scholarly journals Mono-homologous linear DNA recombination by the non-homologous end-joining pathway as a novel and simple gene inactivation method: a proof of concept study inDietziasp. DQ12-45-1b

2018 ◽  
Author(s):  
Shelian Lu ◽  
Yong Nie ◽  
Meng Wang ◽  
Hong-Xiu Xu ◽  
Dong-Ling Ma ◽  
...  
Keyword(s):  
Genetic Manipulation ◽  
Dna Recombination ◽  
Proof Of Concept ◽  
End Joining ◽  
Circular Dna ◽  
Linear Dna ◽  
Genes Encoding ◽  
Non Homologous End Joining ◽  
Nhej Activity

ABSTRACTNon-homologous end-joining (NHEJ) is critical for genome stability because of its roles in double-strand break repair. Ku and ligase D (LigD) are the crucial proteins in this process, and strains expressing Ku and LigD can cyclize linear DNAin vivo.Herein, we established a proof-of-concept mono-homologous linear DNA recombination for gene inactivation or genome editing by which cyclization of linear DNAin vivoby NHEJ could be used to generate non-replicable circular DNA and could allow allelic exchanges between the circular DNA and the chromosome. We achieved this approach inDietziasp. DQ12-45-1b, which expresses Ku and LigD homologs and presents NHEJ activity. By transforming the strain with a linear DNA mono homolog to the sequence in chromosome, we mutated the genome. This method did not require the screening of suitable plasmids and was easy and time-effective. Bioinformatic analysis showed that more than 20% prokaryotic organisms contain Ku and LigD, suggesting the wide distribution of NHEJ activities. Moreover, theEscherichia colistrain also showed NHEJ activity when the Ku and LigD ofDietziasp. DQ12-45-1b were introduced and expressed in it. Therefore, this method may be a widely applicable genome editing tool for diverse prokaryotic organisms, especially for non-model microorganisms.IMPORTANCEThe non-model gram-positive bacteria lack efficient genetic manipulation systems, but they express genes encoding Ku and LigD. The NHEJ pathway inDietziasp. DQ12-45-1b was evaluated and was used to successfully knockout eleven genes in the genome. Since bioinformatic studies revealed that the putative genes encoding Ku and LigD ubiquitously exist in phylogenetically diverse bacteria and archaea, the mono-homologous linear DNA recombination by the NHEJ pathway could be a potentially applicable genetic manipulation method for diverse non-model prokaryotic organisms.

scholarly journals Single-Homology-Arm Linear DNA Recombination by the Nonhomologous End Joining Pathway as a Novel and Simple Gene Inactivation Method: a Proof-of-Concept Study inDietziasp. Strain DQ12-45-1b

2018 ◽  
Vol 84 (19) ◽  
Author(s):  
Shelian Lu ◽  
Yong Nie ◽  
Meng Wang ◽  
Hong-Xiu Xu ◽  
Dong-Ling Ma ◽  
...  
Keyword(s):  
Genetic Manipulation ◽  
Dna Recombination ◽  
Nonhomologous End Joining ◽  
Proof Of Concept ◽  
End Joining ◽  
Circular Dna ◽  
Linear Dna ◽  
Genes Encoding ◽  
Nhej Activity

ABSTRACTNonhomologous end joining (NHEJ) is critical for genome stability because of its roles in double-strand break repair. Ku and ligase D (LigD) are the crucial proteins in this process, and strains expressing Ku and LigD can cyclize linear DNAin vivo. Here, we established a proof-of-concept single-homology-arm linear DNA recombination for gene inactivation or genome editing by which cyclization of linear DNAin vivoby NHEJ could be used to generate nonreplicable circular DNA and could allow allelic exchanges between the circular DNA and the chromosome. We achieved this approach inDietziasp. strain DQ12-45-1b, which expresses Ku and LigD homologs and presents NHEJ activity. By transforming the strain with a linear DNA single homolog to the sequence in the chromosome, we mutated the genome. This method did not require the screening of suitable plasmids and was easy and time-effective. Bioinformatic analysis showed that more than 20% of prokaryotic organisms contain Ku and LigD, suggesting the wide distribution of NHEJ activities. Moreover, anEscherichia colistrain also showed NHEJ activity when the Ku and LigD ofDietziasp. DQ12-45-1b were introduced and expressed in it. Therefore, this method may be a widely applicable genome editing tool for diverse prokaryotic organisms, especially for nonmodel microorganisms.IMPORTANCEMany nonmodel Gram-positive bacteria lack efficient genetic manipulation systems, but they express genes encoding Ku and LigD. The NHEJ pathway inDietziasp. DQ12-45-1b was evaluated and was used to successfully knock out 11 genes in the genome. Since bioinformatic studies revealed that the putative genes encoding Ku and LigD ubiquitously exist in phylogenetically diverse bacteria and archaea, the single-homology-arm linear DNA recombination by the NHEJ pathway could be a potentially applicable genetic manipulation method for diverse nonmodel prokaryotic organisms.

