scholarly journals A method for rapid selection of randomly induced mutations in a gene of interest using CRISPR/Cas9 mediated activation of gene expression

2019 ◽  
Author(s):  
William A. Ng ◽  
Andrew Ma ◽  
Molly Chen ◽  
Bruce H. Reed
Keyword(s):  
Gene Expression ◽  
Screening Strategy ◽  
Loss Of Function ◽  
Wild Type ◽  
Induced Mutations ◽  
Allelic Series ◽  
Lethal Mutations ◽  
F1 Progeny ◽  
Genetic Cross ◽  
Selection Of

AbstractWe have developed a CRISPR/Cas9 based method for isolating randomly induced recessive lethal mutations in a gene of interest (GOI) by selection within the F1 progeny of a single genetic cross. Our method takes advantage of the ability to overexpress a GOI using CRISPR/Cas9 mediated activation of gene expression. In essence, the screening strategy is based upon the idea that if overexpression of a wild type allele can generate a phenotype, then overexpression of a newly induced loss-of-function allele will lack this phenotype. As a proof-of-principle, we used this method to select EMS induced mutations of the Drosophila gene hindsight (hnt). From approximately 45,000 F1 progeny we recovered 8 new EMS induced loss-of-function hnt alleles that we characterized as an allelic series of hypomorphic mutations. This new method can, in theory, be used to recover randomly induced point mutants in a GOI and can be applied to any circumstance where CRISPR/Cas9 mediated activation of gene expression is associated with lethality or a visible phenotype.

scholarly journals A Method for Rapid Selection of Randomly Induced Mutations in a Gene of Interest Using CRISPR/Cas9 Mediated Activation of Gene Expression

2020 ◽  
Vol 10 (6) ◽  
pp. 1893-1901
Author(s):  
William A. Ng ◽  
Andrew Ma ◽  
Molly Chen ◽  
Bruce H. Reed
Keyword(s):  
Gene Expression ◽  
Screening Strategy ◽  
Loss Of Function ◽  
Wild Type ◽  
Induced Mutations ◽  
Allelic Series ◽  
Lethal Mutations ◽  
F1 Progeny ◽  
Genetic Cross ◽  
Selection Of

We have developed a CRISPR/Cas9 based method for isolating randomly induced recessive lethal mutations in a gene of interest (GOI) by selection within the F1 progeny of a single genetic cross. Our method takes advantage of the ability to overexpress a GOI using CRISPR/Cas9 mediated activation of gene expression. In essence, the screening strategy is based upon the idea that if overexpression of a wild type allele can generate a phenotype, then overexpression of a newly induced loss-of-function allele will lack this phenotype. As a proof-of-principle, we used this method to select EMS induced mutations of the Drosophila gene hindsight (hnt). From approximately 45,000 F1 progeny we recovered 8 new EMS induced loss-of-function hnt alleles that we characterized as an allelic series of hypomorphic mutations. This new method can, in theory, be used to recover randomly induced point mutants in a GOI and can be applied to any circumstance where CRISPR/Cas9 mediated activation of gene expression is associated with lethality or a visible phenotype.

scholarly journals Molecular and Phenotypic Analysis of 25 Recessive, Homozygous-Viable Alleles at the Mouse agouti Locus

Genetics ◽  
2002 ◽  
Vol 160 (2) ◽  
pp. 659-674
Author(s):  
Rosalynn J Miltenberger ◽  
Kazumasa Wakamatsu ◽  
Shosuke Ito ◽  
Richard P Woychik ◽  
Liane B Russell ◽  
...  
Keyword(s):  
Loss Of Function ◽  
Wild Type ◽  
Allelic Series ◽  
Phenotypic Analysis ◽  
Protein Coding ◽  
Agouti Locus ◽  
Expression Control ◽  
Gene Expression Control ◽  
Specific Locus

