An efficient and adaptable workflow for editing disease-relevant single nucleotide variants using CRISPR/Cas9

Single nucleotide variants are the commonest genetic alterations in the human genome. At least 60,000 have been reported to be associated with disease. The CRISPR/Cas9 system has transformed genetic research, making it possible to edit single nucleotides and study the function of genetic variants in vitro. While significant advances have improved the efficiency of CRISPR/Cas9, the editing of single nucleotides remains challenging. There are two major obstacles: low efficiency of accurate editing and the isolation of these cells from a pool of cells with other editing outcomes. We present data from 85 transfections of induced pluripotent stem cells and an immortalised cell line, comparing the effects of altering CRISPR/Cas9 design and experimental conditions on rates of single nucleotide substitution. We targeted variants in TP53, which predispose to several cancers, and in TBXT which is implicated in the pathogenesis of the bone cancer, chordoma. We describe a scalable and adaptable workflow for single nucleotide editing that incorporates contemporary techniques including Illumina MiSeq sequencing, TaqMan qPCR and digital droplet PCR for screening transfected cells as well as quality control steps to mitigate against common pitfalls. This workflow can be applied to CRISPR/Cas9 and other genome editing systems to maximise experimental efficiency.

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Association between HOX Transcript Antisense RNA Single-Nucleotide Variants and Recurrent Implantation Failure

International Journal of Molecular Sciences ◽

10.3390/ijms22063021 ◽

2021 ◽

Vol 22 (6) ◽

pp. 3021

Author(s):

Jeong Yong Lee ◽

Eun Hee Ahn ◽

Hyeon Woo Park ◽

Ji Hyang Kim ◽

Young Ran Kim ◽

...

Keyword(s):

Antisense Rna ◽

Coagulation Factors ◽

Healthy Controls ◽

Implantation Failure ◽

Single Nucleotide Variants ◽

Recurrent Implantation Failure ◽

Single Nucleotide ◽

Vitro Fertilization ◽

And Control

Recurrent implantation failure (RIF) refers to the occurrence of more than two failed in vitro fertilization–embryo transfers (IVF-ETs) in the same individual. RIF can occur for many reasons, including embryo characteristics, immunological factors, and coagulation factors. Genetics can also contribute to RIF, with some single-nucleotide variants (SNVs) reported to be associated with RIF occurrence. We examined SNVs in a long non-coding RNA, homeobox (HOX) transcript antisense RNA (HOTAIR), which is known to affect cancer development. HOTAIR regulates epigenetic outcomes through histone modifications and chromatin remodeling. We recruited 155 female RIF patients and 330 healthy controls, and genotyped HOTAIR SNVs, including rs4759314, rs920778, rs7958904, and rs1899663, in all participants. Differences in these SNVs were compared between the patient and control groups. We identified significant differences in the occurrence of heterozygous genotypes and the dominant expression model for the rs1899663 and rs7958904 SNVs between RIF patients and control subjects. These HOTAIR variants were associated with serum hemoglobin (Hgb), luteinizing hormone (LH), total cholesterol (T. chol), and blood urea nitrogen (BUN) levels, as assessed by analysis of variance (ANOVA). We analyzed the four HOTAIR SNVs and found significant differences in haplotype patterns between RIF patients and healthy controls. The results of this study showed that HOTAIR is not only associated with the development of cancer but also with pregnancy-associated diseases. This study represents the first report showing that HOTAIR is correlated with RIF.

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Rare single-nucleotide DAB1 variants and their contribution to Schizophrenia and autism spectrum disorder susceptibility

Human Genome Variation ◽

10.1038/s41439-020-00125-7 ◽

2020 ◽

Vol 7 (1) ◽

Author(s):

Yoshihiro Nawa ◽

Hiroki Kimura ◽

Daisuke Mori ◽

Hidekazu Kato ◽

Miho Toyama ◽

...

