scholarly journals Applications of Genome Editing Technology in Research on Chromosome Aneuploidy Disorders

Cells ◽  
2020 ◽  
Vol 9 (1) ◽  
pp. 239 ◽  
Author(s):  
Silvia Natsuko Akutsu ◽  
Kazumasa Fujita ◽  
Keita Tomioka ◽  
Tatsuo Miyamoto ◽  
Shinya Matsuura
Keyword(s):  
Genome Editing ◽  
Cultured Cells ◽  
Chromosomal Abnormalities ◽  
Induced Pluripotent Stem Cell ◽  
Genetic Alterations ◽  
Chromosome 21 ◽  
Extra Copy ◽  
Chromosomal Segregation ◽  
Induced Pluripotent ◽  
Target Effects

Chromosomal segregation errors in germ cells and early embryonic development underlie aneuploidies, which are numerical chromosomal abnormalities causing fetal absorption, developmental anomalies, and carcinogenesis. It has been considered that human aneuploidy disorders cannot be resolved by radical treatment. However, recent studies have demonstrated that aneuploidies can be rescued to a normal diploid state using genetic engineering in cultured cells. Here, we summarize a series of studies mainly applying genome editing to eliminate an extra copy of human chromosome 21, the cause of the most common constitutional aneuploidy disorder Down syndrome. We also present findings on induced pluripotent stem cell reprogramming, which has been shown to be one of the most promising technologies for converting aneuploidies into normal diploidy without the risk of genetic alterations such as genome editing-mediated off-target effects.

scholarly journals N-Acetylcysteine prevents amyloid-β secretion in neurons derived from human pluripotent stem cells with trisomy 21

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Hiromitsu Toshikawa ◽  
Akihiro Ikenaka ◽  
Li Li ◽  
Yoko Nishinaka-Arai ◽  
Akira Niwa ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Stem Cells ◽  
Pluripotent Stem Cells ◽  
Therapeutic Option ◽  
Amyloid Β ◽  
Chromosome 21 ◽  
Extra Copy ◽  
Increased Risk ◽  
Induced Pluripotent

AbstractDown syndrome (DS) is caused by the trisomy of chromosome 21. Among the many disabilities found in individuals with DS is an increased risk of early-onset Alzheimer's disease (AD). Although higher oxidative stress and an upregulation of amyloid β (Aβ) peptides from an extra copy of the APP gene are attributed to the AD susceptibility, the relationship between the two factors is unclear. To address this issue, we established an in vitro cellular model using neurons differentiated from DS patient-derived induced pluripotent stem cells (iPSCs) and isogenic euploid iPSCs. Neurons differentiated from DS patient-derived iPSCs secreted more Aβ compared to those differentiated from the euploid iPSCs. Treatment of the neurons with an antioxidant, N-acetylcysteine, significantly suppressed the Aβ secretion. These findings suggest that oxidative stress has an important role in controlling the Aβ level in neurons differentiated from DS patient-derived iPSCs and that N-acetylcysteine can be a potential therapeutic option to ameliorate the Aβ secretion.

MODELING PANCREATIC PATHOPHYSIOLOGY USING GENOME EDITING OF ADULT STEM CELL- AND INDUCED PLURIPOTENT STEM CELL (iPSC)-DERIVED ORGANOIDS

2021 ◽  
Author(s):  
Sabrina T. Hirshorn ◽  
Nina Steele ◽  
Yana Zavros
Keyword(s):  
Stem Cell ◽  
Genome Editing ◽  
Pluripotent Stem Cell ◽  
Adult Stem Cells ◽  
Defense Mechanism ◽  
Adult Stem Cell ◽  
Induced Pluripotent Stem Cell ◽  
Organ Development ◽  
Induced Pluripotent

In recent years, organoids have become a novel in vitro method to study gastrointestinal organ development, physiology and disease. An organoid, in short, may be defined as a miniaturized organ that can be grown from adult stem cells in vitro and studied at the microscopic level. Organoids have been used in multitudes of different ways to study the physiology of different human diseases including gastrointestinal cancers such as pancreatic cancer. The development of genome editing based on the bacterial defense mechanism CRISPR/Cas9 has emerged as laboratory tool that provides the opportunity to study the effects of specific genetic changes on organ development, physiology and disease. The CRISPR/Cas9 approach can be combined with organoid technology including the use of induced pluripotent stem cell (iPSC)-derived and tissue-derived organoids. The goal of this review is to provide highlights on the development of organoid technology, and the use of this culture system to study the pathophysiology of specific mutations in the development of pancreatic and gastric cancers.