Abstract Agouti is a paracrine-acting, transient antagonist of melanocortin 1 receptors that specifies the subapical band of yellow on otherwise black hairs of the wild-type coat. To better understand both agouti structure/function and the germline damage caused by chemicals and radiation, an allelic series of 25 recessive, homozygous-viable agouti mutations generated in specific-locus tests were characterized. Visual inspection of fur, augmented by quantifiable chemical analysis of hair melanins, suggested four phenotypic categories (mild, moderate, umbrous-like, severe) for the 18 hypomorphs and a single category for the 7 amorphs (null). Molecular analysis indicated protein-coding alterations in 8 hypomorphs and 6 amorphs, with mild-moderate phenotypes correlating with signal peptide or basic domain mutations, and more devastating phenotypes resulting from C-terminal lesions. Ten hypomorphs and one null demonstrated wild-type coding potential, suggesting that they contain mutations elsewhere in the ≥125-kb agouti locus that either reduce the level or alter the temporal/spatial distribution of agouti transcripts. Beyond the notable contributions to the field of mouse germ cell mutagenesis, analysis of this allelic series illustrates that complete abrogation of agouti function in vivo occurs most often through protein-coding lesions, whereas partial loss of function occurs slightly more frequently at the level of gene expression control.

scholarly journals Analysis of the role of tra-1 in germline sex determination in the nematode Caenorhabditis elegans.

Genetics ◽  
1989 ◽  
Vol 123 (4) ◽  
pp. 755-769 ◽  
Author(s):  
T Schedl ◽  
P L Graham ◽  
M K Barton ◽  
J Kimble
Keyword(s):  
Caenorhabditis Elegans ◽  
Sex Determination ◽  
Germ Line ◽  
Temperature Sensitive ◽  
Loss Of Function ◽  
Wild Type ◽  
Allelic Series ◽  
Isolation And Characterization ◽  
Somatic Gonad

Abstract In wild-type Caenorhabditis elegans there are two sexes, self-fertilizing hermaphrodites (XX) and males (XO). To investigate the role of tra-1 in controlling sex determination in germline tissue, we have examined germline phenotypes of nine tra-1 loss-of-function (lf) mutations. Previous work has shown that tra-1 is needed for female somatic development as the nongonadal soma of tra-1(lf) XX mutants is masculinized. In contrast, the germline of tra-1(lf) XX and XO animals is often feminized; a brief period of spermatogenesis is followed by oogenesis, rather than the continuous spermatogenesis observed in wild-type males. In addition, abnormal gonadal (germ line and somatic gonad) phenotypes are observed which may reflect defects in development or function of somatic gonad regulatory cells. Analysis of germline feminization and abnormal gonadal phenotypes of the various mutations alone or in trans to a deficiency reveals that they cannot be ordered in an allelic series and they do not converge to a single phenotypic endpoint. These observations lead to the suggestion that tra-1 may produce multiple products and/or is autoregulated. One interpretation of the germline feminization is that tra-1(+) is necessary for continued specification of spermatogenesis in males. We also report the isolation and characterization of tra-1 gain-of-function (gf) mutations with novel phenotypes. These include temperature sensitive, recessive germline feminization, and partial somatic loss-of-function phenotypes.

Effect of ARID1a oncogenic loss of function mutations on overall survival in metastatic breast cancer patients.

2020 ◽  
Vol 38 (15_suppl) ◽  
pp. e13068-e13068
Author(s):  
Veronica Mariotti ◽  
Hatem Hussein Soliman
Keyword(s):  
Breast Cancer ◽  
Gene Expression ◽  
Metastatic Breast Cancer ◽  
Cancer Patients ◽  
Metastatic Breast ◽  
Missense Mutations ◽  
Breast Cancer Patients ◽  
Loss Of Function ◽  
Wild Type ◽  
Biologic Effect

