scholarly journals AsCRISPR: a web server for allele-specific sgRNA design in precision medicine

2019 ◽  
Author(s):  
Guihu Zhao ◽  
Jinchen Li ◽  
Yu Tang
Keyword(s):  
Nucleotide Polymorphisms ◽  
Single Nucleotide Variants ◽  
Single Nucleotide ◽  
Guide Rna ◽  
Inherited Diseases ◽  
Bioinformatic Tools ◽  
Point Of Entry ◽  
Allele Specific ◽  
Sgrna Design ◽  
Specific Restriction

AbstractAllele-specific genomic targeting by CRISPR provides a point of entry for personalized gene therapy of dominantly inherited diseases, by selectively disrupting the mutant alleles or disease-causing single nucleotide polymorphisms (SNPs), ideally while leaving normal alleles intact. Moreover, the allele-specific engineering has been increasingly exploited not only in treating inherited diseases and mutation-driven cancers, but also in other important fields such as genome imprinting, haploinsufficiency, genome loci imaging and immunocompatible manipulations. Despite the tremendous utilities of allele-specific targeting by CRISPR, very few bioinformatic tools have been implemented for the allele-specific purpose. We thus developed AsCRISPR (Allele-specific CRISPR), a web tool to aid the design of guide RNA (gRNA) sequences that can discriminate between alleles. It provides users with limited bioinformatics skills to analyze both their own identified variants and heterozygous SNPs deposited in the dbSNP database. Multiple CRISPR nucleases and their engineered variants including newly-developed Cas12b and CasX are included for users’ choice. Meanwhile, AsCRISPR evaluates the on-target efficiencies, specificities and potential off-targets of gRNA candidates, and also displays the allele-specific restriction enzyme sites that might be disrupted upon successful genome edits. In addition, AsCRISPR analyzed with dominant single nucleotide variants (SNVs) retrieved from ClinVar and OMIM databases, and generated a Dominant Database of candidate discriminating gRNAs that may specifically target the alternative allele for each dominant SNV site. A Validated Database was also established, which manually curated the discriminating gRNAs that were experimentally validated in the mounting literatures. AsCRISPR is freely available at http://www.genemed.tech/ascrispr.

scholarly journals CrisPam: SNP-derived PAM analysis web tool and human pathogenic SNPs database for CRISPR allele-specific targeting

2019 ◽  
Author(s):  
Roy Rabinowitz ◽  
Roy Darnell ◽  
Daniel Offen
Keyword(s):  
Dna Cleavage ◽  
Variant Allele ◽  
Nucleotide Polymorphisms ◽  
Single Nucleotide ◽  
Guide Rna ◽  
Dna Targeting ◽  
Specific Targeting ◽  
Novel Technology ◽  
Protospacer Adjacent Motif ◽  
Allele Specific

Abstract Background CRISPR is a promising novel technology for treating genetic conditions. Therefore, it is essential to further develop and promote treatment’s safety and specificity. While the guide-RNA offers position-specific DNA targeting, it may tolerate small changes such as single-nucleotide polymorphisms (SNPs). To that end, an allele-specific targeting approach is in need for future treatments of heterozygous patients, suffering from genetic conditions caused by a SNP. The SNP-derived PAM approach allows highly allele-specific DNA cleavage by incorporating a protospacer adjacent motif (PAM) sequence only at the target allele. Description Here we present CrisPam, a tool that detects SNP-derived PAMs for allele-specific targeting by the CRISPR/Cas system. The algorithm scans the generation of each reported PAM for a given DNA sequence and its variations. A successful result is such that at least one PAM is generated by a SNP. Thus, the PAM shall be part of the variant allele only and the Cas protein will therefore be able to exclusively bind the variant allele for gene-editing, while the wildtype allele remains unchanged. Conclusion CrisPam is available online for researchers and also offers access to the CrisPamDB, a database that contains the CrisPam analysis for any reported pathogenic SNP in humans.

scholarly journals Investigation of allele specific expression in various tissues of broiler chickens using the detection tool VADT

