Whole-genome sequencing of the CRISPR/Cas9-generated cell lines G12LS and C07 carrying a heterozygous and homozygous mutation in the RB1 gene and their parental human embryonic stem cell line H9.
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ABSTRACT: Heterozygous and homozygous mutations were introduced to the human embryonic stem cell line H9 by using the CRISPR/Cas9-system. Since off-target effects can occur and high numbers of SNVs can be acquired during clonal selection, the generated cell lines and the parental cell line were analyzed by whole-genome sequencing.
Project description:The Retinoblastoma 1 (RB1) tumor suppressor, a member of the Retinoblastoma gene family, functions as a pocket protein for the functional binding of E2F transcription factors. About 1/3 of retinoblastoma patients harbor a germline RB1 mutation or deletion, leading to the development of retinoblastoma. Here, we demonstrate generation of a heterozygous deletion of the RB1 gene in the H1 human embryonic stem cell line using CRISPR/Cas9 nickase genome editing. The RB1 heterozygous knockout H1 cell line shows a normal karyotype, maintains a pluripotent state, and is capable of differentiation to the three germline layers.
Project description:Purpose: To identify the genetic basis of posterior polymorphous corneal dystrophy 1 (PPCD1). Methods: Next-generation sequencing was performed on DNA samples from 4 affected and 4 unaffected members of a previously reported family with PPCD1 linked to chromosome 20 between D20S182 and D20S195. Custom capture probes were utilized for targeted region capture of the linked interval. Single nucleotide variants (SNVs) and insertions/deletions (indels) were identified using two bioinformatics pipelines and two annotation databases. Candidate variants met the following criteria: quality score â¥20, read depth â¥5X, heterozygous, novel or rare (minor allele frequency (MAF) ⤠0.05), present in each affected individual and absent in each unaffected individual. Structural variants were detected with two different microarray platforms to identify indels of varying sizes. Results: Sequencing reads aligned to the linked region on chromosome 20, and high coverage was obtained across the sequenced region. The majority of identified variants were detected with both pipelines and annotation databases, although unique variants were identified. Twelve SNVs in 10 genes (2 synonymous variants and 10 noncoding variants) and 9 indels in 7 genes met the filtering criteria and were considered candidate variants for PPCD1. Conclusions: Next-generation sequencing of the PPCD1 interval has identified 17 genes containing novel or rare SNVs and indels that segregate with the affected phenotype in an affected family previously mapped to the PPCD1 locus. We anticipate that screening of these candidate genes in other families previously mapped to the PPCD1 locus will result in the identification of the genetic basis of PPCD1. Four affected and 4 unaffected individuals from a single family were analyzed for copy number variation within the PPCD1 disease locus. Array design and analysis is based on genome build hg19.
Project description:In order to investigate the roles of retinoblastoma family genes during early human development, mutant H9 human embryonic stem cells were generated. Frameshift mutations were introduced in RBL1 and RBL2 genes using the CRISPR/Cas9 technology, and then the shRNA-expression cassette to knockdown RB upon tetracycline treatment was integrated. These cells were cultured in definitive endoderm differentiation conditions for 3 or 6 days with/without tetracycline.
Project description:Familial hypercholesterolemia (FH) is a common inherited disorder that results in premature atherosclerosis. Diagnosis of FH is suspected on the basis of clinical criteria, but confirmation requires genetic testing. In the era of statins, early diagnosis and initiation of treatment can modify disease progression and outcomes. Therefore, cascade screening with a combination of lipid concentration measurements and DNA testing should be used to identify relatives of index cases with a clinical diagnosis of FH. Autosomal dominant FH is related to mutations in the low-density lipoprotein receptor (LDLR), apolipoprotein B-100 (APOB), or proprotein convertase subtilisin/kexin type 9 (PCSK9) genes. Genetic screening of the LDLR gene is challenging to achieve at a feasible cost, especially in people who do not have a founder effect. Nucleotide sequencing of all exons and flanking splicing regions in combination with multiplex ligation probe amplification to detect large insertions or deletions is considered the gold-standard approach to screen for LDLR mutations. Alternatively, the cDNA can be sequenced; however, this procedure is not suitable for use in large populations, because of the need of RNA extraction. Multiplex analysis can be appropriate for population with founder effects or a low number of different mutations. Finally, there are many techniques for a mutation scanning approach, which have some benefits over sequencing, and also with the potential for detecting known and novel mutations. Familial defective Apo B is amenable to genetic diagnosis by screening for a few mutations. Recently, gain-of-function mutations in PCSK9 gene have been demonstrated to cause FH phenotype. Strategies for population screening, cost-effectiveness of genetic screening, ethical aspects, and insurance policies are discussed and need implementation worldwide.