e13068 Background: ARID1a (AT Rich Interactive Domain 1A) is part of the SWI/SNF complex, which regulates gene transcription. Its inactivation has been shown to determine resistance to endocrine therapy via enrichment of basal-like gene expression features [Xu G., 2020]. We recently reported that ARID1a lower gene expression might be associated with worse overall survival (OS) [Mariotti V., 2019]. The aim of this study was to analyze the effect of ARID1a mutations on survival in metastatic breast cancer patients (pts). Methods: Breast cancer patients prospectively consented for inclusion in the ORIEN genomic database with known metastatic disease were analyzed using cBioPortal. Predicted biologic effect of mutations from RNASeq data was determined by OncoKB. OS was calculated from initial breast cancer diagnosis to death from any cause. OS analysis using Kaplan Meier and descriptive statistics were performed on SPSS. Results: We identified 644 pts with metastatic breast cancer. Of these, 88 (12.8%) pts harbored an ARID1a mutation. 62 (70.5%) mutations were missense (biologic effect unknown), the remaining 26 (29.5%) were oncogenic loss of function (OLF) changes (frame-shift, deletion, insertion, or nonsense). Median OS was significantly better in patients harboring missense mutations compared to OLF and wild type mutations [median OS 58.2 months (95% CI 44.5-71.8) with missense mutations vs 22.8 months (95% CI 10.8-34.7) with OLF mutations and 27.48 months (95% CI 24.5-30.3) with wild type]. Demographics, tumor features and chemotherapy use were generally equally distributed among the subgroups (table). Conclusions: In our study the median OS was worse in metastatic breast cancer pts harboring OFL ARID1a mutations compared to pts with wild type ARID1a or harboring missense ARID1a mutations. Further studies are warranted to assess how specific ARID1a mutations might affect survival in metastatic breast cancer pts. [Table: see text]

scholarly journals VALINE-RESISTANCE, A POTENTIAL MARKER IN PLANT CELL GENETICS. II. OPTIMIZATION OF UV MUTAGENESIS AND SELECTION OF VALINE-RESISTANT COLONIES DERIVED FROM TOBACCO MESOPHYLL PROTOPLASTS

Genetics ◽  
1985 ◽  
Vol 109 (2) ◽  
pp. 409-425 ◽  
Author(s):  
M A Grandbastien ◽  
J P Bourgin ◽  
M Caboche
Keyword(s):  
Spontaneous Mutation ◽  
Wild Type Plant ◽  
Acid Uptake ◽  
Wild Type ◽  
Induced Mutations ◽  
Mutant Plant ◽  
Potential Marker ◽  
Valine Resistance ◽  
Selection Of ◽  
Haploid Plants

ABSTRACT The induction and selection of valine-resistant mutants from haploid tobacco (Nicotiana tabacum L.) mesophyll protoplast-derived cells have been studied. Using cells from an original mutant plant obtained previously, we performed reconstruction experiments in order to determine the best conditions for the recovery of resistant cells among a population of sensitive cells. Optimal selective conditions were shown to depend on various factors including cell density, time of addition of valine and seasonal variations affecting the mother plants.—Using cell densities of approximately 104 cells/ml, we defined efficient selective conditions: more than 25% of the putative mutant clones selected from UV-mutagenized protoplasts were reproducibly confirmed to be valine resistant. Further characterization of some regenerated mutant plants indicated that valine-resistance was associated with an uptake deficiency, as in the case of the original mutant plant of the Valr-2 line used for reconstruction experiments. Spontaneous mutation rates for valine-resistance were below accurately detectable levels, i.e., less than 10-6 per cell per generation. Induced mutation frequency varied nonlinearily with UV dose from 10-5 to 5 × 10-4 resistant clones per surviving colony. Two independent loci (vr2 and vr3) were previously shown to be involved in valine-resistance due to amino acid uptake deficiency. Haploid tobacco plants were produced through anther culture from an F1 double-heterozygous plant obtained from a cross between the original mutant plant and a wild-type plant. Study of the level of resistance to valine of protoplast-derived cells allowed the classification of these haploid plants in four types: sensitive, resistant and two intermediary resistant types believed to result from the presence of a mutant allele at only one of the two loci involved. The frequencies of UV-induced mutations in cells derived from haploid plants of one of the intermediary types were compared to those observed in wild-type cells. The results are considered in light of the amphidiploid structure of the tobacco genome.