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
M. Joseph Tomlinson ◽  
Shawn W. Polson ◽  
Jing Qiu ◽  
Juniper A. Lake ◽  
William Lee ◽  
...  
Keyword(s):  
Broiler Chickens ◽  
Nucleotide Polymorphisms ◽  
Rna Seq ◽  
Specific Expression ◽  
Single Nucleotide ◽  
Allele Specific Expression ◽  
Detection Tool ◽  
Commercial Broiler ◽  
Significant Phenomenon ◽  
Allele Specific

AbstractDifferential abundance of allelic transcripts in a diploid organism, commonly referred to as allele specific expression (ASE), is a biologically significant phenomenon and can be examined using single nucleotide polymorphisms (SNPs) from RNA-seq. Quantifying ASE aids in our ability to identify and understand cis-regulatory mechanisms that influence gene expression, and thereby assist in identifying causal mutations. This study examines ASE in breast muscle, abdominal fat, and liver of commercial broiler chickens using variants called from a large sub-set of the samples (n = 68). ASE analysis was performed using a custom software called VCF ASE Detection Tool (VADT), which detects ASE of biallelic SNPs using a binomial test. On average ~ 174,000 SNPs in each tissue passed our filtering criteria and were considered informative, of which ~ 24,000 (~ 14%) showed ASE. Of all ASE SNPs, only 3.7% exhibited ASE in all three tissues, with ~ 83% showing ASE specific to a single tissue. When ASE genes (genes containing ASE SNPs) were compared between tissues, the overlap among all three tissues increased to 20.1%. Our results indicate that ASE genes show tissue-specific enrichment patterns, but all three tissues showed enrichment for pathways involved in translation.

Genes Regulating Immune Response and Amelogenesis Interact in Increasing the Susceptibility to Molar-Incisor Hypomineralization

2018 ◽  
Vol 53 (2) ◽  
pp. 217-227 ◽  
Author(s):  
Diego Girotto Bussaneli ◽  
Manuel Restrepo ◽  
Camila Maria Bullio Fragelli ◽  
Lourdes Santos-Pinto ◽  
Fabiano Jeremias ◽  
...  
Keyword(s):  
Immune Response ◽  
Gene Interactions ◽  
Nucleotide Polymorphisms ◽  
Single Nucleotide ◽  
Nuclear Families ◽  
Molar Incisor Hypomineralization ◽  
Inflammatory Stimuli ◽  
Allele Specific ◽  
Family Based ◽  
And Function

Ameloblasts are sensitive cells whose metabolism and function may be affected by inflammatory stimuli. The aim of this study was to evaluate the possible association between polymorphisms in immune response-related genes and molar-incisor hypomineralization (MIH), and their interaction with polymorphisms in amelogenesis-related genes. DNA samples were obtained from 101 nuclear families that had at least 1 MIH-affected child. Eleven single-nucleotide polymorphisms (SNPs) were investigated in immune response genes using TaqMan® technology allele-specific probes. A transmission disequilibrium test was performed to verify overtransmission of alleles in all MIH families, as well as in families only with mild or severe MIH-affected children. Gene-gene interactions between the immune-related and amelogenesis-related polymorphisms were analyzed by determining whether alleles of those genes were transmitted from heterozygous parents more often in association than individually with MIH-affected children. In severe cases of MIH, significant results were observed for rs10733708 (TGFBR1, OR = 3.5, 95% CI = 1.1–10.6). Statistical evidence for gene-gene interactions between rs6654939 (AMELX) and the SNPs rs2070874 (IL4), rs2275913 (IL17A), rs1800872 (IL10), rs1800587 (IL1A), and rs3771300 (STAT1) was observed. The rs2070874 SNP (IL4) was also significantly overtransmitted from heterozygous parents with the rs7526319 (TUFT1) and the rs2355767 (BMP2) SNPs, suggesting a synergistic effect of the transmission of these alleles with susceptibility to MIH. This family-based study demonstrated an association between variation in TGFBR1 and MIH. Moreover, the polymorphisms in immune response and amelogenesis genes may have an additive effect on the risk of developing MIH.