Keyword(s):

Rare Variants ◽

Adaptor Protein ◽

Autism Spectrum ◽

Case Group ◽

Missense Mutations ◽

Single Nucleotide Variants ◽

Single Nucleotide ◽

Next Generation Sequencing Technology ◽

A Minor

AbstractDisabled 1 (DAB1) is an intracellular adaptor protein in the Reelin signaling pathway and plays an essential role in correct neuronal migration and layer formation in the developing brain. DAB1 has been repeatedly reported to be associated with neurodevelopmental disorders including schizophrenia (SCZ) and autism spectrum disorders (ASD) in genetic, animal, and postmortem studies. Recently, increasing attention has been given to rare single-nucleotide variants (SNVs) found by deep sequencing of candidate genes. In this study, we performed exon-targeted resequencing of DAB1 in 370 SCZ and 192 ASD patients using next-generation sequencing technology to identify rare SNVs with a minor allele frequency <1%. We detected two rare missense mutations (G382C, V129I) and then performed a genetic association study in a sample comprising 1763 SCZ, 380 ASD, and 2190 healthy control subjects. Although no statistically significant association with the detected mutations was observed for either SCZ or ASD, G382C was found only in the case group, and in silico analyses and in vitro functional assays suggested that G382C alters the function of the DAB1 protein. The rare variants of DAB1 found in the present study should be studied further to elucidate their potential functional relevance to the pathophysiology of SCZ and ASD.

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Comparison of biofilm dispersal approaches in Pseudomonas aeruginosa and evaluation of dispersed cells in acute infection mouse model

10.1101/689646 ◽

2019 ◽

Cited By ~ 1

Author(s):

Rohit Ruhal ◽

Fien De Winter ◽

Bart de Jonger ◽

Basil Britto Xavier ◽

Christine lammens ◽

...

Keyword(s):

Mouse Model ◽

Acute Infection ◽

Experimental Conditions ◽

Significance Level ◽

Expression Of Genes ◽

Similar Phenotype ◽

Biofilm Dispersal ◽

Low Efficiency ◽

Dispersed Cells

ABSTRACTBiofilm dispersal is biologically significant process to fully understand its consequences during biofilm based infections. The mechanism for biofilm dispersal may involve response to environmental cues and changes in intracellular secondary messengers. Considering range of cues for biofilm dispersal, it is significant to study if dispersed cells generated by different methods have similar phenotype. In the present study, we have compared four type of biofilm dispersal in P. aeruginosa based in response to environment cues (starvation and nutrient rich), c-di-GMP knockdown and signaling molecule NO. We have determined their dispersal efficiency, susceptibility of dispersed cells for antibiotics, their transcriptomic changes compared to planktonic and biofilm. We also determined if dispersed cells can cause acute infections in mouse models. In vitro experiments showed that NO and c-di-GMP based dispersal methods had high biofilm reduction efficiency of 50 and 75% respectively, however, the nutrient induced dispersion showed low efficiency (≈30%) and more tolerance to colistin. We also showed that in vitro dispersed cells induced >10-fold transcriptomic expression of genes (at significance level of p < 0.005) related to efflux pumps (mexCD-oprJ), antibiotic resistance (arnDET) in dispersed cells induced by NO and nutrient change, while denitrification pathway and virulence (T3SS, VreI, T2SS) genes in all dispersed cells compared to planktonic and biofilm state. When the ability of the dispersed cells was tested in mouse model of lung infection, c-di-GMP and NO based dispersed cells displayed enhanced infections and haematogenous spread to liver and spleen and higher mortality. Although degree of immune response (cytokines) does not differ based on phenotypes inoculated in our experimental conditions. Based on our data most efficient dispersal methods increase murine mortality. This indicates their capability of making biofilm associated infection more complicated. Our data encourage to be more careful for studies suggesting biofilm dispersal during treatment of biofilm infections.

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Multi-omics analysis to identify driving factors in colorectal cancer

Epigenomics ◽

10.2217/epi-2020-0073 ◽

2020 ◽

Vol 12 (18) ◽

pp. 1633-1650

Author(s):

Xi Xu ◽

Chaoju Gong ◽

Yunfeng Wang ◽

Yanyan Hu ◽

Hong Liu ◽

...

Keyword(s):

Colorectal Cancer ◽

Molecular Mechanisms ◽

Copy Number Variations ◽

The Cancer Genome Atlas ◽

Single Nucleotide Variants ◽

Single Nucleotide ◽

Omics Analysis ◽

Competing Endogenous Rna Network ◽

Cancer Genome Atlas

Aim: We aim to identify driving genes of colorectal cancer (CRC) through multi-omics analysis. Materials & methods: We downloaded multi-omics data of CRC from The Cancer Genome Atlas dataset. Integrative analysis of single-nucleotide variants, copy number variations, DNA methylation and differentially expressed genes identified candidate genes that carry CRC risk. Kernal genes were extracted from the weighted gene co-expression network analysis. A competing endogenous RNA network composed of CRC-related genes was constructed. Biological roles of genes were further investigated in vitro. Results: We identified LRRC26 and REP15 as novel prognosis-related driving genes for CRC. LRRC26 hindered tumorigenesis of CRC in vitro. Conclusion: Our study identified novel driving genes and may provide new insights into the molecular mechanisms of CRC.

