337 NONMEIOTIC INTROGRESSION OF QUANTITATIVE TRAIT NUCLEOTIDES AND CORRECTION OF CONGENITAL MUTATIONS IN LIVESTOCK WITH TRANSCRIPTION ACTIVATOR-LIKE EFFECTOR NUCLEASES

2013 ◽  
Vol 25 (1) ◽  
pp. 316
Author(s):  
S. C. Fahrenkrug ◽  
W. Tan ◽  
S. G. Lillico ◽  
D. Stverakova ◽  
C. Proudfoot ◽  
...  
Keyword(s):  
Quantitative Trait ◽  
Gene Editing ◽  
Production Systems ◽  
Gene Knockout ◽  
Direct Injection ◽  
Genetic Enhancement ◽  
Transcription Activator ◽  
Tal Effectors ◽  
Cattle Breeds ◽  
Specific Allele

Genetic enhancement of livestock productivity and welfare are major goals of breeding and genetics programs. However, the introgression of desirable alleles across breeds is slow and inaccurate. The development of gene editing technologies would provide the opportunity to accelerate the genetic improvement of a diversity of livestock breeds. Transcription activator-like effector nucleases (TALEN) are programmable nucleases that join the modular DNA binding domain of transcription activator-like (TAL) effectors with FokI endonuclease. We found that TALEN could be easily manufactured and that 64% displayed activity in swine and cattle primary fibroblasts, with cleavage of 1.5 to 45% of chromosomes in cell populations, as measured by Surveyor assay. Clonal isolation and sequencing revealed that up to 84% of cells contained at least one modified allele, with up to 24% of cells containing biallelic or homozygous chromosomal modification. Co-transfection of a customized TALEN pair with a template containing a specific allele was effective at the nonmeiotic introgression of quantitative trait into naïve cattle breeds. We will also describe the repair of 2 recently described embryonic lethal mutations that are segregating in important dairy cattle breeds (JH1 and HH1). Injection of TALEN mRNA into the cytoplasm of pig and cattle zygotes was capable of inducing gene knockout (KO) in 27 to 75% of embryos analysed (n = 4–59), nearly half of which (8/19) harbored biallelic modification. We will present data describing efforts towards gene conversion by direct injection of livestock embryos. Finally, we will present alternative strategies for the incorporation of gene editing in livestock production systems by cloning or embryo treatment.

scholarly journals TALEN-Mediated Gene Editing of slc24a5 (Solute Carrier Family 24, Member 5) in Kawakawa, Euthynnus affinis

2021 ◽  
Vol 9 (12) ◽  
pp. 1378
Author(s):  
Dipak Pandey ◽  
Takahiro Matsubara ◽  
Taiju Saito ◽  
Yukinori Kazeto ◽  
Koichiro Gen ◽  
...  
Keyword(s):  
Gene Editing ◽  
Early Stage ◽  
Gene Knockout ◽  
Pigment Epithelium ◽  
Retinal Pigment ◽  
The Body ◽  
Induced Mutations ◽  
Transcription Activator ◽  
Cell Stage ◽  
Euthynnus Affinis

Transcription activator-like effector (TALE) nucleases (TALENs) mediated gene editing methods are becoming popular and have revealed the staggering complexity of genome control during development. Here, we present a simple and efficient gene knockout using TALENs in kawakawa, Euthynnus affinis, using slc24a5. We examined slc24a5 gene expression and functional differences between two TALENs that hold the TALE scaffolds, +153/+47 and +136/+63 and target slc24a5. Developmental changes in slc24a5 transcripts were seen in early-stage embryos by real-time PCR; slc24a5 expression was first detected 48 h post fertilization (hpf), which increased dramatically at 72 hpf. Four TALENs, 47- and 63-type of two different target loci (A and B), respectively, were constructed using Platinum TALEN and evaluated in vitro by a single-strand annealing (SSA) assay. TALEN activities were further evaluated in vivo by injecting TALEN mRNAs in the two-cell stage of the zygote. Most of the TALEN-induced mutants showed mosaic patterns in the retinal pigment epithelium (RPE) and fewer melanin pigments on the body at 72 hpf and later when compared to the control, implying the gene’s association with melanin pigment formation. A heteroduplex mobility assay (HMA) and the genome sequence further confirmed the TALEN-induced mutations of substitution, insertion, and deletion at an endogenous locus.

scholarly journals A TAL effector-like protein of an endofungal bacterium increases the stress tolerance and alters the transcriptome of the host