scholarly journals A trans-acting long non-coding RNA represses flowering in Arabidopsis

2021 ◽  
Author(s):  
Yu Jin ◽  
Maxim Ivanov ◽  
Anna Nelson Dittrich ◽  
Andrew Nelson ◽  
Sebastian Marquardt
Keyword(s):  
Gene Expression ◽  
Functional Characterization ◽  
Early Flowering ◽  
Differentially Expressed ◽  
Loss Of Function ◽  
Allelic Series ◽  
Non Coding Rna ◽  
Genome Wide ◽  
Genomic Regions ◽  
Early Flowering Phenotype

Eukaryotic genomes give rise to thousands of long non-coding RNAs (lncRNAs), yet the purpose of lncRNAs remains largely enigmatic. Functional characterization of lncRNAs is challenging due to multiple orthogonal hypothesis for molecular activities of lncRNA loci. Here, we identified a flowering associated intergenic lncRNA (FLAIL) that represses flowering in Arabidopsis. An allelic series of flail loss-of-function mutants generated by CRISPR/Cas9 and T-DNA mutagenesis showed an early flowering phenotype. Gene expression analyses in flail mutants revealed differentially expressed genes linked to the regulation of flowering. A genomic rescue fragment of FLAIL introduced in flail mutants complemented gene expression defects and early flowering, consistent with trans-acting effects of the FLAIL RNA. Knock-down of FLAIL RNA levels using the artificial microRNA approach revealed an early flowering phenotype shared with genomic mutations, indicating a trans-acting role of FLAIL RNA in the repression of flowering time. Genome-wide detection of FLAIL-DNA interactions by ChIRP-seq suggested that FLAIL may directly bind genomic regions. FLAIL bound to genes involved in regulation of flowering that were differentially expressed in flail, consistent with the interpretation of FLAIL as a trans-acting lncRNA directly shaping gene expression. Our findings highlight FLAIL as a trans-acting lncRNA that affects flowering in Arabidopsis, likely through mediating transcriptional regulation of genes directly bound by FLAIL.

scholarly journals Brain transcriptome analysis reveals subtle effects on mitochondrial function and iron homeostasis of mutations in the SORL1 gene implicated in early onset familial Alzheimer’s disease

2020 ◽  
Vol 13 (1) ◽  
Author(s):  
Karissa Barthelson ◽  
Stephen Martin Pederson ◽  
Morgan Newman ◽  
Michael Lardelli
Keyword(s):  
Gene Expression ◽  
Alzheimer’S Disease ◽  
Alzheimer's Disease ◽  
Early Onset ◽  
Iron Homeostasis ◽  
Enrichment Analysis ◽  
Loss Of Function ◽  
Wild Type ◽  
Expression Of Genes ◽  
Insight Into

