scholarly journals Engineering targeted deletions in the mitochondrial genome

2018 ◽  
Author(s):  
Jarryd M. Campbell ◽  
Ester Perales-Clemente ◽  
Hirotaka Ata ◽  
Noemi Vidal-Folch ◽  
Weibin Liu ◽  
...  

Summary ParagraphMitochondria are a network of critical intracellular organelles with diverse functions ranging from energy production to cell signaling. The mitochondrial genome (mtDNA) consists of 37 genes that support oxidative phosphorylation and are prone to dysfunction that can lead to currently untreatable diseases. Further characterization of mtDNA gene function and creation of more accurate models of human disease will require the ability to engineer precise genomic sequence modifications. To date, mtDNA has been inaccessible to direct modification using traditional genome engineering tools due to unique DNA repair contexts in mitochondria1. Here, we report a new DNA modification process using sequence-specific transcription activator-like effector (TALE) proteins to manipulate mtDNA in vivo and in vitro for reverse genetics applications. First, we show mtDNA deletions can be induced in Danio rerio (zebrafish) using site-directed mitoTALE-nickases (mito-nickases). Using this approach, the protein-encoding mtDNA gene nd4 was deleted in injected zebrafish embryos. Furthermore, this DNA engineering system recreated a large deletion spanning from nd5 to atp8, which is commonly found in human diseases like Kearns-Sayre syndrome (KSS) and Pearson syndrome. Enrichment of mtDNA-deleted genomes was achieved using targeted mitoTALE-nucleases (mitoTALENs) by co-delivering both mito-nickases and mitoTALENs into zebrafish embryos. This combined approach yielded deletions in over 90% of injected animals, which were maintained through adulthood in various tissues. Subsequently, we confirmed that large, targeted deletions could be induced with this approach in human cells. In addition, we show that, when provided with a single nick on the mtDNA light strand, the binding of a terminal TALE protein alone at the intended recombination site is sufficient for deletion induction. This “block and nick” approach yielded engineered mitochondrial molecules with single nucleotide precision using two different targeted deletion sites. This precise seeding method to engineer mtDNA variants is a critical step for the exploration of mtDNA function and for creating new cellular and animal models of mitochondrial disease.

2021 ◽  
Vol 11 ◽  
Author(s):  
Ujin Shin ◽  
Vincent Brondani

Nucleases used in genome engineering induce hydrolysis of DNA phosphate backbone in a sequence-specific manner. So far CRISPR-Cas, the RNA-guided nucleases, is the most advanced genome engineering system. The CRISPR nucleases allows recognition of a particular genomic sequence with two distinct molecular interactions: first, by direct interaction between the nuclease and the protospacer-adjacent motif, wherein discrete amino acids interact with DNA base pairs; and second, by hybridization of the guide RNA with the target DNA sequence. Here we report the application of the single strand annealing cellular assay to analyze and quantify nuclease activity of wild type and mutant CRISPR-Cpf1. Using this heterologous marker system based on GFP activity, we observed a comparable PAM recognition selectivity with the NGS analysis. The heterologous marker system has revealed that LbCpf1 is a more specific nuclease than AsCpf1 in a cellular context. We controlled the in vitro activity of the Cpf1 nuclease complexes expressed in mammalian cells and demonstrated that they are responsible of the DNA cleavage at the target site. In addition, we generated and tested LbCpf1 variants with several combinations of mutations at the PAM-recognition positions G532, K538 and Y542. Finally, we showed that the results of the in vitro DNA cleavage assay with the wild type and mutants LbCpf1 corroborate with the selection of 6TG resistant cells associated to the genomic disruption of hprt gene.


2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Sofia M. Saraiva ◽  
Carlha Gutiérrez-Lovera ◽  
Jeannette Martínez-Val ◽  
Sainza Lores ◽  
Belén L. Bouzo ◽  
...  

AbstractTriple negative breast cancer (TNBC) is known for being very aggressive, heterogeneous and highly metastatic. The standard of care treatment is still chemotherapy, with adjacent toxicity and low efficacy, highlighting the need for alternative and more effective therapeutic strategies. Edelfosine, an alkyl-lysophospholipid, has proved to be a promising therapy for several cancer types, upon delivery in lipid nanoparticles. Therefore, the objective of this work was to explore the potential of edelfosine for the treatment of TNBC. Edelfosine nanoemulsions (ET-NEs) composed by edelfosine, Miglyol 812 and phosphatidylcholine as excipients, due to their good safety profile, presented an average size of about 120 nm and a neutral zeta potential, and were stable in biorelevant media. The ability of ET-NEs to interrupt tumor growth in TNBC was demonstrated both in vitro, using a highly aggressive and invasive TNBC cell line, and in vivo, using zebrafish embryos. Importantly, ET-NEs were able to penetrate through the skin barrier of MDA-MB 231 xenografted zebrafish embryos, into the yolk sac, leading to an effective decrease of highly aggressive and invasive tumoral cells’ proliferation. Altogether the results demonstrate the potential of ET-NEs for the development of new therapeutic approaches for TNBC.