2020 ◽  
Vol 117 (29) ◽  
pp. 17122-17129 ◽  
Author(s):  
Morgan E. Carter ◽  
Sara C.D. Carpenter ◽  
Zoë E. Dubrow ◽  
Mark R. Sabol ◽  
Fabio C. Rinaldi ◽  
...  
Keyword(s):  
Pathogenic Bacteria ◽  
Gene Knockout ◽  
Transcription Activator ◽  
Rhizopus Microsporus ◽  
Host Genotype ◽  
Tal Effectors ◽  
Banding Patterns ◽  
Nuclear Localization Signals ◽  
Bacterial Type ◽  
Wild Type Bacterium

Symbioses of bacteria with fungi have only recently been described and are poorly understood. In the symbiosis ofMycetohabitans(formerlyBurkholderia)rhizoxinicawith the fungusRhizopus microsporus, bacterial type III (T3) secretion is known to be essential. Proteins resembling T3-secreted transcription activator-like (TAL) effectors of plant pathogenic bacteria are encoded in the three sequencedMycetohabitansspp. genomes. TAL effectors nuclear-localize in plants, where they bind and activate genes important in disease. The Burkholderia TAL-like (Btl) proteins bind DNA but lack the N- and C-terminal regions, in which TAL effectors harbor their T3 and nuclear localization signals, and activation domain. We characterized a Btl protein, Btl19-13, and found that, despite the structural differences, it can be T3-secreted and can nuclear-localize. Abtl19-13gene knockout did not prevent the bacterium from infecting the fungus, but the fungus became less tolerant to cell membrane stress. Btl19-13 did not alter transcription in a plant-based reporter assay, but 15R. microsporusgenes were differentially expressed in comparisons both of the fungus infected with the wild-type bacterium vs. the mutant and with the mutant vs. a complemented strain. Southern blotting revealedbtlgenes in 14 diverseMycetohabitansisolates. However, banding patterns and available sequences suggest variation, and thebtl19-13phenotype could not be rescued by abtlgene from a different strain. Our findings support the conclusion that Btl proteins are effectors that act on host DNA and play important but varied or possibly host genotype-specific roles in theM. rhizoxinica–R. microsporussymbiosis.

scholarly journals A TAL effector-like protein of symbiotic Mycetohabitans increases stress tolerance and alters the transcriptome of the fungal host Rhizopus microsporus

2020 ◽  
Author(s):  
Morgan E. Carter ◽  
Sara C.D. Carpenter ◽  
Zoë E. Dubrow ◽  
Mark R. Sabol ◽  
Fabio C. Rinaldi ◽  
...  
Keyword(s):  
Pathogenic Bacteria ◽  
Gene Knockout ◽  
Transcription Activator ◽  
Rhizopus Microsporus ◽  
Host Genotype ◽  
Tal Effectors ◽  
Banding Patterns ◽  
Fungal Host ◽  
Nuclear Localization Signals ◽  
Bacterial Type

AbstractSymbioses of bacteria with fungi have only recently been described and are poorly understood. In the symbiosis of Mycetohabitans (formerly Burkholderia) rhizoxinica with the fungus Rhizopus microsporus, bacterial type III (T3) secretion is known to be essential. Proteins resembling T3-secreted transcription activator-like (TAL) effectors of plant pathogenic bacteria are encoded in the three sequenced Mycetohabitans spp. genomes. TAL effectors nuclear localize in plants, where they bind and activate genes important in disease. The Burkholderia TAL-like (Btl) proteins bind DNA but lack the N- and C-terminal regions in which TAL effectors harbor their T3 and nuclear localization signals, and activation domain. We characterized a Btl protein, Btl19-13, and found that, despite the structural differences, it can be T3-secreted and can nuclear localize. A btl19-13 gene knockout did not prevent the bacterium from infecting the fungus, but the fungus became less tolerant to cell membrane stress. Btl19-13 did not alter transcription in a plant-based reporter assay, but 15 R. microsporus genes were differentially expressed in comparisons both of the fungus infected with the wildtype bacterium vs the mutant and with the mutant vs. a complemented strain. Southern blotting revealed btl genes in 14 diverse Mycetohabitans isolates. However, banding patterns and available sequences suggest variation, and the btl19-13 phenotype could not be rescued by a btl gene from a different strain. Our findings support the conclusion that Btl proteins are effectors that act on host DNA and play important but varied or possibly host-genotype-specific roles in the M. rhizoxinica-R. microsporus symbiosis.

