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Article ; Online: De novo PAM generation to reach initially inaccessible target sites for base editing.

Pakari, Kaisa / Wittbrodt, Joachim / Thumberger, Thomas

2023 Volume 150, Issue 2

Abstract: Base editing by CRISPR crucially depends on the presence of a protospacer adjacent motif (PAM) at the correct distance from the editing site. Here, we present and validate an efficient one-shot approach termed 'inception' that expands the editing range. ... ...

Abstract	Base editing by CRISPR crucially depends on the presence of a protospacer adjacent motif (PAM) at the correct distance from the editing site. Here, we present and validate an efficient one-shot approach termed 'inception' that expands the editing range. This is achieved by sequential, combinatorial base editing: de novo generated synonymous, non-synonymous or intronic PAM sites facilitate subsequent base editing at nucleotide positions that were initially inaccessible, further opening the targeting range of highly precise editing approaches. We demonstrate the applicability of the inception concept in medaka (Oryzias latipes) in three settings: loss of function, by introducing a pre-termination STOP codon in the open reading frame of oca2; locally confined multi-codon changes to generate allelic variants with different phenotypic severity in kcnh6a; and the removal of a splice acceptor site by targeting intronic sequences of rx3. Using sequentially acting base editors in the described combinatorial approach expands the number of accessible target sites by 65% on average. This allows the use of well-established tools with NGG PAM recognition for the establishment of thus far unreachable disease models, for hypomorphic allele studies and for efficient targeted mechanistic investigations in a precise and predictable manner.
MeSH term(s)	Clustered Regularly Interspaced Short Palindromic Repeats ; CRISPR-Associated Protein 9/genetics ; CRISPR-Associated Protein 9/metabolism ; CRISPR-Cas Systems/genetics ; Gene Editing ; Oryzias/genetics
Chemical Substances	CRISPR-Associated Protein 9 (EC 3.1.-)
Language	English
Publishing date	2023-01-23
Publishing country	England
Document type	Journal Article
ZDB-ID	90607-4
ISSN	1477-9129 ; 0950-1991
ISSN (online)	1477-9129
ISSN	0950-1991
DOI	10.1242/dev.201115
Database	MEDical Literature Analysis and Retrieval System OnLINE

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Article: A rapid and simple procedure for the isolation and cultivation of fibroblast-like cells from medaka and zebrafish embryos and fin clip biopsies

Beedgen, Lars / Hüllen, Andreas / Gücüm, Sevinç / Thumberger, Thomas / Wittbrodt, Jochen / Thiel, Christian

Laboratory animals. 2022 June, v. 56, no. 3

2022

Abstract: In many human diseases, the molecular pathophysiological mechanisms are not understood, which makes the development and testing of new therapeutic approaches difficult. The generation and characterization of animal models such as mice, rats, fruit flies, ...

Abstract	In many human diseases, the molecular pathophysiological mechanisms are not understood, which makes the development and testing of new therapeutic approaches difficult. The generation and characterization of animal models such as mice, rats, fruit flies, worms or fish offers the possibility for in detail studies of a disease’s development, its course and potential therapies in an organismal context, which considerably minimizes the risk of therapeutic side effects for patients. Nevertheless, due to the high numbers of experimental animals used in research worldwide, attempts to develop alternative test systems will help in reducing their count. In this regard, the cell culture system displays a suitable option due to its potential of delivering nearly unlimited material and the good opportunities for high-throughput studies such as drug testing. Here, we describe a quick and simple method to isolate and cultivate vital fibroblast-like cells from embryos and adults of two popular teleost model organisms, the Japanese rice fish medaka (Oryzias latipes) and the zebrafish (Danio rerio).
Keywords	Danio rerio ; Oryzias latipes ; cell culture ; drugs ; fish ; fruits ; humans ; laboratories ; risk
Language	English
Dates of publication	2022-06
Size	p. 270-278.
Publishing place	SAGE Publications
Document type	Article
ZDB-ID	391008-8
ISSN	1758-1117 ; 0023-6772
ISSN (online)	1758-1117
ISSN	0023-6772
DOI	10.1177/00236772211045483
Database	NAL-Catalogue (AGRICOLA)

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Article ; Online: Precise in vivo functional analysis of DNA variants with base editing using ACEofBASEs target prediction.

Cornean, Alex / Gierten, Jakob / Welz, Bettina / Mateo, Juan Luis / Thumberger, Thomas / Wittbrodt, Joachim

eLife

2022 Volume 11

Abstract: Single nucleotide variants (SNVs) are prevalent genetic factors shaping individual trait profiles and disease susceptibility. The recent development and optimizations of base editors, rubber and pencil genome editing tools now promise to enable direct ... ...