scholarly journals Impact of Pre and Post Variant Filtration Strategies on Imputation

2020 ◽  
Author(s):  
Celine Charon ◽  
Rodrigue Allodji ◽  
Vincent Meyer ◽  
Jean-François Deleuze
Keyword(s):  
Quality Control ◽  
Rare Variants ◽  
Association Studies ◽  
Genome Wide Association Studies ◽  
Nucleotide Polymorphisms ◽  
Direct Effects ◽  
Single Nucleotide Variants ◽  
Single Nucleotide ◽  
Genome Wide ◽  
Conservative Post

Abstract Quality control methods for genome-wide association studies and fine mapping are commonly used for imputation, however, they result in loss of many single nucleotide polymorphisms (SNPs). To investigate the consequences of filtration on imputation, we studied the direct effects on the number of markers, their allele frequencies, imputation quality scores and post-filtration events. We pre-phrased 1,031 genotyped individuals from diverse ethnicities and compared the imputed variants to 1,089 NCBI recorded individuals for additional validation.Without variant pre-filtration based on quality control (QC), we observed no impairment in the imputation of SNPs that failed QC whereas with pre-filtration there was an overall loss of information. Significant differences between frequencies with and without pre-filtration were found only in the range of very rare (5E-04-1E-03) and rare variants (1E-03-5E-03) (p < 1E-04). Increasing the post-filtration imputation quality score from 0.3 to 0.8 reduced the number of single nucleotide variants (SNVs) <0.001 2.5 fold with or without QC pre-filtration and halved the number of very rare variants (5E-04). As a result, to maintain confidence and enough SNVs, we propose here a 2-step post-filtration approach to increase the number of very rare and rare variants compared to conservative post-filtration methods.

scholarly journals A Novel Test System for Genotyping rs43703016 Single-nucleotide Substitutions in the Bovine CSN3 Gene

2019 ◽  
pp. 1-5
Author(s):  
Svetlana Kovalchuk ◽  
Arina Tagmazyan ◽  
Eugene Klimov
Keyword(s):  
Nucleotide Substitution ◽  
Milk Proteins ◽  
Test System ◽  
Nucleotide Polymorphisms ◽  
Dna Testing ◽  
Nucleotide Substitutions ◽  
Single Nucleotide ◽  
Allele Specific ◽  
Selection Of

Aims: Caseins are among the main milk proteins that determine many of its properties. Bovine kappa-casein (CSN3) is associated with the qualitative composition of milk, as well as with the quality of cheese obtained from this milk. The rs43703016 single-nucleotide substitution (g.88532332A>C; Asp148Ala) in exon 4 of the bovine CSN3 gene plays an important role in the production of quality hard cheeses. Various methods for the DNA testing of this substitution have been developed in the last three decades. Emergent DNA technologies provide an opportunity to modernize methods of genotyping single-nucleotide polymorphisms. Results: We have developed and verified a method to differentiate A/C alleles of the rs43703016 substitution in the bovine CSN3 gene by real-time PCR using allele-specific fluorescent probes. Conclusion: Our new method allows fast genotyping of animals, and may be used for selection of cows carrying the CC genotype, which determines good cheese-making properties of milk.

scholarly journals Improving the Precision of Base Editing by Bubble Hairpin Single Guide RNA

mBio ◽  
2021 ◽  
Vol 12 (2) ◽  
Author(s):  
Zhiwei Hu ◽  
Yannan Wang ◽  
Qian Liu ◽  
Yan Qiu ◽  
Zhiyu Zhong ◽  
...  
Keyword(s):  
Genome Editing ◽  
Dna Breaks ◽  
Single Nucleotide ◽  
Base Editing ◽  
Guide Rna ◽  
Guide Rnas ◽  
Double Stranded Dna ◽  
Target Sites ◽  
Guide Sequence ◽  
Sgrna Design

