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.

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

2021 ◽  
Author(s):  
Andrei Kouranov ◽  
Charles Armstrong ◽  
Ashok Shrawat ◽  
Vladimir Sidorov ◽  
Scott Huesgen ◽  
...  
Keyword(s):  
Genetic Diversity ◽  
Plant Breeding ◽  
Genetic Gain ◽  
Somatic Cells ◽  
Naturally Occurring ◽  
Hybrid Plants ◽  
Key Driver ◽  
The Creation ◽  
Hybrid Maize ◽  
Trait Introgression

Abstract Naturally 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 a significant 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. 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.

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.

Characterization of the fission yeast cdc10+ protein that is required for commitment to the cell cycle

1989 ◽  
Vol 92 (1) ◽  
pp. 51-56 ◽  
Author(s):  
V. Simanis ◽  
P. Nurse
Keyword(s):  
Cell Cycle ◽  
Mrna Level ◽  
Apparent Molecular Weight ◽  
Mitotic Cell ◽  
Protein Product ◽  
S Phase ◽  
Mitotic Cell Cycle ◽  
State Changes ◽  
Beta Galactosidase

We have used antiserum raised against a beta-galactosidase-cdc10+ fusion protein to identify the protein product of the cdc10+ start gene of Schizosaccharomyces pombe. This gene is required for progress through the G1 phase of the cell cycle and for activating processes such as the increase in histone mRNA level in preparation for S phase. The protein has an apparent molecular weight of 87,000 and is phosphorylated on multiple serine residues. The protein remains phosphorylated throughout the mitotic cell cycle and shows no significant steady-state changes in level. The antiserum has also detected a protein similar in size to p87cdc10 in human cells.

scholarly journals Effects of mitoTALENs-Directed Double-Strand Breaks on Plant Mitochondrial Genomes

Genes ◽  
2021 ◽  
Vol 12 (2) ◽  
pp. 153
Author(s):  
Shin-ichi Arimura
Keyword(s):  
Double Strand Breaks ◽  
Mitochondrial Genomes ◽  
Transcription Activator ◽  
End Joining ◽  
Strand Breaks ◽  
Large Deletions ◽  
Naturally Occurring ◽  
Copy Numbers ◽  
Nuclear Genomes ◽  
Non Homologous End Joining

Mitochondrial genomes in flowering plants differ from those in animals and yeasts in several ways, including having large and variable sizes, circular, linear and branched structures, long repeat sequences that participate in homologous recombinations, and variable genes orders, even within a species. Understanding these differences has been hampered by a lack of genetic methods for transforming plant mitochondrial genomes. We recently succeeded in disrupting targeted genes in mitochondrial genomes by mitochondria-targeted transcription activator-like effector nucleases (mitoTALENs) in rice, rapeseed, and Arabidopsis. Double-strand breaks created by mitoTALENs were repaired not by non-homologous end-joining (NHEJ) but by homologous recombination (HR) between repeats near and far from the target sites, resulting in new genomic structures with large deletions and different configurations. On the other hand, in mammals, TALENs-induced DSBs cause small insertions or deletions in nuclear genomes and degradation of mitochondrial genomes. These results suggest that the mitochondrial and nuclear genomes of plants and mammals have distinct mechanisms for responding to naturally occurring DSBs. The different responses appear to be well suited to differences in size and copy numbers of each genome.

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 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.

High Resolution Replication of Organoclay Surfaces

1982 ◽  
Vol 40 ◽  
pp. 576-577
Author(s):  
W. W. Barker ◽  
W. E. Rigsby ◽  
V. J. Hurst ◽  
W. J. Humphreys
Keyword(s):  
Clay Mineral ◽  
Organic Molecule ◽  
Organic Molecules ◽  
X Ray Diffraction ◽  
Xrd Pattern ◽  
X Ray ◽  
Naturally Occurring ◽  
Silicate Layers ◽  
Mineral Identification

Experimental clay mineral-organic molecule complexes long have been known and some of them have been extensively studied by X-ray diffraction methods. The organic molecules are adsorbed onto the surfaces of the clay minerals, or intercalated between the silicate layers. Natural organo-clays also are widely recognized but generally have not been well characterized. Widely used techniques for clay mineral identification involve treatment of the sample with H2 O2 or other oxidant to destroy any associated organics. This generally simplifies and intensifies the XRD pattern of the clay residue, but helps little with the characterization of the original organoclay. Adequate techniques for the direct observation of synthetic and naturally occurring organoclays are yet to be developed.

Sign in / Sign up

Export Citation Format

Share Document