scholarly journals Exploiting DNA Endonucleases to Advance Mechanisms of DNA Repair

Biology ◽  
2021 ◽  
Vol 10 (6) ◽  
pp. 530
Author(s):  
Marlo K. Thompson ◽  
Robert W. Sobol ◽  
Aishwarya Prakash
Keyword(s):  
Dna Repair ◽  
Mammalian Cells ◽  
Genome Stability ◽  
Gene Editing ◽  
Adaptive Immune System ◽  
Zinc Finger Nucleases ◽  
Specific Gene ◽  
Target Sequence ◽  
Transcription Activator ◽  
Low Cytotoxicity

The earliest methods of genome editing, such as zinc-finger nucleases (ZFN) and transcription activator-like effector nucleases (TALENs), utilize customizable DNA-binding motifs to target the genome at specific loci. While these approaches provided sequence-specific gene-editing capacity, the laborious process of designing and synthesizing recombinant nucleases to recognize a specific target sequence, combined with limited target choices and poor editing efficiency, ultimately minimized the broad utility of these systems. The discovery of clustered regularly interspaced short palindromic repeat sequences (CRISPR) in Escherichia coli dates to 1987, yet it was another 20 years before CRISPR and the CRISPR-associated (Cas) proteins were identified as part of the microbial adaptive immune system, by targeting phage DNA, to fight bacteriophage reinfection. By 2013, CRISPR/Cas9 systems had been engineered to allow gene editing in mammalian cells. The ease of design, low cytotoxicity, and increased efficiency have made CRISPR/Cas9 and its related systems the designer nucleases of choice for many. In this review, we discuss the various CRISPR systems and their broad utility in genome manipulation. We will explore how CRISPR-controlled modifications have advanced our understanding of the mechanisms of genome stability, using the modulation of DNA repair genes as examples.

Abstract 344: Finding the Achilles’ heel of Muscle Giant---TALEN-mediated Gene-editing in Zebrafish Titin

2015 ◽  
Vol 117 (suppl_1) ◽  
Author(s):  
Yu-Huan Shih ◽  
Xiaolei Xu
Keyword(s):  
Alternative Splicing ◽  
Gene Editing ◽  
Muscle Development ◽  
Exon Skipping ◽  
Transcription Activator ◽  
Detailed Characterization ◽  
A Domains ◽  
Underlying Mechanisms ◽  
Sarcomere Assembly

Background: TITIN (TTN) has more than 300 exons and encodes a gigantic protein that is crucial for heart and muscle development. Mutations in TTN caused a variety of human diseases including cardiomyopathy and muscular dystrophy. Recently, dilated cardiomyopathy-associated mutations on TTN have been found more frequently in exons encoding A-band domains but less in exons encoding the N-terminal Z-disc domains, suggesting that mutations in different exons of TTN cause distinct consequences. To elucidate the underlying mechanisms, we leveraged the Transcription Activator-Like Effects Nuclease (TALEN) technology in zebrafish to introduce truncating mutations in different exons of ttn, and then study their effects on heart and somites. Results: We generated truncational mutations in different exons of zebrafish titins encoding Z-disc, N2B, Novex-3, and A domains, respectively. Because zebrafish contains two titin homologues, ttna and ttnb, we introduced mutations in both genes at the corresponding loci. While both Z-disc and A band mutations on ttna disrupted sarcomere assembly in heart and somites, Z-disc or A band mutations on ttnb only affect somites without affecting the heart. Interestingly, a Z-disc mutation on ttna resulted in milder phenotypes than an A-band mutation, while a Z-disc mutation on ttnb generated severer phenotypes than an A-band mutation. No phenotype was observed in the homozygous fish in either ttna-novex-3 or ttnb-N2B mutant fish. Conclusions: A spectrum of truncational mutations in ttna and ttnb has been generated in zebrafish using the TALEN technology. Mutations in different exons result in different phenotypes. Detailed characterization of these mutants and double mutants will be presented, which shall elicit distinct contribution of alternative splicing and exon skipping as two candidate mechanisms during pathogenesis of Titinopathies.

scholarly journals Gene regulation of adult skeletogenesis in starfish and modifications during gene network co-option