Analysis of the mechanism of DNA recombination using tangles

1995 ◽  
Vol 28 (3) ◽  
pp. 253-313 ◽  
Author(s):  
De Witt Sumners ◽  
Claus Ernst ◽  
Sylvia J. Spengler ◽  
Nicholas R. Cozzarelli
Keyword(s):  
Dna Recombination ◽  
Dna Supercoiling ◽  
Topological Properties ◽  
Circular Dna ◽  
Linear Dna ◽  
Naturally Occurring

The DNA of all organisms has a complex and essential topology. The three topological properties of naturally occurring DNA are supercoiling, catenation, and knotting. Although these properties are denned rigorously only for closed circular DNA, even linear DNA in vivo can have topological properties because it is divided into topologically separate subdomains (Drlica 1987; Roberge & Gasser, 1992). The essentiality of topological properties is demonstrated by the lethal consequence of interfering with topoisomerases, the enzymes that regulate the level of DNA supercoiling and that unlink DNA during its replication (reviewed in Wang, 1991; Bjornsti, 1991; Drlica, 1992; Ullsperger et al. 1995).

scholarly journals Structural basis for proficient oxidized ribonucleotide insertion in double strand break repair

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Joonas A. Jamsen ◽  
Akira Sassa ◽  
Lalith Perera ◽  
David D. Shock ◽  
William A. Beard ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Time Lapse ◽  
Strand Break ◽  
Structural Basis ◽  
End Joining ◽  
Strand Breaks ◽  
Nucleotide Pools ◽  
Nucleotide Insertion ◽  
Non Homologous End Joining

AbstractReactive oxygen species (ROS) oxidize cellular nucleotide pools and cause double strand breaks (DSBs). Non-homologous end-joining (NHEJ) attaches broken chromosomal ends together in mammalian cells. Ribonucleotide insertion by DNA polymerase (pol) μ prepares breaks for end-joining and this is required for successful NHEJ in vivo. We previously showed that pol μ lacks discrimination against oxidized dGTP (8-oxo-dGTP), that can lead to mutagenesis, cancer, aging and human disease. Here we reveal the structural basis for proficient oxidized ribonucleotide (8-oxo-rGTP) incorporation during DSB repair by pol μ. Time-lapse crystallography snapshots of structural intermediates during nucleotide insertion along with computational simulations reveal substrate, metal and side chain dynamics, that allow oxidized ribonucleotides to escape polymerase discrimination checkpoints. Abundant nucleotide pools, combined with inefficient sanitization and repair, implicate pol μ mediated oxidized ribonucleotide insertion as an emerging source of widespread persistent mutagenesis and genomic instability.

scholarly journals An improved method for precise genome editing in zebrafish using CRISPR-Cas9 technique

2021 ◽  
Author(s):  
Eugene V. Gasanov ◽  
Justyna Jędrychowska ◽  
Michal Pastor ◽  
Malgorzata Wiweger ◽  
Axel Methner ◽  
...  
Keyword(s):  
Genome Editing ◽  
High Efficiency ◽  
Target Site ◽  
Proof Of Concept ◽  
Intervention Site ◽  
End Joining ◽  
Guide Rna ◽  
Guide Rnas ◽  
Cas9 Nuclease ◽  
Non Homologous End Joining