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Comprehensive Custom NGS Panel Validation for the Improvement of the Stratification of B-Acute Lymphoblastic Leukemia Patients

Journal of Personalized Medicine ◽

10.3390/jpm10030137 ◽

2020 ◽

Vol 10 (3) ◽

pp. 137

Author(s):

Adrián Montaño ◽

Jesús Hernández-Sánchez ◽

Maribel Forero-Castro ◽

María Matorra-Miguel ◽

Eva Lumbreras ◽

...

Keyword(s):

Acute Lymphoblastic Leukemia ◽

Wide Spectrum ◽

Lymphoblastic Leukemia ◽

Point Mutations ◽

Genetic Alterations ◽

Copy Number Variations ◽

Single Step ◽

Fusion Genes ◽

Single Nucleotide Variants ◽

Single Nucleotide

Background: B-acute lymphoblastic leukemia (B-ALL) is a hematological neoplasm of the stem lymphoid cell of the B lineage, characterized by the presence of genetic alterations closely related to the course of the disease. The number of alterations identified in these patients grows as studies of the disease progress, but in clinical practice, the conventional techniques frequently used are only capable of detecting the most common alterations. However, techniques, such as next-generation sequencing (NGS), are being implemented to detect a wide spectrum of new alterations that also include point mutations. Methods: In this study, we designed and validated a comprehensive custom NGS panel to detect the main genetic alterations present in the disease in a single step. For this purpose, 75 B-ALL diagnosis samples from patients previously characterized by standard-of-care diagnostic techniques were sequenced. Results: The use of the custom NGS panel allowed the correct detection of the main genetic alterations present in B-ALL patients, including the presence of an aneuploid clone in 14 of the samples and some of the recurrent fusion genes in 35 of the samples. The panel was also able to successfully detect a number of secondary alterations, such as single nucleotide variants (SNVs) and copy number variations (CNVs) in 66 and 46 of the samples analyzed, respectively, allowing for further refinement of the stratification of patients. The custom NGS panel could also detect alterations with a high level of sensitivity and reproducibility when the findings obtained by NGS were compared with those obtained from other conventional techniques. Conclusions: The use of this custom NGS panel allows us to quickly and efficiently detect the main genetic alterations present in B-ALL patients in a single assay (SNVs and insertions/deletions (INDELs), recurrent fusion genes, CNVs, aneuploidies, and single nucleotide polymorphisms (SNPs) associated with pharmacogenetics). The application of this panel would thus allow us to speed up and simplify the molecular diagnosis of patients, helping patient stratification and management.

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Neuroblastoma with flat genomic profile: a question of representativity?

BMJ Case Reports ◽

10.1136/bcr-2018-225568 ◽

2018 ◽

pp. bcr-2018-225568

Author(s):

Anders Valind ◽

Ingrid Öra ◽

Fredrik Mertens ◽

David Gisselsson

Keyword(s):

Single Nucleotide Polymorphism Array ◽

Reference Sample ◽

Genetic Alterations ◽

Nucleotide Polymorphism ◽

Single Nucleotide Variants ◽

Array Analysis ◽

Allelic Profile ◽

Single Nucleotide ◽

Whole Exome ◽

Genome Copy Number

Neuroblastoma is one of the most common paediatric malignancies. Detection of somatic genetic alterations in this tumour is instrumental for its risk stratification and treatment. On the other hand, an absence of detected chromosomal imbalances in neuroblastoma biopsies is difficult to interpret because it is unclear whether this situation truly reflects the tumour genome or if it is due to suboptimal sampling. We here present a neuroblastoma in the left adrenal of a newborn. The tumour was subjected to single-nucleotide polymorphism array analysis of five tumour regions with >80% tumour cells in histological mirror sections. This revealed no aberrations compared with a normal reference sample from the patient. Whole exome sequencing identified two single-nucleotide variants present in most tumour regions, corroborating that the tumour resulted from monoclonal expansion. Our data provide proof-of-principle that rare cases of neuroblastoma can have a normal whole genome copy number and allelic profile.