2006 ◽  
Vol 80 (8) ◽  
pp. 4179-4182 ◽  
Author(s):  
Pierre Rivailler ◽  
Amitinder Kaur ◽  
R. Paul Johnson ◽  
Fred Wang

ABSTRACT A pathogenic isolate of rhesus cytomegalovirus (rhCMV 180.92) was cloned, sequenced, and annotated. Comparisons with the published rhCMV 68.1 genome revealed 8 open reading frames (ORFs) in isolate 180.92 that are absent in 68.1, 10 ORFs in 68.1 that are absent in 180.92, and 34 additional ORFs that were not previously annotated. Most of the differences appear to be due to genetic rearrangements in both isolates from a region that is frequently altered in human CMV (hCMV) during in vitro passage. These results indicate that the rhCMV ORF repertoire is larger than previously recognized. Like hCMV, understanding of the complete coding capacity of rhCMV is complicated by genomic instability and may require comparisons with additional isolates in vitro and in vivo.


2017 ◽  
Vol 37 (6) ◽  
pp. 3565-3571 ◽  
Author(s):  
Xiaoli Wang ◽  
Wenmei Zhang ◽  
Yan Ding ◽  
Xingrong Guo ◽  
Yahong Yuan ◽  
...  

2011 ◽  
Vol 137 (1) ◽  
pp. 64-69 ◽  
Author(s):  
Judy Yuet-Wa Chan ◽  
Johnny Chi-Man Koon ◽  
Xiaozhou Liu ◽  
Michael Detmar ◽  
Biao Yu ◽  
...  

1989 ◽  
Vol 9 (11) ◽  
pp. 4835-4845
Author(s):  
S J Anderson ◽  
S Miyake ◽  
D Y Loh

We identified a regulatory region of the murine V beta promoter by both in vivo and in vitro analyses. The results of transient transfection assays indicated that the dominant transcription-activating element within the V beta 8.3 promoter is the palindromic motif identified previously as the conserved V beta decamer. Elimination of this element, by linear deletion or specific mutation, reduced transcriptional activity from this promoter by 10-fold. DNase I footprinting, gel mobility shift, and methylation interference assays confirmed that the palindrome acts as the binding site of a specific nuclear factor. In particular, the V beta promoter motif functioned in vitro as a high-affinity site for a previously characterized transcription activator, ATF. A consensus cyclic AMP response element (CRE) but not a consensus AP-1 site, can substitute for the decamer in vivo. These data suggest that cyclic AMP response element-binding protein (ATF/CREB) or related proteins activate V beta transcription.


Molecules ◽  
2018 ◽  
Vol 24 (1) ◽  
pp. 8 ◽  
Author(s):  
Mayra Antúnez-Mojica ◽  
Andrés Rojas-Sepúlveda ◽  
Mario Mendieta-Serrano ◽  
Leticia Gonzalez-Maya ◽  
Silvia Marquina ◽  
...  

By using a zebrafish embryo model to guide the chromatographic fractionation of antimitotic secondary metabolites, seven podophyllotoxin-type lignans were isolated from a hydroalcoholic extract obtained from the steam bark of Bursera fagaroides. The compounds were identified as podophyllotoxin (1), β-peltatin-A-methylether (2), 5′-desmethoxy-β-peltatin-A-methylether (3), desmethoxy-yatein (4), desoxypodophyllotoxin (5), burseranin (6), and acetyl podophyllotoxin (7). The biological effects on mitosis, cell migration, and microtubule cytoskeleton remodeling of lignans 1–7 were further evaluated in zebrafish embryos by whole-mount immunolocalization of the mitotic marker phospho-histone H3 and by a tubulin antibody. We found that lignans 1, 2, 4, and 7 induced mitotic arrest, delayed cell migration, and disrupted the microtubule cytoskeleton in zebrafish embryos. Furthermore, microtubule cytoskeleton destabilization was observed also in PC3 cells, except for 7. Therefore, these results demonstrate that the cytotoxic activity of 1, 2, and 4 is mediated by their microtubule-destabilizing activity. In general, the in vivo and in vitro models here used displayed equivalent mitotic effects, which allows us to conclude that the zebrafish model can be a fast and cheap in vivo model that can be used to identify antimitotic natural products through bioassay-guided fractionation.


