scholarly journals Analysis of single nucleotide variants in CRISPR-Cas9 edited zebrafish embryos shows no evidence of off-target inflation

2019 ◽  
Author(s):  
Marie R Mooney ◽  
Erica E Davis ◽  
Nicholas Katsanis
Keyword(s):  
De Novo ◽  
Point Mutations ◽  
Enzyme Engineering ◽  
Single Nucleotide Variants ◽  
Guide Rna ◽  
Whole Exome ◽  
Two Generations ◽  
Sgrna Design ◽  
Favorable Conditions

AbstractTherapeutic applications of CRISPR-Cas9 gene editing have spurred innovation in Cas9 enzyme engineering and single guide RNA (sgRNA) design algorithms to minimize potential off-target events. While recent work in rodents outlines favorable conditions for specific editing and uses a trio design to control for the contribution of natural genome variation, the potential for CRISPR-Cas9 to induce de novo mutations in vivo remains a topic of interest. In zebrafish, we performed whole exome sequencing (WES) on two generations of offspring derived from the same founding pair: 54 exomes from control and CRISPR-Cas9 edited embryos in the first generation (F0), and 16 exomes from the progeny of inbred F0 pairs in the second generation (F1). We did not observe an increase in the number of transmissible variants in edited individuals in F1, nor in F0 edited mosaic individuals, arguing that in vivo editing does not precipitate an inflation of deleterious point mutations.

scholarly journals Extending the Coding Potential of Viral Genomes with Overlapping Antisense ORFs: A Case for the De Novo Creation of the Gene Encoding the Antisense Protein ASP of HIV-1

Viruses ◽  
2022 ◽  
Vol 14 (1) ◽  
pp. 146
Author(s):  
Angelo Pavesi ◽  
Fabio Romerio
Keyword(s):  
De Novo ◽  
Point Mutations ◽  
Selective Advantage ◽  
Genomic Region ◽  
Fine Tuning ◽  
Sequence Evolution ◽  
Viral Genomes ◽  
Stop Codons ◽  
Hiv 1

Gene overprinting occurs when point mutations within a genomic region with an existing coding sequence create a new one in another reading frame. This process is quite frequent in viral genomes either to maximize the amount of information that they encode or in response to strong selective pressure. The most frequent scenario involves two different reading frames in the same DNA strand (sense overlap). Much less frequent are cases of overlapping genes that are encoded on opposite DNA strands (antisense overlap). One such example is the antisense ORF, asp in the minus strand of the HIV-1 genome overlapping the env gene. The asp gene is highly conserved in pandemic HIV-1 strains of group M, and it is absent in non-pandemic HIV-1 groups, HIV-2, and lentiviruses infecting non-human primates, suggesting that the ~190-amino acid protein that is expressed from this gene (ASP) may play a role in virus spread. While the function of ASP in the virus life cycle remains to be elucidated, mounting evidence from several research groups indicates that ASP is expressed in vivo. There are two alternative hypotheses that could be envisioned to explain the origin of the asp ORF. On one hand, asp may have originally been present in the ancestor of contemporary lentiviruses, and subsequently lost in all descendants except for most HIV-1 strains of group M due to selective advantage. Alternatively, the asp ORF may have originated very recently with the emergence of group M HIV-1 strains from SIVcpz. Here, we used a combination of computational and statistical approaches to study the genomic region of env in primate lentiviruses to shed light on the origin, structure, and sequence evolution of the asp ORF. The results emerging from our studies support the hypothesis of a recent de novo addition of the antisense ORF to the HIV-1 genome through a process that entailed progressive removal of existing internal stop codons from SIV strains to HIV-1 strains of group M, and fine tuning of the codon sequence in env that reduced the chances of new stop codons occurring in asp. Altogether, the study supports the notion that the HIV-1 asp gene encodes an accessory protein, providing a selective advantage to the virus.

scholarly journals De novo structural mutation rates and gamete-of-origin biases revealed through genome sequencing of 2,396 families

