scholarly journals Ecology Shapes Microbial Immune Strategy: Temperature and Oxygen as Determinants of the Incidence of CRISPR Adaptive Immunity

2018 ◽  
Author(s):  
Jake L. Weissman ◽  
Rohan M. R. Laljani ◽  
William F. Fagan ◽  
Philip L. F. Johnson
Keyword(s):  
Predictive Model ◽  
End Joining ◽  
Viral Pathogens ◽  
Defense Strategies ◽  
Machine Learning Approach ◽  
Associated Proteins ◽  
Non Homologous End Joining ◽  
Using Data ◽  
Restriction Modification

AbstractBacteria and archaea are locked in a near-constant battle with their viral pathogens. Despite previous mechanistic characterization of numerous prokaryotic defense strategies, the underlying ecological drivers of different strategies remain largely unknown and predicting which species will take which strategies remains a challenge. Here, we focus on the CRISPR immune strategy and develop a phylogenetically-corrected machine learning approach to build a predictive model of CRISPR incidence using data on over 100 traits across over 2600 species. We discover a strong but hitherto-unknown negative interaction between CRISPR and aerobicity, which we hypothesize may result from interference between CRISPR associated proteins and non-homologous end-joining DNA repair due to oxidative stress. Our predictive model also quantitatively confirms previous observations of an association between CRISPR and temperature. Finally, we contrast the environmental associations of different CRISPR system types (I, II, III) and restriction modification systems, all of which act as intracellular immune systems.

scholarly journals Characterization of gamma irradiation-induced mutations in Arabidopsis mutants deficient in non-homologous end joining

2020 ◽  
Vol 61 (5) ◽  
pp. 639-647
Author(s):  
Yan Du ◽  
Yoshihiro Hase ◽  
Katsuya Satoh ◽  
Naoya Shikazono
Keyword(s):  
Gamma Rays ◽  
Wild Type ◽  
End Joining ◽  
Complex Type ◽  
Single Base ◽  
In The Wild ◽  
Non Homologous End Joining ◽  
Dna Ends ◽  
Wild Type Control

Abstract To investigate the involvement of the non-homologous end joining (NHEJ) pathway in plant mutagenesis by ionizing radiation, we conducted a genome-wide characterization of the mutations induced by gamma rays in NHEJ-deficient Arabidopsis mutants (AtKu70−/− and AtLig4−/−). Although both mutants were more sensitive to gamma rays than the wild-type control, the AtKu70−/− mutant was slightly more sensitive than the AtLig4−/− mutant. Single-base substitutions (SBSs) were the predominant mutations in the wild-type control, whereas deletions (≥2 bp) and complex-type mutations [i.e. more than two SBSs or short insertion and deletions (InDels) separated by fewer than 10 bp] were frequently induced in the mutants. Single-base deletions were the most frequent deletions in the wild-type control, whereas the most common deletions in the mutants were 11–30 bp. The apparent microhomology at the rejoined sites of deletions peaked at 2 bp in the wild-type control, but was 3–4 bp in the mutants. This suggests the involvement of alternative end joining and single-strand annealing pathways involving increased microhomology for rejoining DNA ends. Complex-type mutations comprising short InDels were frequently detected in the mutants, but not in the wild-type control. Accordingly, NHEJ is more precise than the backup pathways, and is the main pathway for rejoining the broken DNA ends induced by ionizing radiation in plants.

scholarly journals Heterologous Cas9 and non-homologous end joining as a Potentially Organism-Agnostic Knockout (POAK) system in bacteria

2019 ◽  
Author(s):  
Isaac N Plant
Keyword(s):  
Dna Cleavage ◽  
Sugar Metabolism ◽  
End Joining ◽  
Gene Deletions ◽  
Transformation Protocol ◽  
E Coli ◽  
Rapid Sequence ◽  
Error Prone Repair ◽  
Non Homologous End Joining

Making targeted gene deletions is essential for studying organisms, but is difficult in many prokaryotes due to the inefficiency of homologous recombination based methods. Here, I describe an easily modifiable, single-plasmid system that can be used to make rapid, sequence targeted, markerless knockouts in both a Gram-negative and a Gram-positive organism. The system is comprised of targeted DNA cleavage by Cas9 and error-prone repair by Non-Homologous End Joining (NHEJ) proteins. I confirm previous results showing that Cas9 and NHEJ can make knockouts when NHEJ is expressed before Cas9. Then, I show that Cas9 and NHEJ can be used to make knockouts when expressed simultaneously. I term the new method Potentially Organism-Agnostic Knockout (POAK) system and characterize its function in Escherichia coli and Weissella confusa. First, I develop a novel transformation protocol for W. confusa. Next, I show that, as in E. coli, POAK can create knockouts in W. confusa. Characterization of knockout efficiency across galK in both E. coli and W. confusa showed that while all gRNAs are effective in E. coli, only some gRNAs are effective in W. confusa, and cut site position within a gene does not determine knockout efficiency for either organism. I examine the sequences of knockouts in both organisms and show that POAK produces similar edits in both E. coli and W. confusa. Finally, as an example of the importance of being able to make knockouts quickly, I target W. confusa sugar metabolism genes to show that two sugar importers are not necessary for metabolism of their respective sugars. Having demonstrated that simultaneous expression of Cas9 and NHEJ is sufficient for making knockouts in two minimally related bacteria, POAK represents a hopeful avenue for making knockouts in other under-utilized bacteria.