Sarcoma ◽  
2012 ◽  
Vol 2012 ◽  
pp. 1-11 ◽  
Author(s):  
Alexander B. Mohseny ◽  
Pancras C. W. Hogendoorn ◽  
Anne-Marie Cleton-Jansen

High-grade osteosarcoma is an aggressive tumor most commonly affecting adolescents. The early age of onset might suggest genetic predisposition; however, the vast majority of the tumors are sporadic. Early onset, most often lack of a predisposing condition or lesion, only infrequent (<2%) prevalence of inheritance, extensive genomic instability, and a wide histological heterogeneity are just few factors to mention that make osteosarcoma difficult to study. Therefore, it is sensible to design and use models representative of the human disease. Here we summarize multiple osteosarcoma models establishedin vitroandin vivo, comment on their utilities, and highlight newest achievements, such as the use of zebrafish embryos. We conclude that to gain a better understanding of osteosarcoma, simplification of this extremely complex tumor is needed. Therefore, we parse the osteosarcoma problem into parts and propose adequate models to study them each separately. A better understanding of osteosarcoma provides opportunities for discovering and assaying novel effective treatment strategies.


2020 ◽  
Author(s):  
Nelson V. Simwela ◽  
Katie R. Hughes ◽  
Michael T. Rennie ◽  
Michael P. Barrett ◽  
Andrew P. Waters

AbstractCurrent malaria control efforts rely significantly on artemisinin combinational therapies which have played massive roles in alleviating the global burden of the disease. Emergence of resistance to artemisinins is therefore, not just alarming but requires immediate intervention points such as development of new antimalarial drugs or improvement of the current drugs through adjuvant or combination therapies. Artemisinin resistance is primarily conferred by Kelch13 propeller mutations which are phenotypically characterised by generalised growth quiescence, altered haemoglobin trafficking and downstream enhanced activity of the parasite stress pathways through the ubiquitin proteasome system (UPS). Previous work on artemisinin resistance selection in a rodent model of malaria, which we and others have recently validated using reverse genetics, has also shown that mutations in deubiquitinating enzymes, DUBs (upstream UPS component) modulates susceptibility of malaria parasites to both artemisinin and chloroquine. The UPS or upstream protein trafficking pathways have, therefore, been proposed to be not just potential drug targets, but also possible intervention points to overcome artemisinin resistance. Here we report the activity of small molecule inhibitors targeting mammalian DUBs in malaria parasites. We show that generic DUB inhibitors can block intraerythrocytic development of malaria parasites in vitro and possess antiparasitic activity in vivo and can be used in combination with additive effect. We also show that inhibition of these upstream components of the UPS can potentiate the activity of artemisinin in vitro as well as in vivo to the extent that ART resistance can be overcome. Combinations of DUB inhibitors anticipated to target different DUB activities and downstream 20s proteasome inhibitors are even more effective at improving the potency of artemisinins than either inhibitors alone providing proof that targeting multiple UPS activities simultaneously could be an attractive approach to overcoming artemisinin resistance. These data further validate the parasite UPS as a target to both enhance artemisinin action and potentially overcome resistance. Lastly, we confirm that DUB inhibitors can be developed into in vivo antimalarial drugs with promise for activity against all of human malaria and could thus further exploit their current pursuit as anticancer agents in rapid drug repurposing programs.Graphical abstract


Blood ◽  
1988 ◽  
Vol 71 (3) ◽  
pp. 766-770
Author(s):  
PT Curtin ◽  
YW Kan

We have previously described an English family with gamma delta beta- thalassemia in which a large deletion stops 25 kilobases (kb) upstream from the beta-globin gene locus, and yet the beta-globin gene is inactive in vivo. Affected family members had a beta-thalassemia minor phenotype with a normal hemoglobin A2 level. Gene mapping showed that these subjects were heterozygous for a chromosome bearing a large deletion that began in the G gamma-globin gene, extended through the epsilon-globin gene, and continued upstream for at least 75 kb. The A gamma-, delta-, and beta-globin gene loci on this chromosome were intact. To examine the possibility that an additional defect was present in the beta-globin gene, we cloned, sequenced, and examined the expression of the beta-globin gene from the affected chromosome. No mutation was found in the beta-globin gene sequence from 990 base-pairs 5′ to the cap site to 350 basepairs 3′ to the polyadenylation signal. The gene was subcloned into an expression vector and introduced into HeLa cells. Analysis of RNA derived from these cells, using a ribonuclease protection assay, revealed qualitatively and quantitatively normal transcription. Thus a structurally and functionally normal beta-globin gene is inactive in the presence of a large deletion more than 25 kb upstream. The loss of beta-globin gene function may be due to disturbance of chromatin conformation caused by the deletion or may be the result of loss of upstream sequences that are necessary for beta-globin gene expression in vivo.


Sign in / Sign up

Export Citation Format

Share Document