2020 ◽  
Author(s):  
Jonathan R. Belyeu ◽  
Harrison Brand ◽  
Harold Wang ◽  
Xuefang Zhao ◽  
Brent S. Pedersen ◽  
...  
Keyword(s):  
De Novo ◽  
Genome Structure ◽  
Point Mutations ◽  
Large Family ◽  
Germline Mutations ◽  
Autism Spectrum ◽  
Parental Age ◽  
Single Nucleotide Variants ◽  
Multiple Testing Correction ◽  
Structural Mutations

AbstractEach human genome includes de novo mutations that arose during gametogenesis. While these germline mutations represent a fundamental source of new genetic diversity, they can also create deleterious alleles that impact fitness. The germline mutation rate for single nucleotide variants and factors that significantly influence this rate, such as parental age, are now well established. However, far less is known about the frequency, distribution, and features that impact de novo structural mutations. We report a large, family-based study of germline mutations, excluding aneuploidy, that affect genome structure among 572 genomes from 33 families in a multigenerational CEPH-Utah cohort and 2,363 cases of non-familial autism spectrum disorder (ASD), 1,938 unaffected siblings, and both parents (9,599 genomes in total). We find that de novo structural mutations detected by alignment-based, short-read WGS occurred at an overall rate of at least 0.160 events per genome in unaffected individuals and was significantly higher (0.206 per genome) in ASD cases. In both probands and unaffected samples, nearly 73% of de novo structural mutations arose in paternal gametes, and predict most de novo structural mutations to be caused by mutational mechanisms that do not require sequence homology. After multiple testing correction we did not observe a statistically significant correlation between parental age and the rate of de novo structural variation in offspring. These results highlight that a spectrum of mutational mechanisms contribute to germline structural mutations, and that these mechanisms likely have markedly different rates and selective pressures than those leading to point mutations.

Molecular genetic analysis of Saccharomyces cerevisiae C1-tetrahydrofolate synthase mutants reveals a noncatalytic function of the ADE3 gene product and an additional folate-dependent enzyme

1990 ◽  
Vol 10 (11) ◽  
pp. 5679-5687
Author(s):  
C K Barlowe ◽  
D R Appling
Keyword(s):  
Saccharomyces Cerevisiae ◽  
De Novo ◽  
Point Mutations ◽  
Gene Replacement ◽  
Molecular Genetic Analysis ◽  
Yeast Cells ◽  
Yeast Saccharomyces Cerevisiae ◽  
Purine Synthesis

In eucaryotes, 10-formyltetrahydrofolate (formyl-THF) synthetase, 5,10-methenyl-THF cyclohydrolase, and NADP(+)-dependent 5,10-methylene-THF dehydrogenase activities are present on a single polypeptide termed C1-THF synthase. This trifunctional enzyme, encoded by the ADE3 gene in the yeast Saccharomyces cerevisiae, is thought to be responsible for the synthesis of the one-carbon donor 10-formyl-THF for de novo purine synthesis. Deletion of the ADE3 gene causes adenine auxotrophy, presumably as a result of the lack of cytoplasmic 10-formyl-THF. In this report, defined point mutations that affected one or more of the catalytic activities of yeast C1-THF synthase were generated in vitro and transferred to the chromosomal ADE3 locus by gene replacement. In contrast to ADE3 deletions, point mutations that inactivated all three activities of C1-THF synthase did not result in an adenine requirement. Heterologous expression of the Clostridium acidiurici gene encoding a monofunctional 10-formyl-THF synthetase in an ade3 deletion strain did not restore growth in the absence of adenine, even though the monofunctional synthetase was catalytically competent in vivo. These results indicate that adequate cytoplasmic 10-formyl-THF can be produced by an enzyme(s) other than C1-THF synthase, but efficient utilization of that 10-formyl-THF for purine synthesis requires a nonenzymatic function of C1-THF synthase. A monofunctional 5,10-methylene-THF dehydrogenase, dependent on NAD+ for catalysis, has been identified and purified from yeast cells (C. K. Barlowe and D. R. Appling, Biochemistry 29:7089-7094, 1990). We propose that the characteristics of strains expressing full-length but catalytically inactive C1-THF synthase could result from the formation of a purine-synthesizing multienzyme complex involving the structurally unchanged C1-THF synthase and that production of the necessary one-carbon units in these strains is accomplished by an NAD+ -dependent 5,10-methylene-THF dehydrogenase.