Abstract To prevent or delay the onset of Alzheimer’s disease (AD), we must understand its molecular basis. The great majority of AD cases arise sporadically with a late onset after 65 years of age (LOAD). However, rare familial cases of AD can occur due to dominant mutations in a small number of genes that cause an early onset prior to 65 years of age (EOfAD). As EOfAD and LOAD share similar pathologies and disease progression, analysis of EOfAD genetic models may give insight into both subtypes of AD. Sortilin-related receptor 1 (SORL1) is genetically associated with both EOfAD and LOAD and provides a unique opportunity to investigate the relationships between both forms of AD. Currently, the role of SORL1 mutations in AD pathogenesis is unclear. To understand the molecular consequences of SORL1 mutation, we performed targeted mutagenesis of the orthologous gene in zebrafish. We generated an EOfAD-like mutation, V1482Afs, and a putatively null mutation, to investigate whether EOfAD-like mutations in sorl1 display haploinsufficiency by acting through loss-of-function mechanisms. We performed mRNA-sequencing on whole brains, comparing wild type fish with their siblings heterozygous for EOfAD-like or putatively loss-of-function mutations in sorl1, or transheterozygous for these mutations. Differential gene expression analysis identified a small number of differentially expressed genes due to the sorl1 genotypes. We also performed enrichment analysis on all detectable genes to obtain a more complete view on changes to gene expression by performing three methods of gene set enrichment analysis, then calculated an overall significance value using the harmonic mean p-value. This identified subtle effects on expression of genes involved in energy production, mRNA translation and mTORC1 signalling in both the EOfAD-like and null mutant brains, implying that these effects are due to sorl1 haploinsufficiency. Surprisingly, we also observed changes to expression of genes occurring only in the EOfAD-mutation carrier brains, suggesting gain-of-function effects. Transheterozygosity for the EOfAD-like and null mutations (i.e. lacking wild type sorl1), caused apparent effects on iron homeostasis and other transcriptome changes distinct from the single-mutation heterozygous fish. Our results provide insight into the possible early brain molecular effects of an EOfAD mutation in human SORL1. Differential effects of heterozygosity and complete loss of normal SORL1 expression are revealed.

scholarly journals Effects of Coronavirus Persistence on the Genome Structure and Subsequent Gene Expression, Pathogenicity and Adaptation Capability

Cells ◽  
2020 ◽  
Vol 9 (10) ◽  
pp. 2322
Author(s):  
Ching-Hung Lin ◽  
Cheng-Yao Yang ◽  
Meilin Wang ◽  
Shan-Chia Ou ◽  
Chen-Yu Lo ◽  
...  
Keyword(s):  
Gene Expression ◽  
Persistent Infection ◽  
Genome Structure ◽  
Regulatory Sequence ◽  
Unanswered Question ◽  
Wild Type ◽  
Cellular Environment ◽  
Fundamental Research ◽  
Coronavirus Infection ◽  
Selection Of

Coronaviruses are able to establish persistence. However, how coronaviruses react to persistence and whether the selected viruses have altered their characteristics remain unclear. In this study, we found that the persistent infection of bovine coronavirus (BCoV), which is in the same genus as SARS-COV-2, led to alterations of genome structure, attenuation of gene expression, and the synthesis of subgenomic mRNA (sgmRNA) with a previously unidentified pattern. Subsequent analyses revealed that the altered genome structures were associated with the attenuation of gene expression. In addition, the genome structure at the 5′ terminus and the cellular environment during the persistence were responsible for the sgmRNA synthesis, solving the previously unanswered question regarding the selection of transcription regulatory sequence for synthesis of BCoV sgmRNA 12.7. Although the BCoV variants (BCoV-p95) selected under the persistence replicated efficiently in cells without persistent infection, its pathogenicity was still lower than that of wild-type (wt) BCoV. Furthermore, in comparison with wt BCoV, the variant BCoV-p95 was not able to efficiently adapt to the challenges of alternative environments, suggesting wt BCoV is genetically robust. We anticipate that the findings derived from this fundamental research can contribute to the disease control and treatments against coronavirus infection including SARS-CoV-2.

scholarly journals Reconstructing a wild-type Pseudomonas aeruginosa reference strain PAO1

2021 ◽  
Author(s):  
Samuel Lee ◽  
Larry Gallagher ◽  
Colin Manoil
Keyword(s):  
Reference Strain ◽  
Genetic Selection ◽  
Loss Of Function ◽  
Wild Type ◽  
Regulatory Mutation ◽  
Phenotypic Analysis ◽  
Strain Pao1 ◽  
Suppressor Mutations ◽  
Wild Type Parent ◽  
Selection Of