scholarly journals NHJ-1 regulates canonical non-homologous end joining in Caenorhabditis elegans

2019 ◽  
Author(s):  
Aleksandar Vujin ◽  
Steven J. Jones ◽  
Monique Zetka
Keyword(s):  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Dapi Staining ◽  
Strand Breaks ◽  
C Elegans ◽  
Meiotic Dsbs ◽  
Ring Complex ◽  
Non Homologous End Joining

AbstractCanonical non-homologous end joining (cNHEJ) is a near-universally conserved pathway for the repair of DNA double-strand breaks (DSBs). While the cNHEJ pathway encompasses more than a dozen factors in vertebrates and is similarly complex in other eukaryotes, in the nematode C. elegans the entire known cNHEJ toolkit consists of two proteins that comprise the Ku ring complex, cku-70 and cku-80, and the terminal ligase lig-4. Here, we report the discovery of nhj-1 as the fourth cNHEJ factor in C. elegans. Observing a difference in the phenotypic response to ionizing radiation (IR) between two lines of the wild type N2 strain, we mapped the locus causative of IR-sensitivity to a candidate on chromosome V. Using CRISPR-Cas9 mutagenesis, we show that disrupting the nhj-1 sequence induces IR-sensitivity in an IR-resistant background. Double mutants of nhj-1 and the cNHEJ factors lig-4 or cku-80 do not exhibit additive IR-sensitivity, arguing that nhj-1 is a member of the cNHEJ pathway. Furthermore, like the loss of lig-4, the loss of nhj-1 in the com-1 genetic background, in which meiotic DSBs are repaired by cNHEJ instead of homologous recombination, increased the number of DAPI-staining bodies in diakinesis, consistent with increased chromosome fragmentation in the absence of cNHEJ repair. Finally, we show that NHJ-1 localizes to many somatic nuclei in the L1 larva, but not the primordial germline, which is in accord with a role in the predominantly somatically active cNHEJ. Although nhj-1 shares no sequence homology with other known eukaryotic cNHEJ factors and is taxonomically restricted to the Rhadbitid family, its discovery underscores the evolutionary plasticity of even highly conserved pathways, and may represent a springboard for further characterization of cNHEJ in C. elegans.

scholarly journals CRISPR-Cas9-Based Mutagenesis of the Mucormycosis-Causing Fungus Lichtheimia corymbifera

2020 ◽  
Vol 21 (10) ◽  
pp. 3727
Author(s):  
Sandugash Ibragimova ◽  
Csilla Szebenyi ◽  
Rita Sinka ◽  
Elham I. Alzyoud ◽  
Mónika Homa ◽  
...  
Keyword(s):  
Genetic Manipulation ◽  
Pathogenic Fungus ◽  
Strand Break ◽  
Targeted Mutagenesis ◽  
End Joining ◽  
Targeted Gene Disruption ◽  
Lichtheimia Corymbifera ◽  
Non Homologous End Joining ◽  
Opportunistic Pathogenic

Lichtheimia corymbifera is considered as one of the most frequent agents of mucormycosis. The lack of efficient genetic manipulation tools hampers the characterization of the pathomechanisms and virulence factors of this opportunistic pathogenic fungus. Although such techniques have been described for certain species, the performance of targeted mutagenesis and the construction of stable transformants have remained a great challenge in Mucorales fungi. In the present study, a plasmid-free CRISPR-Cas9 system was applied to carry out a targeted gene disruption in L. corymbifera. The described method is based on the non-homologous end-joining repair of the double-strand break caused by the Cas9 enzyme. Using this method, short, one-to-five nucleotide long-targeted deletions could be induced in the orotidine 5′-phosphate decarboxylase gene (pyrG) and, as a result, uracil auxotrophic strains were constructed. These strains are applicable as recipient strains in future gene manipulation studies. As we know, this is the first genetic modification of this clinically relevant fungus.

scholarly journals Multi-layered chromatin proteomics identifies cell vulnerabilities in DNA repair

2021 ◽  
Author(s):  
Gianluca Sigismondo ◽  
Lavinia Arseni ◽  
Thomas G Hofmann ◽  
Martina Seiffert ◽  
Jeroen Krijgsveld
Keyword(s):  
Genome Stability ◽  
Parp Inhibitor ◽  
Strand Break ◽  
Parp Inhibitors ◽  
High Temporal Resolution ◽  
End Joining ◽  
Post Translational Modifications ◽  
Dna Double Strand Break ◽  
Associated Proteins ◽  
Non Homologous End Joining