scholarly journals A cell cycle-dependent CRISPR-Cas9 activation system based on an anti-CRISPR protein shows improved genome editing accuracy

2020 ◽  
Vol 3 (1) ◽  
Author(s):  
Daisuke Matsumoto ◽  
Hirokazu Tamamura ◽  
Wataru Nomura
Keyword(s):  
Cell Cycle ◽  
Genome Editing ◽  
Target Genes ◽  
Genetic Alterations ◽  
Homology Directed Repair ◽  
Activation System ◽  
A Cell ◽  
Cycle Dependent ◽  
Natural Inhibitors ◽  
Target Effects

Abstract The development of genome editing systems based on the Cas9 endonuclease has greatly facilitated gene knockouts and targeted genetic alterations. Precise editing of target genes without off-target effects is crucial to prevent adverse effects in clinical applications. Although several methods have been reported to result in less off-target effects associated with the CRISPR technology, these often exhibit lower editing efficiency. Therefore, efficient, accurate, and innocuous CRISPR technology is still required. Anti-CRISPR proteins are natural inhibitors of CRISPR-Cas systems derived from bacteriophages. Here, the anti-CRISPR protein, AcrIIA4, was fused with the N terminal region of human Cdt1 that is degraded specifically in S and G2, the phases of the cell cycle when homology-directed repair (HDR) is dominant. Co-expression of SpyCas9 and AcrIIA4-Cdt1 not only increases the frequency of HDR but also suppress off-targets effects. Thus, the combination of SpyCas9 and AcrIIA4-Cdt1 is a cell cycle-dependent Cas9 activation system for accurate and efficient genome editing.

scholarly journals In vitro genome editing rescues parkinsonism phenotypes in induced pluripotent stem cells-derived dopaminergic neurons carrying LRRK2 p.G2019S mutation

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Kuo-Hsuan Chang ◽  
Cheng-Yen Huang ◽  
Chih-Hsin Ou-Yang ◽  
Chang-Han Ho ◽  
Han-Yi Lin ◽  
...  
Keyword(s):  
Stem Cells ◽  
Induced Pluripotent Stem Cells ◽  
Genome Editing ◽  
Pluripotent Stem Cells ◽  
Dopaminergic Neurons ◽  
Expression Patterns ◽  
Peroxisome Proliferator ◽  
Dna Breaks ◽  
Induced Pluripotent ◽  
Target Effects

Abstract Background The c.G6055A (p.G2019S) mutation in leucine-rich repeat kinase 2 (LRRK2) is the most prevalent genetic cause of Parkinson’s disease (PD). CRISPR/Cas9-mediated genome editing by homology-directed repair (HDR) has been applied to correct the mutation but may create small insertions and deletions (indels) due to double-strand DNA breaks. Adenine base editors (ABEs) could convert targeted A·T to G·C in genomic DNA without double-strand breaks. However, the correction efficiency of ABE in LRRK2 c.G6055A (p.G2019S) mutation remains unknown yet. This study aimed to compare the mutation correction efficiencies and off-target effects between HDR and ABEs in induced pluripotent stem cells (iPSCs) carrying LRRK2 c.G6055A (p.G2019S) mutation. Methods A set of mutation-corrected isogenic lines by editing the LRRK2 c.G6055A (p.G2019S) mutation in a PD patient-derived iPSC line using HDR or ABE were established. The mutation correction efficacies, off-target effects, and indels between HDR and ABE were compared. Comparative transcriptomic and proteomic analyses between the LRRK2 p.G2019S iPSCs and isogenic control cells were performed to identify novel molecular targets involved in LRRK2-parkinsonism pathways. Results ABE had a higher correction rate (13/53 clones, 24.5%) than HDR (3/47 clones, 6.4%). Twenty-seven HDR clones (57.4%), but no ABE clones, had deletions, though 14 ABE clones (26.4%) had off-target mutations. The corrected isogenic iPSC-derived dopaminergic neurons exhibited reduced LRRK2 kinase activity, decreased phospho-α-synuclein expression, and mitigated neurite shrinkage and apoptosis. Comparative transcriptomic and proteomic analysis identified different gene expression patterns in energy metabolism, protein degradation, and peroxisome proliferator-activated receptor pathways between the mutant and isogenic control cells. Conclusions The results of this study envision that ABE could directly correct the pathogenic mutation in iPSCs for reversing disease-related phenotypes in neuropathology and exploring novel pathophysiological targets in PD.