AbstractCurrent methods of CRISPR-Cas9-mediated site-specific mutagenesis create deletions and small insertions at the target site which are repaired by imprecise non-homologous end-joining. Targeting of the Cas9 nuclease relies on a short guide RNA (gRNA) corresponding to the genome sequence approximately at the intended site of intervention. We here propose an improved version of CRISPR-Cas9 genome editing that relies on two complementary guide RNAs instead of one. Two guide RNAs delimit the intervention site and allow the precise deletion of several nucleotides at the target site. As proof of concept, we generated heterozygous deletion mutants of the kcng4b, gdap1, and ghitm genes in the zebrafish Danio rerio using this method. A further analysis by high-resolution DNA melting demonstrated a high efficiency and a low background of unpredicted mutations. The use of two complementary gRNAs improves CRISPR-Cas9 specificity and allows the creation of predictable and precise mutations in the genome of D. rerio.

scholarly journals Restoration of RPGR expression in vivo using CRISPR/Cas9 gene editing

Gene Therapy ◽  
2021 ◽  
Author(s):  
Jessica D. Gumerson ◽  
Amal Alsufyani ◽  
Wenhan Yu ◽  
Jingqi Lei ◽  
Xun Sun ◽  
...  
Keyword(s):  
Expression Vectors ◽  
End Joining ◽  
Reading Frame ◽  
Guide Rna ◽  
Domain Specific ◽  
Somatic Gene ◽  
Non Homologous End Joining ◽  
Cone Opsins ◽  
Inherited Retinal Degeneration

AbstractMutations in the gene for Retinitis Pigmentosa GTPase Regulator (RPGR) cause the X-linked form of inherited retinal degeneration, and the majority are frameshift mutations in a highly repetitive, purine-rich region of RPGR known as the OFR15 exon. Truncation of the reading frame in this terminal exon ablates the functionally important C-terminal domain. We hypothesized that targeted excision in ORF15 by CRISPR/Cas9 and the ensuing repair by non-homologous end joining could restore RPGR reading frame in a portion of mutant photoreceptors thereby correcting gene function in vivo. We tested this hypothesis in the rd9 mouse, a naturally occurring mutant line that carries a frameshift mutation in RPGRORF15, through a combination of germline and somatic gene therapy approaches. In germline gene-edited rd9 mice, probing with RPGR domain-specific antibodies demonstrated expression of full length RPGRORF15 protein. Hallmark features of RPGR mutation-associated early disease phenotypes, such as mislocalization of cone opsins, were no longer present. Subretinal injections of the same guide RNA (sgRNA) carried in AAV sgRNA and SpCas9 expression vectors restored reading frame of RPGRORF15 in a subpopulation of cells with broad distribution throughout the retina, confirming successful correction of the mutation. These data suggest that a simplified form of genome editing mediated by CRISPR, as described here, could be further developed to repair RPGRORF15 mutations in vivo.

scholarly journals Autophosphorylation and Self-Activation of DNA-Dependent Protein Kinase

Genes ◽  
2021 ◽  
Vol 12 (7) ◽  
pp. 1091
Author(s):  
Aya Kurosawa
Keyword(s):  
Protein Kinase ◽  
Conformational Changes ◽  
Strand Break ◽  
In Vivo Studies ◽  
End Joining ◽  
Dependent Protein Kinase ◽  
Dependent Protein ◽  
Non Homologous End Joining

The DNA-dependent protein kinase catalytic subunit (DNA-PKcs), a member of the phosphatidylinositol 3-kinase-related kinase family, phosphorylates serine and threonine residues of substrate proteins in the presence of the Ku complex and double-stranded DNA. Although it has been established that DNA-PKcs is involved in non-homologous end-joining, a DNA double-strand break repair pathway, the mechanisms underlying DNA-PKcs activation are not fully understood. Nevertheless, the findings of numerous in vitro and in vivo studies have indicated that DNA-PKcs contains two autophosphorylation clusters, PQR and ABCDE, as well as several autophosphorylation sites and conformational changes associated with autophosphorylation of DNA-PKcs are important for self-activation. Consistent with these features, an analysis of transgenic mice has shown that the phenotypes of DNA-PKcs autophosphorylation mutations are significantly different from those of DNA-PKcs kinase-dead mutations, thereby indicating the importance of DNA-PKcs autophosphorylation in differentiation and development. Furthermore, there has been notable progress in the high-resolution analysis of the conformation of DNA-PKcs, which has enabled us to gain a visual insight into the steps leading to DNA-PKcs activation. This review summarizes the current progress in the activation of DNA-PKcs, focusing in particular on autophosphorylation of this kinase.