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Atsuko Yamazaki ◽  
Shumpei Yamakawa ◽  
Yoshiaki Morino ◽  
Yasunori Sasakura ◽  
Hiroshi Wada
Keyword(s):  
Sea Urchins ◽  
Gene Knockout ◽  
Expression Patterns ◽  
Homeobox Gene ◽  
Regulatory System ◽  
Nutrient Sensing ◽  
Regulatory Genes ◽  
Transcription Activator ◽  
Patiria Pectinifera ◽  
Skeleton Formation

AbstractThe larval skeleton of the echinoderm is believed to have been acquired through co-option of a pre-existing gene regulatory network (GRN); that is, the mechanism for adult skeleton formation in the echinoderm was deployed in early embryogenesis during echinoderm diversification. To explore the evolutionary changes that occurred during co-option, we examined the mechanism for adult skeletogenesis using the starfish Patiria pectinifera. Expression patterns of skeletogenesis-related genes (vegf, vegfr, ets1/2, erg, alx1, ca1, and clect) suggest that adult skeletogenic cells develop from the posterior coelom after the start of feeding. Treatment with inhibitors and gene knockout using transcription activator-like effector nucleases (TALENs) suggest that the feeding-nutrient sensing pathway activates Vegf signaling via target of rapamycin (TOR) activity, leading to the activation of skeletogenic regulatory genes in starfish. In the larval skeletogenesis of sea urchins, the homeobox gene pmar1 activates skeletogenic regulatory genes, but in starfish, localized expression of the pmar1-related genes phbA and phbB was not detected during the adult skeleton formation stage. Based on these data, we provide a model for the adult skeletogenic GRN in the echinoderm and propose that the upstream regulatory system changed from the feeding-TOR-Vegf pathway to a homeobox gene-system during co-option of the skeletogenic GRN.

Gene editing as a promising approach for respiratory diseases

2018 ◽  
Vol 55 (3) ◽  
pp. 143-149 ◽  
Author(s):  
Yichun Bai ◽  
Yang Liu ◽  
Zhenlei Su ◽  
Yana Ma ◽  
Chonghua Ren ◽  
...  
Keyword(s):  
Respiratory Diseases ◽  
Gene Editing ◽  
Gene Mutations ◽  
Well Being ◽  
Short Review ◽  
Zinc Finger Nucleases ◽  
Transcription Activator ◽  
Mortality And Morbidity ◽  
Chronic Respiratory Diseases ◽  
Difficulty Breathing

Respiratory diseases, which are leading causes of mortality and morbidity in the world, are dysfunctions of the nasopharynx, the trachea, the bronchus, the lung and the pleural cavity. Symptoms of chronic respiratory diseases, such as cough, sneezing and difficulty breathing, may seriously affect the productivity, sleep quality and physical and mental well-being of patients, and patients with acute respiratory diseases may have difficulty breathing, anoxia and even life-threatening respiratory failure. Respiratory diseases are generally heterogeneous, with multifaceted causes including smoking, ageing, air pollution, infection and gene mutations. Clinically, a single pulmonary disease can exhibit more than one phenotype or coexist with multiple organ disorders. To correct abnormal function or repair injured respiratory tissues, one of the most promising techniques is to correct mutated genes by gene editing, as some gene mutations have been clearly demonstrated to be associated with genetic or heterogeneous respiratory diseases. Zinc finger nucleases (ZFN), transcription activator-like effector nucleases (TALEN) and clustered regulatory interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) systems are three innovative gene editing technologies developed recently. In this short review, we have summarised the structure and operating principles of the ZFNs, TALENs and CRISPR/Cas9 systems and their preclinical and clinical applications in respiratory diseases.

scholarly journals Carboxylated branched poly(β-amino ester) nanoparticles enable robust cytosolic protein delivery and CRISPR-Cas9 gene editing

2019 ◽  
Vol 5 (12) ◽  
pp. eaay3255 ◽  
Author(s):  
Yuan Rui ◽  
David R. Wilson ◽  
John Choi ◽  
Mahita Varanasi ◽  
Katie Sanders ◽  
...  
Keyword(s):  
Protein Delivery ◽  
Gene Editing ◽  
Gene Knockout ◽  
Cell Types ◽  
Endosomal Escape ◽  
Biological Research ◽  
Amino Ester ◽  
Cytosolic Protein