scholarly journals Generation of two ISL1-tdTomato reporter human induced pluripotent stem cell lines using CRISPR-Cas9 genome editing

2021 ◽  
pp. 102363
Author(s):  
Satomi Tsukamoto ◽  
Koji Nakade ◽  
Tamami Wakabayashi ◽  
Kenichi Nakashima ◽  
Miho Takami ◽  
...  
Keyword(s):  
Stem Cell ◽  
Genome Editing ◽  
Cell Lines ◽  
Pluripotent Stem Cell ◽  
Induced Pluripotent Stem Cell ◽  
Stem Cell Lines ◽  
Induced Pluripotent

Abstract 38: Engineered Anisotropic Scaffolds Promote the Function of Cocultured Cardiomyocytes Derived From Human Pluripotent Stem Cells

2016 ◽  
Vol 119 (suppl_1) ◽  
Author(s):  
Maureen Wanjare ◽  
Joseph Jung-Woong Kim ◽  
Ngan Huang
Keyword(s):  
Pore Diameter ◽  
Cultured Cells ◽  
Three Dimensional ◽  
Induced Pluripotent Stem Cell ◽  
Average Pore ◽  
Cardiac Tissues ◽  
Electrospun Scaffolds ◽  
In Series ◽  
Induced Pluripotent

Since the heart is effectively an anisotropic organ in which the cardiomyocytes (CM) are locally aligned in series, it is important to engineer cardiac tissues that promote CM alignment in order to closely mimic the architecture of the native tissue, as well as better mimic the cellular composition of the heart. The objective of this study was to define the role of anisotropic extracellular matrix cues on the organization and survival of human induced pluripotent stem cell-derived CMs (hiPSC-CMs) by co-culturing hiPSC-CMs and primary endothelial cells (ECs) on parallel-aligned microfibrillar scaffolds. The hiPSC-CMs were generated from hiPSCs using small molecule Wnt pathway agonists and antagonists. Subsequently, the hiPSC-CMs were sequentially seeded on day 15 after EC attachment. We cultured monocultures and cocultures on electrospun three-dimensional (3D) scaffolds of polycaprolactone (PCL) and polyethylene oxide (PEO) polymer blends with an average fiber diameter of 14 μm. Aligned scaffolds were fabricated by stretching the randomly oriented scaffolds by 300% of the original scaffold length. Randomly oriented fibrillar scaffolds had an average pore diameter of 17 μm when compared to the 36 μm pore diameter of aligned scaffolds. Our results indicate that alignment of co-cultured cells at a 5:1 hiPSC-CMs : EC ratio was promoted by anistropic 3D electrospun scaffolds when compared to similar random 3D electrospun scaffolds. Additionally, cocultured cells on aligned fibrillar scaffolds had a mean angle of orientation of 30.8°, relative to the direction of fibrils, which was similar to that of hiPSC-CM monocultures on aligned scaffolds (32.8°). In contrast, the degree of alignment of hiPSC-CMs on randomly oriented fibrillary scaffolds was 43.4°, which suggests a non-oriented population of cells. Aligned scaffolds also produced more synchronized cardiomyocyte contraction than random scaffold orientations, although both induced spontaneous contraction frequency of ~1Hz. This study highlights the importance of nanotopographical cues and intercellular interactions in mediating the morphology and contractility of hiPSC-CMs for treatment of cardiovascular diseases such as myocardial infarction.