scholarly journals Leaky severe combined immunodeficiency in mice lacking non-homologous end joining factors XLF and MRI

2020 ◽  
Author(s):  
Sergio Castañeda-Zegarra ◽  
Qindong Zhang ◽  
Amin Alirezaylavasani ◽  
Marion Fernandez-Berrocal ◽  
Rouan Yao ◽  
...  
Keyword(s):  
Severe Combined Immunodeficiency ◽  
Embryonic Lethality ◽  
Combined Immunodeficiency ◽  
End Joining ◽  
Mild Phenotype ◽  
Reduced Body ◽  
Non Homologous End Joining ◽  
Mature Lymphocyte ◽  
And Function

AbstractNon-homologous end-joining (NHEJ) is a DNA repair pathway required to detect, process, and ligate DNA double-stranded breaks (DSBs) throughout the cell cycle. The NHEJ pathway is necessary for V(D)J recombination in developing B and T lymphocytes. During NHEJ, Ku70 and Ku80 form a heterodimer that recognizes DSBs and promotes recruitment and function of downstream factors PAXX, MRI, DNA-PKcs, Artemis, XLF, XRCC4, and LIG4. Mutations in several known NHEJ genes result in severe combined immunodeficiency (SCID). Inactivation of Mri, Paxx or Xlf in mice results in normal or mild phenotype, while combined inactivation of Xlf/Mri, Xlf/Paxx, or Xlf/Dna-pkcs leads to late embryonic lethality. Here, we describe three new mouse models. We demonstrate that deletion of Trp53 rescues embryonic lethality in mice with combined deficiencies of Xlf and Mri. Furthermore, Xlf-/-Mri-/-Trp53+/- and Xlf-/-Paxx-/-Trp53+/- mice possess reduced body weight, severely reduced mature lymphocyte counts, and accumulation of progenitor B cells. We also report that combined inactivation of Mri/Paxx results in live-born mice with modest phenotype, and combined inactivation of Mri/Dna-pkcs results in embryonic lethality. Therefore, we conclude that XLF is functionally redundant with MRI and PAXX during lymphocyte development in vivo. Moreover, Mri genetically interacts with Dna-pkcs and Paxx.

scholarly journals Tuba1a is uniquely important for axon guidance through midline commissural structures

2020 ◽  
Author(s):  
Georgia Buscaglia ◽  
Jayne Aiken ◽  
Katelyn J. Hoff ◽  
Kyle R. Northington ◽  
Emily A. Bates
Keyword(s):  
Axon Guidance ◽  
Growth Cone ◽  
Intracellular Transport ◽  
Genetic Manipulation ◽  
Morphological Changes ◽  
Loss Of Function ◽  
Developmental Guidance ◽  
Genes Encoding ◽  
Developing Neurons

AbstractDeveloping neurons undergo dramatic morphological changes to appropriately migrate and extend axons to make synaptic connections. The microtubule cytoskeleton, made of α/β-tubulin dimers, drives neurite outgrowth, promotes neuronal growth cone responses, and facilitates intracellular transport of critical cargoes during neurodevelopment. TUBA1A constitutes the majority of α-tubulin in the developing brain and mutations to TUBA1A in humans cause severe brain malformations accompanied by varying neurological defects, collectively termed tubulinopathies. Studies of TUBA1A function in vivo have been limited by the presence of multiple genes encoding highly similar tubulin proteins, which prevents TUBA1A-specific antibody generation and makes genetic manipulation challenging. Here we present a novel tagging method for studying and manipulating TUBA1A in cells without impairing tubulin function. Using this tool, we show that a TUBA1A loss-of-function mutation TUBA1AN102D (TUBA1AND), reduced the amount of TUBA1A protein and prevented incorporation of TUBA1A into microtubule polymers. Reduced Tuba1a α-tubulin in heterozygous Tuba1aND/+ mice significantly impacted axon extension and impaired formation of forebrain commissures. Neurons with reduced Tuba1a caused by Tuba1aND had altered microtubule dynamics and slower neuron outgrowth compared to controls. Neurons deficient in Tuba1a failed to localize microtubule associated protein-1b (Map1b) to the developing growth cone, likely impacting reception of developmental guidance cues. Overall, we show that reduced Tuba1a is sufficient to support neuronal migration, but not axon guidance, and provide mechanistic insight as to how TUBA1A tunes microtubule function to support neurodevelopment.