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Effects of FSHR and FSHB Variants on Hormonal Profile and Reproductive Outcomes of Infertile Women With Endometriosis

Frontiers in Endocrinology ◽

10.3389/fendo.2021.760616 ◽

2021 ◽

Vol 12 ◽

Author(s):

Bianca Bianco ◽

Flavia Altheman Loureiro ◽

Camila Martins Trevisan ◽

Carla Peluso ◽

Denise Maria Christofolini ◽

...

Keyword(s):

Cross Sectional Study ◽

Reproductive Outcomes ◽

Single Nucleotide Variants ◽

Cross Sectional ◽

Single Nucleotide ◽

Hormonal Profile ◽

Ivf Outcomes ◽

Mild Disease ◽

Dimensionality Reduction Method

BackgroundSingle nucleotide variants (SNVs) FSHB:c.-211G>T, FSHR:c.919G>A, and FSHR:c.2039G>A were reported to be associated with the variability in FSH and LH levels, and in vitro fertilization (IVF) outcomes. In this study, we aimed to evaluate the effects of FSHB:c.-211G>T, FSHR:c.919G>A, and FSHR:c.2039G>A variants, alone and combined, on the hormonal profile and reproduction outcomes of women with endometriosis.MethodsA cross-sectional study was performed comprising 213 infertile Brazilian women with endometriosis who underwent IVF treatment. Genotyping was performed using TaqMan real-time PCR. Variables were compared according to the genotypes of each variant and genetic models, and the combined effects of the SNVs were evaluated using the multifactorial dimensionality reduction method.ResultsFSHB:c.-211G>T affected LH levels in women with overall endometriosis and minimal/mild disease. FSHR:c.919G>A affected FSH levels in women with overall endometriosis and the number of oocytes retrieved in those with moderate/severe endometriosis. Moreover, the FSHR:c.2039G>A affected FSH levels in women with overall endometriosis, LH levels and total amount of rFSH in those with minimal/mild disease, and number of follicles and number of oocytes retrieved in those with moderate/severe endometriosis. No effect on hormone profile or reproductive outcomes was observed when the genotypes were combined.ConclusionsVariants of the FSHB and FSHR genes separately interfered with the hormonal profiles and IVF outcomes of women with endometriosis.

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Heteroduplex DNA correction in Saccharomyces cerevisiae is mismatch specific and requires functional PMS genes

Molecular and Cellular Biology ◽

10.1128/mcb.9.10.4432-4440.1989 ◽

1989 ◽

Vol 9 (10) ◽

pp. 4432-4440

Author(s):

B Kramer ◽

W Kramer ◽

M S Williamson ◽

S Fogel

Keyword(s):

Saccharomyces Cerevisiae ◽

Mismatch Repair ◽

Dna Mismatch Repair ◽

Evolutionary Relationship ◽

Close Correlation ◽

Single Nucleotide ◽

Dna Mismatch ◽

Low Efficiency ◽

Specificity Pattern

In vitro-constructed heteroduplex DNAs with defined mismatches were corrected in Saccharomyces cerevisiae cells with efficiencies that were dependent on the mismatch. Single-nucleotide loops were repaired very efficiently; the base/base mismatches G/T, A/C, G/G, A/G, G/A, A/A, T/T, T/C, and C/T were repaired with a high to intermediate efficiency. The mismatch C/C and a 38-nucleotide loop were corrected with low efficiency. This substrate specificity pattern resembles that found in Escherichia coli and Streptococcus pneumoniae, suggesting an evolutionary relationship of DNA mismatch repair in pro- and eucaryotes. Repair of the listed mismatches was severely impaired in the putative S. cerevisiae DNA mismatch repair mutants pms1 and pms2. Low-efficiency repair also characterized pms3 strains, except that correction of single-nucleotide loops occurred with an efficiency close to that of PMS wild-type strains. A close correlation was found between the repair efficiencies determined in this study and the observed postmeiotic segregation frequencies of alleles with known DNA sequence. This suggests an involvement of DNA mismatch repair in recombination and gene conversion in S. cerevisiae.

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