Project description:CRISPRs and TALENs are efficient systems for gene editing in many organisms including plants. In many cases the CRISPR-Cas or TALEN modules are expressed in the plant cell only transiently. Theoretically, transient expression of the editing modules should limit unexpected effects compared to stable transformation. However, very few studies have measured the off-target and unpredicted effects of editing strategies on the plant genome, and none of them have compared these two major editing systems. We conducted a comprehensive genome-wide investigation of off-target mutations using either a CRISPR-Cas9 or a TALEN strategy. We observed a similar number of SNVs and InDels for the two editing strategies compared to control non-transfected plants, with an average of 8.25 SNVs and 19.5 InDels for the CRISPR-edited plants, and an average of 17.5 SNVs and 32 InDels for the TALEN-edited plants. Interestingly, a comparable number of SNVs and InDels could be detected in the PEG-treated control plants. This shows that except for the on-target modifications, the gene editing tools used in this study did not show a significant off-target activity nor unpredicted effects on the genome, and that the PEG treatment in itself was probably the main source of mutations found in the edited plants.
Project description:Hyposmia is a common nonmotor symptom in Parkinson's disease (PD) and has been variably detected in monogenic parkinsonism. SYNJ1 has been recently identified as the gene defective in a novel form of autosomal-recessive, early-onset atypical parkinsonism, designed as PARK20. To assess olfaction in PARK20, we administered the University of Pennsylvania Smell Identification Test (UPSIT) in four groups of subjects: SYNJ1 homozygous (HOM = 3) and heterozygous (HET = 4); sporadic PD (PD = 68); and healthy control subjects (CTR = 61). A linear regression model was constructed to assess the association between raw UPSIT score (outcome) and group (HOM, HET, PD, and CTR), adjusting for age, gender, and current smoking status. Likewise in PD patients, odor identification is impaired in homozygous SYNJ1 mutation carriers. Although the limited sample size precludes definite conclusions about olfaction in SYNJ1-related parkinsonism, our findings suggest new insights into PARK20 phenotype and pathophysiology.
Project description:Human nude SCID is a rare autosomal recessive inborn error of immunity (IEI) characterized by congenital athymia, alopecia, and nail dystrophy. Few cases have been reported to date. However, the recent introduction of newborn screening for IEIs and high-throughput sequencing has led to the identification of novel and atypical cases. Moreover, immunological alterations have been recently described in patients carrying heterozygous mutations. The aim of this paper is to describe the extended phenotype associated with FOXN1 homozygous, compound heterozygous, or heterozygous mutations. We collected clinical and laboratory information of a cohort of 11 homozygous, 2 compound heterozygous, and 5 heterozygous patients with recurrent severe infections. All, except one heterozygous patient, had signs of CID or SCID. Nail dystrophy and alopecia, that represent the hallmarks of the syndrome, were not always present, while almost 50% of the patients developed Omenn syndrome. One patient with hypomorphic compound heterozygous mutations had a late-onset atypical phenotype. A SCID-like phenotype was observed in 4 heterozygous patients coming from the same family. A spectrum of clinical manifestations may be associated with different mutations. The severity of the clinical phenotype likely depends on the amount of residual activity of the gene product, as previously observed for other SCID-related genes. The severity of the manifestations in this heterozygous family may suggest a mechanism of negative dominance of the specific mutation or the presence of additional mutations in noncoding regions.
Project description:The most prevalent form of epileptic encephalopathy is Dravet syndrome (DRVT), which is triggered by the pathogenic variant SCN1A in 80% of cases. iPSCs with different SCN1A mutations have been constructed by several groups to model DRVT syndrome. However, no studies involving DRVT-iPSCs with rare genetic variants have been conducted. Here, we established two DRVT-iPSC lines harboring a homozygous mutation in the CPLX1 gene and heterozygous mutation in SCN9A gene. Therefore, the derivation of these iPSC lines provides a unique cellular platform to dissect the molecular mechanisms underlying the cellular dysfunctions consequent to CPLX1 and SCN9A mutations.
Project description:Whole-genome sequencing of the CRISPR/Cas9-generated cell lines G12LS and C07 carrying a heterozygous and homozygous mutation in the RB1 gene and their parental human embryonic stem cell line H9.