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 Genotypes and Phenotypes of MEF2C Haploinsufficiency Syndrome: New Cases and Novel Point Mutations

2021 ◽  
Vol 9 ◽  
Author(s):  
Lin Wan ◽  
Xinting Liu ◽  
Linyan Hu ◽  
Huimin Chen ◽  
Yulin Sun ◽  
...  
Keyword(s):  
De Novo ◽  
Neurodevelopmental Disorder ◽  
Point Mutations ◽  
Clinical Manifestations ◽  
Poor Performance ◽  
Genetic Alterations ◽  
Mendelian Inheritance ◽  
Whole Exome ◽  
Patients With Epilepsy ◽  
Free Status

Aim: MEF2C haploinsufficiency syndrome (MCHS) is a severe neurodevelopmental disorder. We describe the clinical phenotypes and genotypes of seven patients with MCHS to enhance the understanding of clinical manifestations and genetic alterations associated with MCHS.Method: Seven patients (6 females and 1 male, aged between 2 years 5 months and 6 years) who had MEF2C mutations, and their parents underwent trio-based whole-exome sequencing; subsequently, their clinical features were assessed. A literature review of patients with MCHS was performed by searching the PubMed and Online Mendelian Inheritance in Man databases.Results: Seven mutations were identified, of which six were unreported in the past; of the reported cases, five patients had de novo mutations but two had an undefined inheritance pattern. All patients presented delays in developmental milestones, severe intellectual disabilities and lack of speech. Six patients exhibited infantile hypotonia, five patients experienced stereotypic movements and were unable to walk, four patients exhibited poor eye contact indicative of autism and two showed poor performance. While six patients experienced seizure, five among them became seizure free after receiving anti-seizure medicine. Three patients showed a regression in their development, whereas the mothers of two patients exhibited mosaicism but were healthy without any abovementioned symptoms.Interpretation: Regression was not a common phenomenon but occurred in MCHS. The prognosis of MCHS patients with epilepsy was good, but most patients can achieve a seizure-free status. Healthy people may have low-level mosaicism and carry a pathogenic MEF2C mutation.

scholarly journals A Novel WT1 Mutation Identified in a 46,XX Testicular/Ovotesticular DSD Patient Results in the Retention of Intron 9

Biology ◽  
2021 ◽  
Vol 10 (12) ◽  
pp. 1248
Author(s):  
Dmytro Sirokha ◽  
Olexandra Gorodna ◽  
Yakov Vitrenko ◽  
Nataliya Zelinska ◽  
Rafal Ploski ◽  
...  
Keyword(s):  
Splice Site ◽  
Rna Binding ◽  
De Novo ◽  
Zinc Fingers ◽  
Point Mutations ◽  
Whole Exome ◽  
Alternative Donor ◽  
Wt1 Mutation ◽  
Wt1 Protein ◽  
Genetic Rearrangements