Abstract	Single nucleotide variants (SNVs) are prevalent genetic factors shaping individual trait profiles and disease susceptibility. The recent development and optimizations of base editors, rubber and pencil genome editing tools now promise to enable direct functional assessment of SNVs in model organisms. However, the lack of bioinformatic tools aiding target prediction limits the application of base editing in vivo. Here, we provide a framework for adenine and cytosine base editing in medaka (
MeSH term(s)	Adenine ; Animals ; CRISPR-Cas Systems ; Cytosine ; DNA ; Gene Editing ; Mutation ; Zebrafish/genetics
Chemical Substances	Cytosine (8J337D1HZY) ; DNA (9007-49-2) ; Adenine (JAC85A2161)
Language	English
Publishing date	2022-04-04
Publishing country	England
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	2687154-3
ISSN	2050-084X ; 2050-084X
ISSN (online)	2050-084X
ISSN	2050-084X
DOI	10.7554/eLife.72124
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Article ; Online: In vivo identification and validation of novel potential predictors for human cardiovascular diseases.

Hammouda, Omar T / Wu, Meng Yue / Kaul, Verena / Gierten, Jakob / Thumberger, Thomas / Wittbrodt, Joachim

PloS one

2021 Volume 16, Issue 12, Page(s) e0261572

Abstract: Genetics crucially contributes to cardiovascular diseases (CVDs), the global leading cause of death. Since the majority of CVDs can be prevented by early intervention there is a high demand for the identification of predictive causative genes. While ... ...

Abstract	Genetics crucially contributes to cardiovascular diseases (CVDs), the global leading cause of death. Since the majority of CVDs can be prevented by early intervention there is a high demand for the identification of predictive causative genes. While genome wide association studies (GWAS) correlate genes and CVDs after diagnosis and provide a valuable resource for such causative candidate genes, often preferentially those with previously known or suspected function are addressed further. To tackle the unaddressed blind spot of understudied genes, we particularly focused on the validation of human heart phenotype-associated GWAS candidates with little or no apparent connection to cardiac function. Building on the conservation of basic heart function and underlying genetics from fish to human we combined CRISPR/Cas9 genome editing of the orthologs of human GWAS candidates in isogenic medaka with automated high-throughput heart rate analysis. Our functional analyses of understudied human candidates uncovered a prominent fraction of heart rate associated genes from adult human patients impacting on the heart rate in embryonic medaka already in the injected generation. Following this pipeline, we identified 16 GWAS candidates with potential diagnostic and predictive power for human CVDs.
MeSH term(s)	Animals ; Animals, Genetically Modified ; CRISPR-Cas Systems/genetics ; Cardiovascular Diseases/diagnosis ; Cardiovascular Diseases/genetics ; Cardiovascular Diseases/pathology ; Gene Editing ; Genome-Wide Association Study ; Heart Rate/genetics ; Humans ; Myosin Light Chains/genetics ; Oryzias/genetics ; Promoter Regions, Genetic/genetics
Chemical Substances	Myosin Light Chains
Language	English
Publishing date	2021-12-17
Publishing country	United States
Document type	Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't ; Validation Study
ISSN	1932-6203
ISSN (online)	1932-6203
DOI	10.1371/journal.pone.0261572
Database	MEDical Literature Analysis and Retrieval System OnLINE

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Article ; Online: Fish primary embryonic pluripotent cells assemble into retinal tissue mirroring in vivo early eye development.

Zilova, Lucie / Weinhardt, Venera / Tavhelidse, Tinatini / Schlagheck, Christina / Thumberger, Thomas / Wittbrodt, Joachim

eLife

2021 Volume 10

Abstract: Organoids derived from pluripotent stem cells promise the solution to current challenges in basic and biomedical research. Mammalian organoids are however limited by long developmental time, variable success, and lack of direct comparison to an in vivo ... ...

Abstract	Organoids derived from pluripotent stem cells promise the solution to current challenges in basic and biomedical research. Mammalian organoids are however limited by long developmental time, variable success, and lack of direct comparison to an in vivo reference. To overcome these limitations and address species-specific cellular organization, we derived organoids from rapidly developing teleosts. We demonstrate how primary embryonic pluripotent cells from medaka and zebrafish efficiently assemble into anterior neural structures, particularly retina. Within 4 days, blastula-stage cell aggregates reproducibly execute key steps of eye development: retinal specification, morphogenesis, and differentiation. The number of aggregated cells and genetic factors crucially impacted upon the concomitant morphological changes that were intriguingly reflecting the in vivo situation. High efficiency and rapid development of fish-derived organoids in combination with advanced genome editing techniques immediately allow addressing aspects of development and disease, and systematic probing of impact of the physical environment on morphogenesis and differentiation.
MeSH term(s)	Animals ; Cell Differentiation ; Embryonic Stem Cells/cytology ; Embryonic Stem Cells/metabolism ; Humans ; Morphogenesis ; Organogenesis ; Organoids/cytology ; Organoids/metabolism ; Oryzias ; Pluripotent Stem Cells/physiology ; Retina/cytology ; Retina/growth & development ; Retina/metabolism ; Zebrafish
Language	English
Publishing date	2021-07-12
Publishing country	England
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	2687154-3
ISSN	2050-084X ; 2050-084X
ISSN (online)	2050-084X
ISSN	2050-084X
DOI	10.7554/eLife.66998
Database	MEDical Literature Analysis and Retrieval System OnLINE

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Article ; Online: A rapid and simple procedure for the isolation and cultivation of fibroblast-like cells from medaka and zebrafish embryos and fin clip biopsies.

