scholarly journals Circularization of genes and chromosome by CRISPR in human cells

2018 ◽  
Author(s):  
Henrik Devitt Møller ◽  
Lin Lin ◽  
Xi Xiang ◽  
Trine Skov Petersen ◽  
Jinrong Huang ◽  
...  
Keyword(s):  
Genetic Engineering ◽  
Genetic Disorders ◽  
Ring Chromosome ◽  
Genetic Alterations ◽  
Human Cells ◽  
Chromosome Engineering ◽  
Guide Rnas ◽  
Ring Chromosomes ◽  
Biosensor System ◽  
Technology Applications

AbstractExtrachromosomal circular DNA (eccDNA) and ring chromosomes are genetic alterations found in humans with genetic disorders and diseases such as cancer. However, there is a lack of genetic engineering tool to recapitulate these features. Here, we report the discovery that delivery of pairs of CRISPR/Cas9 guide RNAs into human cells generate functional eccDNAs and ring chromosomes. We generated a dual-fluorescence eccDNA biosensor system, which allows us to study CRISPR deletion, inversion, and circularization of genes inside cells. Analysis after CRISPR editing at intergenic and genic loci in human embryonic kidney 293T cells and human mammary fibroblasts reveal that CRISPR deleted DNA readily form eccDNA in human cells. DNA in sizes from a few hundred base pairs up to a 47.4 megabase-sized ring chromosome (chr18) can be circularized. Our discoveries advance and expand CRISPR-Cas9 technology applications for genetic engineering, modeling of human diseases, and chromosome engineering.One Sentence Summary: CRISPR circularization of DNA offers new tools for studying eccDNA biogenesis, function, chromosome engineering, and synthetic biology.

scholarly journals Ring chromosome 20 syndrome – A rare chromosomal cause of refractory epilepsy in children

2017 ◽  
Vol 04 (01) ◽  
pp. 087-089 ◽  
Author(s):  
Umesh Kalane ◽  
Chaitanya Datar ◽  
Shilpa Kalane
Keyword(s):  
Chromosomal Abnormalities ◽  
Refractory Epilepsy ◽  
Genetic Disorders ◽  
Epileptiform Activity ◽  
Intractable Epilepsy ◽  
Ring Chromosome ◽  
Normal Karyotype ◽  
Epileptiform Discharges ◽  
Chromosome 20 ◽  
Work Up

AbstractGenetic disorders and chromosomal abnormalities have been shown to represent 2–3% of all cases of epilepsy. Ring chromosome 20 syndrome is a rare chromosomal abnormality and a rare cause of intractable epilepsy. Exact prevalence of ring chromosome 20 is not known. We report a case of a 10-year old boy who had had intractable epilepsy since 2 years of age. Birth history was insignificant and there was no obvious dysmorphism. His motor milestones were normal but cognition and speech were delayed. Electroencephalography showed progressive worsening from initial bi-frontal epileptiform activity to generalized discharges. Neuroimaging and metabolic work up was normal. Karyotype study showed ring chromosome 20. Diagnosis of ring chromosome 20 or r(20) syndrome was made. Ring chromosome 20 syndrome is a rare cause of refractory epilepsy A patient who present with intractable epilepsy with frontal epileptiform discharges, mental developmental delay, without dysmorphic features should be suspected of chromosomal abnormalities especially ring chromosome 20.

scholarly journals Human Ring Chromosomes – New Insights for their Clinical Significance

2013 ◽  
Vol 16 (1) ◽  
pp. 13-19 ◽  
Author(s):  
R.S. Guilherme ◽  
E Klein ◽  
A.B. Hamid ◽  
S Bhatt ◽  
M Volleth ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Clinical Signs ◽  
Ring Chromosome ◽  
Signs And Symptoms ◽  
Molecular Techniques ◽  
Ring Chromosomes ◽  
Multicolor Banding ◽  
Clinical Signs And Symptoms ◽  
Clinical Problems

Abstract Twenty-nine as yet unreported ring chromosomes were characterized in detail by cytogenetic and molecular techniques. For FISH (fluorescence in situ hybridization) previously published high resolution approaches such as multicolor banding (MCB), subcentromere-specific multi-color-FISH (cenM-FISH) and two to three-color-FISH applying locus-specific probes were used. Overall, ring chromosome derived from chromosomes 4 (one case), 10 (one case), 13 (five cases), 14, (three cases), 18 (two cases), 21 (eight cases), 22 (three cases), X (five cases) and Y (one case) were studied. Eight cases were detected prenatally, eight due developmental delay and dysmorphic signs, and nine in connection with infertility and/or Turner syndrome. In general, this report together with data from the literature, supports the idea that ring chromosome patients fall into two groups: group one with (severe) clinical signs and symptoms due to the ring chromosome and group two with no obvious clinical problems apart from infertility.