scholarly journals Linear-double-stranded DNA (ldsDNA) based AND logic computation in mammalian cells

2018 ◽  
Author(s):  
Weijun Su ◽  
Chunze Zhang ◽  
Shuai Li
Keyword(s):  
Synthetic Biology ◽  
Mammalian Cells ◽  
Rapid Development ◽  
Genetic Circuits ◽  
End Joining ◽  
Double Stranded Dna ◽  
Logic Computation ◽  
Key Enzyme ◽  
Non Homologous End Joining

AbstractSynthetic biology employs engineering principles to redesign biological system for clinical or industrial purposes. The development and application of novel genetic devices for genetic circuits construction will facilitate the rapid development of synthetic biology. Here we demonstrate that mammalian cells could perform two- and three-input linear-double-stranded DNA (ldsDNA) based Boolean AND logic computation. Through hydrodynamic ldsDNA delivery, two-input ldsDNA-base AND-gate computation could be achieved in vivo. Inhibition of DNA-PKcs expression, a key enzyme in non-homologous end joining (NHEJ), could significantly downregulate the intensity of output signals from ldsDNA-based AND-gate. We further reveal that in mammalian cells ldsDNAs could undergo end processing and then perform AND-gate calculation to generate in-frame output proteins. Moreover, we show that ldsDNAs or plasmids with identical overlapping sequences could also serve as inputs of AND-gate computation. Our work establishes novel genetic devices and principles for genetic circuits construction, thus may open a new gate for the development of new disease targeting strategies and new protein genesis methodologies.

scholarly journals Leaky Severe Combined Immunodeficiency in Mice Lacking Non-homologous End Joining Factors XLF and MRI

2020 ◽  
Author(s):  
Sergio Castaneda-Zegarra ◽  
Qindong Zhang ◽  
Amin Alirezaylavasani ◽  
Marion Fernandez-Berrocal ◽  
Rouan Yao ◽  
...  
Keyword(s):  
Severe Combined Immunodeficiency ◽  
Embryonic Lethality ◽  
Combined Immunodeficiency ◽  
End Joining ◽  
Mild Phenotype ◽  
Reduced Body ◽  
Non Homologous End Joining ◽  
Mature Lymphocyte ◽  
And Function

Non-homologous end-joining (NHEJ) is a DNA repair pathway required to detect, process, and ligate DNA double-stranded breaks (DSBs) throughout the cell cycle. The NHEJ pathway is necessary for V(D)J recombination in developing B and T lymphocytes. During NHEJ, Ku70 and Ku80 form a heterodimer that recognizes DSBs and promotes recruitment and function of downstream factors PAXX, MRI, DNA-PKcs, Artemis, XLF, XRCC4, and LIG4. Mutations in several known NHEJ genes result in severe combined immunodeficiency (SCID). Inactivation of Mri, Paxx or Xlf in mice results in normal or mild phenotype, while combined inactivation of Xlf/Mri, Xlf/Paxx, or Xlf/Dna-pkcs leads to late embryonic lethality. Here, we describe three new mouse models. We demonstrate that deletion of Trp53 rescues embryonic lethality in mice with combined deficiencies of Xlf and Mri. Furthermore, Xlf-/-Mri-/-Trp53+/- and Xlf-/-Paxx-/-Trp53+/- mice possess reduced body weight, severely reduced mature lymphocyte counts, and accumulation of progenitor B cells. We also report that combined inactivation of Mri/Paxx results in live-born mice with modest phenotype, and combined inactivation of Mri/Dna-pkcs results in embryonic lethality. Therefore, we conclude that XLF is functionally redundant with MRI and PAXX during lymphocyte development in vivo. Moreover, Mri genetically interacts with Dna-pkcs and Paxx.

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