ABSTRACT Base editing is a powerful genome editing approach that enables single-nucleotide changes without double-stranded DNA breaks (DSBs). However, off-target effects as well as other undesired editings at on-target sites remain obstacles for its application. Here, we report that bubble hairpin single guide RNAs (BH-sgRNAs), which contain a hairpin structure with a bubble region on the 5′ end of the guide sequence, can be efficiently applied to both cytosine base editor (CBE) and adenine base editor (ABE) and significantly decrease off-target editing without sacrificing on-target editing efficiency. Meanwhile, such a design also improves the purity of C-to-T conversions induced by base editor 3 (BE3) at on-target sites. Our results present a distinctive and effective strategy to improve the specificity of base editing. IMPORTANCE Base editors are DSB-free genome editing tools and have been widely used in diverse living systems. However, it is reported that these tools can cause substantial off-target editings. To meet this challenge, we developed a new approach to improve the specificity of base editors by using hairpin sgRNAs with a bubble. Furthermore, our sgRNA design also dramatically reduced indels and unwanted base substitutions at on-target sites. We believe that the BH-sgRNA design is a significant improvement over existing sgRNAs of base editors, and our design promises to be adaptable to various base editors. We expect that it will make contributions to improving the safety of gene therapy.

cDNA sequence of five mouse guanine deaminase (Gda) alleles and mapping to mouse chromosome 19

Genome ◽  
2002 ◽  
Vol 45 (2) ◽  
pp. 276-281
Author(s):  
Raymond D Giese ◽  
Floyd F Snyder
Keyword(s):  
Mouse Chromosome ◽  
Nucleotide Polymorphisms ◽  
Mus Spretus ◽  
Chromosome 19 ◽  
Single Nucleotide ◽  
Guanine Deaminase ◽  
Key Words Mouse ◽  
Allele Specific ◽  
Guanine Base ◽  
Codon Change

Guanine deaminase catalyses the conversion of guanine to xanthine and ammonia, thereby irreversibly removing the guanine base from the pool of guanine-containing metabolites. We have identified five alleles at the mouse guanine deaminase locus by cDNA sequencing. These alleles were defined by single-nucleotide polymorphisms at a total of 19 positions. For each allele the representative strains are as follows: Gdaa, C57BL/6J and DBA/2J; Gdab, A/J; Gdac, MOLF/Ei; Gdad, CAST/Ei; and Gdae, SPRET-1. The only codon change resulting in an amino acid substitution was found at nucleotide 523, where GAT was replaced by AAT in Mus spretus resulting in the deduced substitution of Asp-174 by Asn. The single-nucleotide difference between the a and b alleles was also typed by allele-specific oligonucleotide amplification for 17 common strains of Mus musculus susbp. musculus. By typing the A×B and B×A recombinant inbred (RI) strain sets, Gda was mapped to mouse chromosome 19, a region syntenic with human chromosome 9q11–q22.Key Words: mouse guanine deaminase, alleles, mapping.

Genetics of Schizophrenia and Bipolar Disorder

2017 ◽  
Author(s):  
Alexander Charney ◽  
Pamela Sklar
Keyword(s):  
Bipolar Disorder ◽  
Copy Number ◽  
Psychotic Disorders ◽  
Copy Number Variants ◽  
Nucleotide Polymorphisms ◽  
Common Variants ◽  
Single Nucleotide Variants ◽  
Single Nucleotide ◽  
Number Variation

Schizophrenia and bipolar disorder are the classic psychotic disorders. Both diseases are strongly familial, but have proven recalcitrant to genetic methodologies for identifying the etiology until recently. There is now convincing genetic evidence that indicates a contribution of many DNA changes to the risk of becoming ill. For schizophrenia, there are large contributions of rare copy number variants and common single nucleotide variants, with an overall highly polygenic genetic architecture. For bipolar disorder, the role of copy number variation appears to be much less pronounced. Specific common single nucleotide polymorphisms are associated, and there is evidence for polygenicity. Several surprises have emerged from the genetic data that indicate there is significantly more molecular overlap in copy number variants between autism and schizophrenia, and in common variants between schizophrenia and bipolar disorder.

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