scholarly journals Correction of amyotrophic lateral sclerosis related phenotypes in induced pluripotent stem cell-derived motor neurons carrying a hexanucleotide expansion mutation in C9orf72 by CRISPR/Cas9 genome editing using homology-directed repair

2020 ◽  
Vol 29 (13) ◽  
pp. 2200-2217 ◽  
Author(s):  
Nidaa A Ababneh ◽  
Jakub Scaber ◽  
Rowan Flynn ◽  
Andrew Douglas ◽  
Paola Barbagallo ◽  
...  
Keyword(s):  
Amyotrophic Lateral Sclerosis ◽  
Stem Cell ◽  
Genome Editing ◽  
Motor Neurons ◽  
Pluripotent Stem Cell ◽  
Induced Pluripotent Stem Cell ◽  
Dementia Patient ◽  
Homology Directed Repair ◽  
Induced Pluripotent ◽  
Lateral Sclerosis

Abstract The G4C2 hexanucleotide repeat expansion (HRE) in C9orf72 is the commonest cause of familial amyotrophic lateral sclerosis (ALS). A number of different methods have been used to generate isogenic control lines using clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 and non-homologous end-joining by deleting the repeat region, with the risk of creating indels and genomic instability. In this study, we demonstrate complete correction of an induced pluripotent stem cell (iPSC) line derived from a C9orf72-HRE positive ALS/frontotemporal dementia patient using CRISPR/Cas9 genome editing and homology-directed repair (HDR), resulting in replacement of the excised region with a donor template carrying the wild-type repeat size to maintain the genetic architecture of the locus. The isogenic correction of the C9orf72 HRE restored normal gene expression and methylation at the C9orf72 locus, reduced intron retention in the edited lines and abolished pathological phenotypes associated with the C9orf72 HRE expansion in iPSC-derived motor neurons (iPSMNs). RNA sequencing of the mutant line identified 2220 differentially expressed genes compared with its isogenic control. Enrichment analysis demonstrated an over-representation of ALS relevant pathways, including calcium ion dependent exocytosis, synaptic transport and the Kyoto Encyclopedia of Genes and Genomes ALS pathway, as well as new targets of potential relevance to ALS pathophysiology. Complete correction of the C9orf72 HRE in iPSMNs by CRISPR/Cas9-mediated HDR provides an ideal model to study the earliest effects of the hexanucleotide expansion on cellular homeostasis and the key pathways implicated in ALS pathophysiology.

scholarly journals CRISPR Del/Rei: A simple, flexible and efficient pipeline for scarless genome editing

2021 ◽  
Author(s):  
Marah H. Wahbeh ◽  
Kyra L. Feuer ◽  
Sara Abdollahi ◽  
Christian Yovo ◽  
Eman Rabie ◽  
...  
Keyword(s):  
Genome Editing ◽  
Dna Sequences ◽  
Editing Efficiency ◽  
Manual Selection ◽  
Variant Alleles ◽  
Induced Pluripotent ◽  
The Individual ◽  
User Friendly ◽  
Selection Of ◽  
Target Effects

Scarless genome editing is an important tool for the accurate recapitulation of genetic variation in human disease models. Various CRISPR/Cas9-based scarless editing methods have been reported. However, some of these methods have low editing efficiency (1-5%) and require manual selection of hundreds of clones to reach the desired number. Other protocols use large selection cassettes with laborious vector assembly and specialized reagents and equipment, or have poorly understood off-target effects. To address these limitations, we developed a simple, highly efficient scarless editing strategy to edit DNA sequences in induced pluripotent stem cells, which we call CRISPR Del/Rei. This novel editing strategy consists of a two-step deletion-reinsertion strategy that produces isogenic clones in ~8 weeks using accessible, user-friendly reagents. The editing efficiency ranges from ~15–100% for Step 1 and ~5–20% for Step 2 after selection, which greatly reduces the amount of required manual clone isolation. Screening the transfected bulk cells and the individual clones is rapid and simple, consisting of PCR and gel electrophoresis. Despite the two editing steps, off-target effects are rare. Additionally, the experiment is well-controlled because the same protocol generates isogenic clones carrying all variant alleles. In this way, CRISPR Del/Rei serves as a valuable addition to the evolving CRISPR/Cas9 gene-editing toolset.

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