Idiopathic basal ganglia calcification (IBGC) is characterized by bilateral calcification of the basal ganglia associated with a spectrum of neuropsychiatric and motor syndromes. In this study, we ...set out to determine the frequency of the recently identified IBGC gene
SLC20A2
in 27 IBGC cases from the Mayo Clinic Florida Brain Bank using both Sanger sequencing and TaqMan copy number analysis to cover the complete spectrum of possible mutations. We identified
SLC20A2
pathogenic mutations in two of the 27 cases of IBGC (7 %). Sequencing analysis identified a p.S113* nonsense mutation in
SLC20A2
in one case. TaqMan copy number analysis of
SLC20A2
further revealed a genomic deletion in a second case, which was part of a large previously reported Canadian IBGC family with dystonia. Subsequent whole-genome sequencing in this family revealed a 563,256-bp genomic deletion with precise breakpoints on chromosome 8 affecting multiple genes including
SLC20A2
and the known dystonia-related gene
THAP1
. The deletion co-segregated with disease in all family members. The deletion of
THAP1
in addition to
SLC20A2
in the Canadian IBGC family may contribute to the severe and early onset dystonia in this family. The identification of an
SLC20A2
genomic deletion in a familial form of IBGC demonstrates that reduced SLC20A2 in the absence of mutant protein is sufficient to cause neurodegeneration and that previously reported
SLC20A2
mutation frequencies may be underestimated.
Biallelic mutations in neuraminidase 1 (
) are associated with cherry-red spots. Whole genome sequencing contributes to eliminating pseudo-homozygous mutations when large-scale deletion of one allele ...in
and other genes occurs.
Bilateral cherry-red spots in the macula were the only detectable sign in an 11-year-old girl with reduced visual acuity over the last two years. Targeted exome sequencing of genes for inherited eye diseases identified a homozygous c.544A>G (p.Ser182Gly) variation in the
gene. This variant was also present in her mother in the heterozygous state but not in her father. Whole genome sequencing identified a heterozygous 27.5 kb deletion involving the whole coding exons of
in her father. Sanger sequencing disclosed the breakpoint of the deletion. This heterozygous deletion was also detected in the patient, so the c.544A>G mutation should be heterozygous in the patient.
The results of this case remind us of the limitations of routine exome sequencing and the need to perform segregation studies and deletion/duplication analysis or WGS if parental studies do not support exome findings. In addition, patients with sialidosis may present with ocular manifestations without systemic signs early in the disease course.
Background
CRISPR‐Cas9‐based technologies have revolutionized experimental manipulation of mammalian genomes. None‐the‐less, limitations of the delivery and efficacy of these technologies restrict ...their application in primary cells.
Aims
To create an optimized protocol for penetrant, reproducible, and fast targeted genome editing in cell cultures derived from primary cells, using patient‐derived glioblastoma stem‐like cells (GSCs) and human neural stem/progenitor cells (NSCs) for proof‐of‐concept experiments.
Methods and results
We employed transient nucleofection of Cas9:sgRNA ribonucleoprotein complexes composed of chemically synthesized 2′‐O‐methyl 3′phosphorothioate‐modified sgRNAs and purified Cas9 protein. Insertion‐deletion mutation (indel) frequency and size distribution were measured via computational deconvolution of Sanger sequencing trace data. We found that this optimized technique routinely allows for >90% indel formation in only 3 days, without the need to create clonal lines for simple loss‐of‐function experiments. Using Western blotting, we observed near‐total protein loss of target genes in cell pools. Additionally, we found that this approach allows for the creation of targeted genomic deletions. Furthermore, by using RNA‐seq in edited NSCs to assess gene expression changes resulting from knockout of tumor suppressors commonly altered in glioblastoma, we also demonstrated the utility of this method for quickly creating a series of gene knockouts that allow for the study of oncogenic activities.
Conclusion
Our data suggest that this relatively simple method can be used for highly efficient and fast gene knockout, as well as for targeted genomic deletions, even in hyperdiploid cells (such as GSCs). This represents an extremely useful tool for the cancer research community when wishing to inactivate not only coding genes, but also non‐coding RNAs, UTRs, enhancers, and promoters. This method can be readily applied to diverse cell types by varying the nucleofection conditions.
Major EV-B populations characterized by 5′ terminal deletions (5′TD) have been shown to be associated with the development of myocarditis and type 1 diabetes in mice or humans. To date, the dynamics ...of EV-B 5′TD-RNA forms’ emergence during the course of infection and their impact on cellular functions remain unclear. Using a RACE-PCR approach in CVB3/28-infected mouse organs, we showed an early (3 days post infection, DPI) emergence of major 5′TD populations associated with minor full-length RNA forms. Viral replication activities with infectious particle production were associated with heart, liver, and pancreas acute inflammatory lesions, whereas clearance of viral RNA without organ lesions was observed in the brain, lung, intestines, and muscles from 3 to 7 DPI. At 28 DPI, low viral RNA levels, +/-RNA ratios < 5 associated with viral protein 1 expression revealed a persistent infection in the heart and pancreas. This persistent infection was characterized by molecular detection of only 5′TD RNA forms that were associated with dystrophin cleavage in the heart and insulin production impairment in beta-pancreatic cells. These results demonstrated that major EV-B 5′TD RNA forms can be early selected during systemic infection and that their maintenance may drive EV-induced acute and persistent infections with target cell dysfunctions.