scholarly journals Engineering of Systematic Elimination of a Targeted Chromosome in Human Cells

2017 ◽  
Vol 2017 ◽  
pp. 1-5 ◽  
Author(s):  
Hiroshi Sato ◽  
Hiroki Kato ◽  
Haruyoshi Yamaza ◽  
Keiji Masuda ◽  
Huong Thi Nguyen Nguyen ◽  
...  
Keyword(s):  
Normal Cell ◽  
Expression Vector ◽  
Gene Dosage ◽  
Genetic Disorders ◽  
Human Cells ◽  
Chromosome 21 ◽  
Disease Phenotypes ◽  
Simplex Virus ◽  
Selection Of ◽  
Trisomic Cell

Embryonic trisomy leads to abortion or congenital genetic disorders in humans. The most common autosomal chromosome abnormalities are trisomy of chromosomes 13, 18, and 21. Although alteration of gene dosage is thought to contribute to disorders caused by extra copies of chromosomes, genes associated with specific disease phenotypes remain unclear. To generate a normal cell from a trisomic cell as a means of etiological analysis or candidate therapy for trisomy syndromes, we developed a system to eliminate a targeted chromosome from human cells. Chromosome 21 was targeted by integration of a DNA cassette in HeLa cells that harbored three copies of chromosome 21. The DNA cassette included two inverted loxP sites and a herpes simplex virus thymidine kinase (HSV-tk) gene. This system causes missegregation of chromosome 21 after expression of Cre recombinase and subsequently enables the selection of cells lacking the chromosome by culturing in a medium that includes ganciclovir (GCV). Cells harboring only two copies of chromosome 21 were efficiently induced by transfection of a Cre expression vector, indicating that this approach is useful for eliminating a targeted chromosome.

Predicting the efficiency of prime editing guide RNAs in human cells

2020 ◽  
Author(s):  
Hui Kwon Kim ◽  
Goosang Yu ◽  
Jinman Park ◽  
Seonwoo Min ◽  
Sungtae Lee ◽  
...  
Keyword(s):  
Human Cells ◽  
Guide Rnas

scholarly journals Characterization of Cas12a nucleases reveals diverse PAM profiles between closely-related orthologs

2020 ◽  
Vol 48 (10) ◽  
pp. 5624-5638
Author(s):  
Thomas Jacobsen ◽  
Fani Ttofali ◽  
Chunyu Liao ◽  
Srinivas Manchalu ◽  
Benjamin N Gray ◽  
...  
Keyword(s):  
Selective Pressure ◽  
Amino Acid Identity ◽  
Human Cells ◽  
High Sequence Identity ◽  
Guide Rnas ◽  
Adaptive Immune ◽  
Translation Systems ◽  
Adaptive Immune Systems ◽  
Single Effector

Abstract CRISPR-Cas systems comprise diverse adaptive immune systems in prokaryotes whose RNA-directed nucleases have been co-opted for various technologies. Recent efforts have focused on expanding the number of known CRISPR-Cas subtypes to identify nucleases with novel properties. However, the functional diversity of nucleases within each subtype remains poorly explored. Here, we used cell-free transcription-translation systems and human cells to characterize six Cas12a single-effector nucleases from the V-A subtype, including nucleases sharing high sequence identity. While these nucleases readily utilized each other's guide RNAs, they exhibited distinct PAM profiles and apparent targeting activities that did not track based on phylogeny. In particular, two Cas12a nucleases encoded by Prevotella ihumii (PiCas12a) and Prevotella disiens (PdCas12a) shared over 95% amino-acid identity yet recognized distinct PAM profiles, with PiCas12a but not PdCas12a accommodating multiple G’s in PAM positions -2 through -4 and T in position -1. Mutational analyses transitioning PiCas12a to PdCas12a resulted in PAM profiles distinct from either nuclease, allowing more flexible editing in human cells. Cas12a nucleases therefore can exhibit widely varying properties between otherwise related orthologs, suggesting selective pressure to diversify PAM recognition and supporting expansion of the CRISPR toolbox through ortholog mining and PAM engineering.