The 46,XX testicular DSD (disorder/difference of sexual development) and 46,XX ovotesticular DSD (46,XX TDSD and 46,XX OTDSD) phenotypes are caused by genetic rearrangements or point mutations resulting in imbalance between components of the two antagonistic, pro-testicular and pro-ovarian pathways; however, the genetic causes of 46,XX TDSD/OTDSD are not fully understood, and molecular diagnosis for many patients with the conditions is unavailable. Only recently few mutations in the WT1 (WT1 transcription factor; 11p13) gene were described in a group of 46,XX TDSD and 46,XX OTDSD individuals. The WT1 protein contains a DNA/RNA binding domain consisting of four zinc fingers (ZnF) and a three-amino acid (KTS) motif that is present or absent, as a result of alternative splicing, between ZnF3 and ZnF4 (±KTS isoforms). Here, we present a patient with 46,XX TDSD/OTDSD in whom whole exome sequencing revealed a heterozygous de novo WT1 c.1437A>G mutation within an alternative donor splice site which is used for −KTS WT1 isoform formation. So far, no mutation in this splice site has been identified in any patient group. We demonstrated that the mutation results in the retention of intron 9 in the mature mRNA of the 46,XX TDSD/OTDSD patient. In cases when the erroneous mRNA is translated, exclusively the expression of a truncated WT1 +KTS protein lacking ZnF4 and no −KTS protein occurs from the mutated allele of the patient. We discuss potential mechanisms and pathways which can be disturbed upon two conditions: Absence of Zn4F and altered +KTS/−KTS ratio.

Genetic landscape of Rett syndrome-like phenotypes revealed by whole exome sequencing

2019 ◽  
Vol 56 (6) ◽  
pp. 396-407 ◽  
Author(s):  
Kazuhiro Iwama ◽  
Takeshi Mizuguchi ◽  
Eri Takeshita ◽  
Eiji Nakagawa ◽  
Tetsuya Okazaki ◽  
...  
Keyword(s):  
Exome Sequencing ◽  
Whole Exome Sequencing ◽  
Rett Syndrome ◽  
De Novo ◽  
Diagnostic Yield ◽  
Single Nucleotide Variants ◽  
Serine Threonine Kinase ◽  
Whole Exome ◽  
Genetic Landscape ◽  
Strong Candidate

BackgroundRett syndrome (RTT) is a characteristic neurological disease presenting with regressive loss of neurodevelopmental milestones. Typical RTT is generally caused by abnormality of methyl-CpG binding protein 2 (MECP2). Our objective to investigate the genetic landscape of MECP2-negative typical/atypical RTT and RTT-like phenotypes using whole exome sequencing (WES).MethodsWe performed WES on 77 MECP2-negative patients either with typical RTT (n=11), atypical RTT (n=22) or RTT-like phenotypes (n=44) incompatible with the RTT criteria.ResultsPathogenic or likely pathogenic single-nucleotide variants in 28 known genes were found in 39 of 77 (50.6%) patients. WES-based CNV analysis revealed pathogenic deletions involving six known genes (including MECP2) in 8 of 77 (10.4%) patients. Overall, diagnostic yield was 47 of 77 (61.0 %). Furthermore, strong candidate variants were found in four novel genes: a de novo variant in each of ATPase H+ transporting V0 subunit A1 (ATP6V0A1), ubiquitin-specific peptidase 8 (USP8) and microtubule-associated serine/threonine kinase 3 (MAST3), as well as biallelic variants in nuclear receptor corepressor 2 (NCOR2).ConclusionsOur study provides a new landscape including additional genetic variants contributing to RTT-like phenotypes, highlighting the importance of comprehensive genetic analysis.

Cytosine, but not adenine, base editors induce genome-wide off-target mutations in rice

Science ◽  
2019 ◽  
pp. eaaw7166 ◽  
Author(s):  
Shuai Jin ◽  
Yuan Zong ◽  
Qiang Gao ◽  
Zixu Zhu ◽  
Yanpeng Wang ◽  
...  
Keyword(s):  
High Fidelity ◽  
Single Nucleotide Variants ◽  
Disease Treatment ◽  
Single Nucleotide ◽  
Genetic Changes ◽  
Base Editing ◽  
Guide Rna ◽  
Genome Wide ◽  
Adenine Base

Cytosine and adenine base editors (CBEs and ABEs) are promising new tools for achieving the precise genetic changes required for disease treatment and trait improvement. However, genome-wide and unbiased analyses of their off-target effects in vivo are still lacking. Our whole genome sequencing (WGS) analysis of rice plants treated with BE3, high-fidelity BE3 (HF1-BE3), or ABE revealed that BE3 and HF1-BE3, but not ABE, induce substantial genome-wide off-target mutations, which are mostly the C→T type of single nucleotide variants (SNVs) and appear to be enriched in genic regions. Notably, treatment of rice with BE3 or HF1-BE3 in the absence of single-guide RNA also results in the rise of genome-wide SNVs. Thus, the base editing unit of BE3 or HF1-BE3 needs to be optimized in order to attain high fidelity.