•Dual fluorescence selection system can enrich genomic deletions efficiently.•It can enrich cells with 170bp small deletions at a frequency as high as 74%.•It can enrich cells with 5kb large ...deletions at a frequency of 46%.
Production of nuclear donor cells with a high percentage of desired modifications is a critical step in the successful generation of genetically modified pigs through somatic cell nuclear transfer (SCNT). The CRISPR/Cas9 system has been used for efficient modification of the nuclear DNA in eukaryotic cells, including porcine cells. However, in vitro modified cells are often phenotypically indistinguishable from unmodified cells, hampering their enrichment. Here we investigate a dual fluorescence selection system for the efficient enrichment of porcine embryonic fibroblasts (PEFs) with CRISPR/Cas9-induced chromosomal deletions. Enrichment of cells with 170bp deletions reached a frequency of 74%, whilst enrichment of cells with a larger 5kb deletions achieved a frequency of 46%. This demonstrates the utility of a dual fluorescence reporter as an attractive tool for improving the efficiency of generating genome edited pigs.
Deletion of oncosuppressors occurs frequently in the cancer genome. A great deal of effort has been made to therapeutically restore the lost function of oncosuppressors, with little clinically ...translatable success, however. Reassuringly, besides the disappointing restoration endeavors, oncosuppressor loss can be therapeutically exploited in several other ways, such as the “synthetic lethality” strategies and the “therapeutic vulnerability” created by codeletion of neighboring genes. The study by Liu et al showed that codeletion of p53 and a neighboring essential gene POLR2A rendered colon cancer cells highly sensitive to further inhibition of POLR2A both in vitro and in vivo. In recent years, several studies have reported similar phenomenon in a wide range of cancer types. In this focus article, we will introduce several kinds of anticancer opportunities created by the loss of oncosuppressors and discuss their mechanisms. Given the frequency of oncosuppressor loss in cancer, its therapeutic exploitation rather merits further investigation and may open a new window for oncotherapy.
Bacteria of the genus Xenorhabdus are symbionts of soil entomopathogenic nematodes of the genus Steinernema. This symbiotic association constitutes an insecticidal complex active against a wide range ...of insect pests. Unlike other Xenorhabdus species, Xenorhabdus poinarii is avirulent when injected into insects in the absence of its nematode host. We sequenced the genome of the X. poinarii strain G6 and the closely related but virulent X. doucetiae strain FRM16. G6 had a smaller genome (500–700 kb smaller) than virulent Xenorhabdus strains and lacked genes encoding potential virulence factors (hemolysins, type 5 secretion systems, enzymes involved in the synthesis of secondary metabolites, and toxin–antitoxin systems). The genomes of all the X. poinarii strains analyzed here had a similar small size. We did not observe the accumulation of pseudogenes, insertion sequences or decrease in coding density usually seen as a sign of genomic erosion driven by genetic drift in host-adapted bacteria. Instead, genome reduction of X. poinarii seems to have been mediated by the excision of genomic blocks from the flexible genome, as reported for the genomes of attenuated free pathogenic bacteria and some facultative mutualistic bacteria growing exclusively within hosts. This evolutionary pathway probably reflects the adaptation of X. poinarii to specific host.
The present study investigated the efficiency of CRISPR/Cas9 in creating genomic deletions as the basis of its application in removing selection marker genes or the intergenic regions. Three loci, ...representing a transgene and two rice genes, were targeted at two sites each, in separate experiments, and the deletion of the defined fragments was investigated by PCR and sequencing. Genomic deletions were found at a low rate among the transformed callus lines that could be isolated, cultured, and regenerated into plants harboring the deletion. However, randomly regenerated plants showed mixed genomic effects, and generally did not harbor heritable genomic deletions. To determine whether point mutations occurred at each targeted site, a total of 114 plants consisting of primary transgenic lines and their progeny were analyzed. Ninety-three plants showed targeting, 60 of which were targeted at both sites. The presence of point mutations at both sites was correlated with the guide RNA efficiency. In summary, genomic deletions through dual-targeting by the paired-guide RNAs were generally observed in callus, while de novo point mutations at one or both sites occurred at high rates in transgenic plants and their progeny, generating a variety of insertion–deletions or single-nucleotide variations. In this study, point mutations were exceedingly favored over genomic deletions; therefore, for the recovery of plant lines harboring targeted deletions, identifying early transformed clones harboring the deletions, and isolating them for plant regeneration is recommended.
PDF