A RING CHROMOSOME IN AN F1 HYBRID BETWEEN WHEAT AND AGROPYRON

1959 ◽  
Vol 37 (6) ◽  
pp. 1271-1276 ◽  
Author(s):  
Koichiro Tsunewaki
Keyword(s):  
Ring Chromosome ◽  
Root Tip ◽  
Root Tips ◽  
F1 Hybrid ◽  
Ring Chromosomes ◽  
Differential Elimination ◽  
Tip Cells ◽  
Root Tip Cells ◽  
Mitotic Behavior

A plant having 41 normal rod-shaped chromosomes and a ring chromosome was found among hexaploid.F1 hybrids from a wheat–Agropyron cross. Cytological investigations were carried out to determine the mitotic behavior of this ring chromosome.The investigations revealed that most of the possible products of the breakage–fusion–bridge cycle known to occur in a ring chromosome were present in root tip cells. The fact that a rod-shaped chromosome is not derived from a ring chromosome in the cycle was confirmed, because no metaphase cells examined had 42 or more rod-shaped chromosomes.About 80% of the ring chromosomes were eliminated from the root tips of the seedling after 26 days. The size of the ring chromosome did not appear to influence the rate of elimination. The polyploid nature of the plant may account for the rapid, non-differential elimination of this chromosome.

scholarly journals Ring Chromosome 20 Associated with Refractory Epilepsy: A Case Report

2012 ◽  
Vol 2 (3) ◽  
pp. 141-144
Author(s):  
A Radha Rama Devi ◽  
Usha R Dutta
Keyword(s):  
Behavioral Problems ◽  
Refractory Epilepsy ◽  
Ring Chromosome ◽  
Epileptic Seizures ◽  
Dysmorphic Features ◽  
Mild Developmental Delay ◽  
Chromosome 20 ◽  
First Case ◽  
South Indian ◽  
Ring Chromosomes

Ring chromosome 20 is a rare chromosomal abnormality characterized mainly by refractory epileptic seizures, cognitive and behavioral problems, and absence of definite dysmorphic features. We report a 5-year-old boy with refractory epilepsy and minimal dysmorphic features who first presented with mild developmental delay at 11 months of age. The karyotype of the child was 46,XY,r(20)(p13q13.3). Till date there are 69 cases of ring chromosome 20 reported in the literature, including mosaics and supernumerary ring chromosomes. To our knowledge, this is the first case of ring chromosome 20 with refractory epilepsy reported from the south Indian population.DOI: http://dx.doi.org/10.3126/ajms.v2i3.5607Asian Journal of Medical Sciences 2 (2011) 141-144

scholarly journals The non-coding genome in genetic brain disorders: new targets for therapy?

2021 ◽  
Author(s):  
Eva Medico-Salsench ◽  
Faidra Karkala ◽  
Kristina Lanko ◽  
Tahsin Stefan Barakat
Keyword(s):  
Gene Regulation ◽  
Large Scale ◽  
Human Genetics ◽  
Genetic Disorders ◽  
Regulatory Elements ◽  
Genetic Alterations ◽  
Brain Diseases ◽  
Brain Disorders ◽  
Intrinsic Complexity ◽  
Spatio Temporal

Abstract The non-coding genome, consisting of more than 98% of all genetic information in humans and once judged as ‘Junk DNA’, is increasingly moving into the spotlight in the field of human genetics. Non-coding regulatory elements (NCREs) are crucial to ensure correct spatio-temporal gene expression. Technological advancements have allowed to identify NCREs on a large scale, and mechanistic studies have helped to understand the biological mechanisms underlying their function. It is increasingly becoming clear that genetic alterations of NCREs can cause genetic disorders, including brain diseases. In this review, we concisely discuss mechanisms of gene regulation and how to investigate them, and give examples of non-coding alterations of NCREs that give rise to human brain disorders. The cross-talk between basic and clinical studies enhances the understanding of normal and pathological function of NCREs, allowing better interpretation of already existing and novel data. Improved functional annotation of NCREs will not only benefit diagnostics for patients, but might also lead to novel areas of investigations for targeted therapies, applicable to a wide panel of genetic disorders. The intrinsic complexity and precision of the gene regulation process can be turned to the advantage of highly specific treatments. We further discuss this exciting new field of ‘enhancer therapy’ based on recent examples.

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