Next Generation Sequencing and Cytogenetic Based Evaluation of Indian Pierre Robin Sequence Families Reveals CNV Regions of Modest Effect and a Novel LOXL3 Mutation

2021 ◽  
pp. 105566562110527
Author(s):  
Anubhuti Sood ◽  
Uzma Shamim ◽  
Om P. Kharbanda ◽  
Madhulika Kabra ◽  
Neerja Gupta ◽  
...  
Keyword(s):  
De Novo ◽  
Tertiary Care ◽  
Family Members ◽  
Point Mutations ◽  
Copy Number Variations ◽  
Care Hospital ◽  
Pierre Robin Sequence ◽  
Single Nucleotide Variants ◽  
Robin Sequence ◽  
Pierre Robin

Background Pierre Robin Sequence (PRS) affects approximately 1 per 8500 to 14000 new-borns worldwide. Although the clinical entity is well defined, the pathogenesis of PRS is debated. The present study aims to understand the contribution of genomic imbalances and genetic variants in patients clinically diagnosed of PRS. Methodology A total of 7 independent patients with nonsyndromic PRS thoroughly evaluated by a medical geneticist at a tertiary care hospital, were included in the study. Blood samples were collected from these patients and their family members. Array CGH was performed on all 7 patients and their respective family members for detection of underlying cytogenetic defects. Whole exome sequencing (WES) was performed for 5 families to capture single nucleotide variants or small indels. Results Cytogenetic analyses did not detect any previously reported gross chromosomal aberrations for PRS in the patient cohort. However, copy number variations (CNVs) of size <1 Mb were detected in patients which may have implications in PRS. The present study provided evidence for the occurrence of de novo deletions at 7p14.1 locus in PRS patients: further validating the candidate loci susceptibility in oral clefts. WES data identified LOXL3 as candidate gene, carrying novel deleterious variant, which is suggestive of the role of point mutations in the pathogenesis of PRS. Conclusion The present study offered considerable insight into the contribution of cytogenetic defects and novel point mutation in the etiology of nonsyndromic PRS. Studies comprising large number of cases are required to fully elucidate the genetic mechanisms underlying the PRS phenotype.

MYT1L mutation in a patient causes intellectual disability and early onset of obesity: a case report and review of the literature

2019 ◽  
Vol 32 (4) ◽  
pp. 409-413 ◽  
Author(s):  
Abeer Al Tuwaijri ◽  
Majid Alfadhel
Keyword(s):  
Intellectual Disability ◽  
Early Onset ◽  
De Novo ◽  
Copy Number Variants ◽  
Speech Delay ◽  
Single Nucleotide Variants ◽  
Genome Wide ◽  
Whole Exome ◽  
Syndromic Obesity ◽  
The Central Nervous System

Abstract Background Obesity has become one of the greatest health risks worldwide. Recently, there was an explosion of information regarding the role of the central nervous system (CNS) in the development of monogenic and syndromic obesity. Case presentation Over the last decade, terminal and interstitial submicroscopic deletions of copy number variants (CNVs) in 2p25.3 and single nucleotide variants (SNVs) in myelin transcription factor 1 like (MYT1L) were detected by genome-wide array analysis and whole exome sequencing (WES) in patients with a nonspecific clinical phenotype that commonly includes intellectual disability (ID), early onset of obesity and speech delay. Here, we report the first Saudi female patient with mild to moderate ID, early onset of obesity and speech delay associated with a de novo pathogenic SNV in the MYT1L gene (c. 1585G>A [Gly529Arg]), which causes an amino acid change from Gly to Arg at position 529 that leads to mental retardation, autosomal dominant 39.

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