The DNA damage response (DDR) is essential to maintain genome stability, and its deregulation predisposes to carcinogenesis while encompassing attractive targets for cancer therapy. Chromatin governs the DDR via interplay among all chromatin layers including DNA, histones post-translational modifications (hPTMs), and chromatin-associated proteins. Here we employ multi-layered proteomics to characterize chromatin-mediated interactions of repair proteins, signatures of hPTMs, and the DNA-bound proteome during DNA double-strand break repair at high temporal resolution. We functionally attribute novel chromatin-associated proteins to repair by non-homologous end-joining or homologous recombination (HR) revealing histone reader ATAD2, microtubule organizer TPX2 and histone methyltransferase G9A as regulators of HR and PARP inhibitor sensitivity. Furthermore, we dynamically profile numerous hPTMs at γH2AX-mononucleosomes during the DDR. Integration of these complementary data implicated G9A-mediated monomethylation of H3K56 in HR. Collectively, we provide a dynamic chromatin-centered view of DDR, while representing a valuable resource for the use of PARP inhibitors in cancer.

scholarly journals Demonstration of targeted crossovers in hybrid maize using CRISPR technology

2022 ◽  
Vol 5 (1) ◽  
Author(s):  
Andrei Kouranov ◽  
Charles Armstrong ◽  
Ashok Shrawat ◽  
Vladimir Sidorov ◽  
Scott Huesgen ◽  
...  
Keyword(s):  
Mitotic Cell ◽  
Mitotic Cell Cycle ◽  
End Joining ◽  
Naturally Occurring ◽  
Hybrid Plants ◽  
Key Driver ◽  
Non Homologous End Joining ◽  
Hybrid Maize ◽  
Trait Introgression

AbstractNaturally occurring chromosomal crossovers (CO) during meiosis are a key driver of genetic diversity. The ability to target CO at specific allelic loci in hybrid plants would provide an advantage to the plant breeding process by facilitating trait introgression, and potentially increasing the rate of genetic gain. We present the first demonstration of targeted CO in hybrid maize utilizing the CRISPR Cas12a system. Our experiments showed that stable and heritable targeted CO can be produced in F1 somatic cells using Cas12a at a significantly higher rate than the natural CO in the same interval. Molecular characterization of the recombinant plants demonstrated that the targeted CO were driven by the non-homologous end joining (NHEJ) or HDR repair pathways, presumably during the mitotic cell cycle. These results are a step towards the use of RNA-guided nuclease technology to simplify the creation of targeted genome combinations in progeny and accelerate breeding.

908-P: Predictive Model for Glycemic Level of People with Diabetes Mellitus Using Data Extracted from Electronic Medical Records in Primary Care

Diabetes ◽  
2020 ◽  
Vol 69 (Supplement 1) ◽  
pp. 908-P
Author(s):  
SOSTENES MISTRO ◽  
THALITA V.O. AGUIAR ◽  
VANESSA V. CERQUEIRA ◽  
KELLE O. SILVA ◽  
JOSÉ A. LOUZADO ◽  
...  
Keyword(s):  
Diabetes Mellitus ◽  
Primary Care ◽  
Predictive Model ◽  
Electronic Medical Records ◽  
Medical Records ◽  
Glycemic Level ◽  
Using Data

scholarly journals Average fractional polarization of extragalactic sources at Planck frequencies

2018 ◽  
Vol 618 ◽  
pp. A29 ◽  
Author(s):  
T. Trombetti ◽  
C. Burigana ◽  
G. De Zotti ◽  
V. Galluzzi ◽  
M. Massardi
Keyword(s):  
High Sensitivity ◽  
Point Sources ◽  
Distribution Analysis ◽  
Microwave Background ◽  
Residual Noise ◽  
Upper Limits ◽  
Dusty Galaxies ◽  
Using Data ◽  
Polarization Fraction

Recent detailed simulations have shown that an insufficiently accurate characterization of the contamination of unresolved polarized extragalactic sources can seriously bias measurements of the primordial cosmic microwave background (CMB) power spectrum if the tensor-to-scalar ratio r ∼ 0.001, as predicted by models currently of special interest (e.g., Starobinsky’s R2 and Higgs inflation). This has motivated a reanalysis of the median polarization fraction of extragalactic sources (radio-loud AGNs and dusty galaxies) using data from the Planck polarization maps. Our approach, exploiting the intensity distribution analysis, mitigates or overcomes the most delicate aspects of earlier analyses based on stacking techniques. By means of simulations, we have shown that the residual noise bias on the median polarization fraction, Πmedian, of extragalactic sources is generally ≲0.1%. For radio sources, we have found Πmedian ≃ 2.83%, with no significant dependence on either frequency or flux density, in good agreement with the earlier estimate and with high-sensitivity measurements in the frequency range 5–40 GHz. No polarization signal is detected in the case of dusty galaxies, implying 90% confidence upper limits of Πdusty ≲ 2.2% at 353 GHz and of ≲3.9% at 217 GHz. The contamination of CMB polarization maps by unresolved point sources is discussed.

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