Efficient cytosolic protein delivery is necessary to fully realize the potential of protein therapeutics. Current methods of protein delivery often suffer from low serum tolerance and limited in vivo efficacy. Here, we report the synthesis and validation of a previously unreported class of carboxylated branched poly(β-amino ester)s that can self-assemble into nanoparticles for efficient intracellular delivery of a variety of different proteins. In vitro, nanoparticles enabled rapid cellular uptake, efficient endosomal escape, and functional cytosolic protein release into cells in media containing 10% serum. Moreover, nanoparticles encapsulating CRISPR-Cas9 ribonucleoproteins (RNPs) induced robust levels of gene knock-in (4%) and gene knockout (>75%) in several cell types. A single intracranial administration of nanoparticles delivering a low RNP dose (3.5 pmol) induced robust gene editing in mice bearing engineered orthotopic murine glioma tumors. This self-assembled polymeric nanocarrier system enables a versatile protein delivery and gene editing platform for biological research and therapeutic applications.

scholarly journals Immunodeficient Rabbit Models: History, Current Status and Future Perspectives

2020 ◽  
Vol 10 (20) ◽  
pp. 7369
Author(s):  
Jun Song ◽  
Brooke Pallas ◽  
Dongshan Yang ◽  
Jifeng Zhang ◽  
Yash Agarwal ◽  
...  
Keyword(s):  
Gene Editing ◽  
Interleukin 2 ◽  
Current Status ◽  
Loss Of Function ◽  
Future Perspectives ◽  
Transcription Activator ◽  
Immune Genes ◽  
Forkhead Box ◽  
Recombination Activating Gene ◽  
Model Family

Production of immunodeficient (ID) models in non-murine animal species had been extremely challenging until the advent of gene-editing tools: first zinc finger nuclease (ZFN), then transcription activator-like effector nuclease (TALEN), and most recently clustered regularly interspaced short palindromic repeats-associated protein 9 (CRISPR)/Cas9. We and others used those gene-editing tools to develop ID rabbits carrying a loss of function mutation in essential immune genes, such as forkhead box protein N1 (FOXN1), recombination activating gene 1/2 (RAG1/2), and interleukin 2 receptor subunit gamma (IL2RG). Like their mouse counterparts, ID rabbits have profound defects in their immune system and are prone to bacterial and pneumocystis infections without prophylactic antibiotics. In addition to their use as preclinical models for primary immunodeficient diseases, ID rabbits are expected to contribute significantly to regenerative medicine and cancer research, where they serve as recipients for allo- and xeno-grafts, with notable advantages over mouse models, including a longer lifespan and a much larger body size. Here we provide a concise review of the history and current status of the development of ID rabbits, as well as future perspectives of this new member in the animal model family.

scholarly journals Tomato bHLH132 Transcription Factor Controls Growth and Defense and Is Activated by Xanthomonas euvesicatoria Effector XopD During Pathogenesis

2019 ◽  
Vol 32 (12) ◽  
pp. 1614-1622 ◽  
Author(s):  
Jung-Gun Kim ◽  
Mary Beth Mudgett
Keyword(s):  
Transcription Factor ◽  
Protease Activity ◽  
Transcriptional Profiling ◽  
Bhlh Transcription Factor ◽  
Transcription Activator ◽  
Tal Effectors ◽  
Sumo Protease ◽  
Tal Effector ◽  
Helix Loop Helix ◽  
Xanthomonas Euvesicatoria

Effector-dependent manipulation of host transcription is a key virulence mechanism used by Xanthomonas species causing bacterial spot disease in tomato and pepper. Transcription activator-like (TAL) effectors employ novel DNA-binding domains to directly activate host transcription, whereas the non-TAL effector XopD uses a small ubiquitin-like modifier (SUMO) protease activity to represses host transcription. The targets of TAL and non-TAL effectors provide insight to the genes governing susceptibility and resistance during Xanthomonas infection. In this study, we investigated the extent to which the X. euvesicatoria non-TAL effector strain Xe85-10 activates tomato transcription to gain new insight to the transcriptional circuits and virulence mechanisms associated with Xanthomonas euvesicatoria pathogenesis. Using transcriptional profiling, we identified a putative basic helix-loop-helix (bHLH) transcription factor, bHLH132, as a pathogen-responsive gene that is moderately induced by microbe-associated molecular patterns and defense hormones and is highly induced by XopD during X. euvesicatoria infection. We also found that activation of bHLH132 transcription requires the XopD SUMO protease activity. Silencing bHLH132 mRNA expression results in stunted tomato plants with enhanced susceptibility to X. euvesicatoria infection. Our work suggests that bHLH132 is required for normal vegetative growth and development as well as resistance to X. euvesicatoria. It also suggests new transcription-based models describing XopD virulence and recognition in tomato.

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