The P. aeruginosa reference strain PAO1 has been used to delineate much of the physiology, metabolism and fundamental biology of the species. The wild-type parent of PAO1 was lost, and PAO1 carries a regulatory mutation introduced for positive genetic selection that affects antibiotic resistance, virulence, quorum sensing and other traits. The mutation is a loss-of-function change in an oxidoreductase gene (mexS), which constitutively activates a stress response controlled by a positive regulator (MexT). Fitness defects associated with the constitutive response have led to the inadvertent selection of mexT– suppressor mutations, creating genetic heterogeneity in PAO1 sublines studied in different laboratories. To help circumvent complications due to the mexS–minus phenotypes, we created a wild-type version of PAO1 (called LPAO) by “reverting” its mexS to the functional allele likely to have been in its parent. Phenotypic analysis revealed that the mexS– allele in PAO1 makes growth sensitive to salt (NaCl) and is lethal when combined with mutations inactivating the major sodium antiporter (ShaABCDEF). The salt sensitivity of PAO1 may underlie some complex mexS– phenotypes and help explain the selection of mexT– suppressor mutations. To facilitate genetic comparisons of PAO1, LPAO and other P. aeruginosa strains, we developed a transformation procedure to transfer selectable alleles, such as transposon insertion alleles, between strains. Overall, the study helps explain phenotypic heterogeneity of PAO1-derived strains and provides resources to help recognize and eliminate difficulties due to it. IMPORTANCE The P. aeruginosa reference strain PAO1 carries a regulatory mutation that may affect processes characterized in it. To eliminate complications due to the mutation, we constructed a version of the missing wild-type parent strain and developed methods to transfer mutations between PAO1 and the new strain. The methods are likely to be applicable to other isolates of P. aeruginosa as well.

scholarly journals Human neural networks with sparse TDP-43 pathology reveal NPTX2 misregulation in ALS/FTLD

2021 ◽  
Author(s):  
Marian Hruska-Plochan ◽  
Katharina M Hembach ◽  
Silvia Ronchi ◽  
Vera I Wiersma ◽  
Zuzanna Maniecka ◽  
...  
Keyword(s):  
Functional Networks ◽  
Loss Of Function ◽  
Wild Type ◽  
Cellular Models ◽  
Self Organized ◽  
Rna Targets ◽  
Age Dependent ◽  
Manual Selection ◽  
Selection Of ◽  
Human Specific

Human cellular models of neurodegeneration require reproducibility and longevity, which is necessary for simulating these age-dependent diseases. Such systems are particularly needed for TDP-43 proteinopathies, which involve human-specific mechanisms that cannot be directly studied in animal models. To explore the emergence and consequences of TDP-43 pathologies, we generated iPSC-derived, colony morphology neural stem cells (iCoMoNSCs) via manual selection of neural precursors. Single-cell transcriptomics (scRNA-seq) and comparison to independent NSCs, showed that iCoMoNSCs are uniquely homogenous and self-renewing. Differentiated iCoMoNSCs formed a self-organized multicellular system consisting of synaptically connected and electrophysiologically active neurons, which matured into long-lived functional networks. Neuronal and glial maturation in iCoMoNSC-derived cultures was similar to that of cortical organoids. Overexpression of wild-type TDP-43 in a minority of iCoMoNSC-derived neurons led to progressive fragmentation and aggregation, resulting in loss of function and neurotoxicity. scRNA-seq revealed a novel set of misregulated RNA targets coinciding in both TDP-43 overexpressing neurons and patient brains exhibiting loss of nuclear TDP-43. The strongest misregulated target encoded for the synaptic protein NPTX2, which was consistently misaccumulated in ALS and FTLD patient neurons with TDP-43 pathology. Our work directly links TDP-43 misregulation and NPTX2 accumulation, thereby highlighting a new pathway of neurotoxicity.

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