 99%

Overcoming the effect of ASH1L haploinsufficiency on stem cells amenability to genome editing and differentiation into the neuronal lineage – a technical report

Cheon

Ritchie

Vacharasin

et al. 2021

Preprint

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Genome editing and neuronal differentiation protocols have proliferated in the last decade. Mutations in genes that control pluripotency could lead to a potential obstacle with regards to the survival and differentiation potential of the genome-edited cell lines. Here we describe a protocol for the generation, and differentiation, of cell lines containing CRISPR/Cas9 induced mutations in the histone methyltransferase ASH1L. This chromatin modifier was previously implicated in hematopoietic stem cell pluripotency and is a major genetic risk factor for autism spectrum disorders (ASD). We find that haploinsufficiency of ASH1L leads to decreased NANOG gene expression leading to reduce cell survival and increased spontaneous differentiation. We report a method that provides improved single-cell survival with higher colony formation efficiency in ASH1L mutant stem cells. Additionally, we describe a modified dual-SMAD inhibition neuronal induction methodology that permits the successful generation of human neurons with mutations in ASH1L, in a smaller scale than previously reported methods. With our modified CRISPR-genome editing and neuronal differentiation protocols, it is possible to generate genome-edited stem cells containing mutations in genes that impact pluripotency and could affect subsequent cell lineage-specific differentiation. Our detailed technical report presents cost-effective strategies that will benefit researchers focusing on both translational and basic science using stem cell experimental systems.

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Toward a CRISPR Picture: Use of CRISPR/Cas9 to Model Diseases in Human Stem Cells In Vitro

Cited by 13 publications

References 31 publications

Modeling of Aniridia-Related Keratopathy by CRISPR/Cas9 Genome Editing of Human Limbal Epithelial Cells and Rescue by Recombinant PAX6 Protein

Modeling of Aniridia-Related Keratopathy by CRISPR/Cas9 Genome Editing of Human Limbal Epithelial Cells and Rescue by Recombinant PAX6 Protein

Stem Cells and Regenerative Medicine

Overcoming the effect of ASH1L haploinsufficiency on stem cells amenability to genome editing and differentiation into the neuronal lineage – a technical report

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