Beedgen, Lars / Hüllen, Andreas / Gücüm, Sevinç / Thumberger, Thomas / Wittbrodt, Jochen / Thiel, Christian

Laboratory animals

2021 Volume 56, Issue 3, Page(s) 270–278

Abstract	In many human diseases, the molecular pathophysiological mechanisms are not understood, which makes the development and testing of new therapeutic approaches difficult. The generation and characterization of animal models such as mice, rats, fruit flies, worms or fish offers the possibility for in detail studies of a disease's development, its course and potential therapies in an organismal context, which considerably minimizes the risk of therapeutic side effects for patients. Nevertheless, due to the high numbers of experimental animals used in research worldwide, attempts to develop alternative test systems will help in reducing their count. In this regard, the cell culture system displays a suitable option due to its potential of delivering nearly unlimited material and the good opportunities for high-throughput studies such as drug testing. Here, we describe a quick and simple method to isolate and cultivate vital fibroblast-like cells from embryos and adults of two popular teleost model organisms, the Japanese rice fish medaka (
MeSH term(s)	Animals ; Biopsy ; Fibroblasts ; Humans ; Mice ; Oryzias ; Rats ; Surgical Instruments ; Zebrafish
Language	English
Publishing date	2021-09-22
Publishing country	England
Document type	Journal Article
ZDB-ID	391008-8
ISSN	1758-1117 ; 0023-6772
ISSN (online)	1758-1117
ISSN	0023-6772
DOI	10.1177/00236772211045483
Database	MEDical Literature Analysis and Retrieval System OnLINE

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Article ; Online: Boosting targeted genome editing using the hei-tag.

Thumberger, Thomas / Tavhelidse-Suck, Tinatini / Gutierrez-Triana, Jose Arturo / Cornean, Alex / Medert, Rebekka / Welz, Bettina / Freichel, Marc / Wittbrodt, Joachim

eLife

2022 Volume 11

Abstract: Precise, targeted genome editing by CRISPR/Cas9 is key for basic research and translational approaches in model and non-model systems. While active in all species tested so far, editing efficiencies still leave room for improvement. The bacterial Cas9 ... ...

Abstract	Precise, targeted genome editing by CRISPR/Cas9 is key for basic research and translational approaches in model and non-model systems. While active in all species tested so far, editing efficiencies still leave room for improvement. The bacterial Cas9 needs to be efficiently shuttled into the nucleus as attempted by fusion with nuclear localization signals (NLSs). Additional peptide tags such as FLAG- or myc-tags are usually added for immediate detection or straightforward purification. Immediate activity is usually granted by administration of preassembled protein/RNA complexes. We present the 'hei-tag (
MeSH term(s)	Animals ; CRISPR-Cas Systems/genetics ; Cytosine ; Gene Editing/methods ; Mammals ; Nuclear Localization Signals ; RNA, Messenger/genetics
Chemical Substances	Nuclear Localization Signals ; RNA, Messenger ; Cytosine (8J337D1HZY)
Language	English
Publishing date	2022-03-25
Publishing country	England
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	2687154-3
ISSN	2050-084X ; 2050-084X
ISSN (online)	2050-084X
ISSN	2050-084X
DOI	10.7554/eLife.70558
Database	MEDical Literature Analysis and Retrieval System OnLINE

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Article ; Online: Swift Large-scale Examination of Directed Genome Editing.

Hammouda, Omar T / Böttger, Frank / Wittbrodt, Joachim / Thumberger, Thomas

PloS one

2019 Volume 14, Issue 3, Page(s) e0213317

Abstract: In the era of CRISPR gene editing and genetic screening, there is an increasing demand for quick and reliable nucleic acid extraction pipelines for rapid genotyping of large and diverse sample sets. Despite continuous improvements of current workflows, ... ...

Abstract	In the era of CRISPR gene editing and genetic screening, there is an increasing demand for quick and reliable nucleic acid extraction pipelines for rapid genotyping of large and diverse sample sets. Despite continuous improvements of current workflows, the handling-time and material costs per sample remain major limiting factors. Here we present a robust method for low-cost DIY-pipet tips addressing these needs; i.e. using a cellulose filter disc inserted into a regular pipet tip. These filter-in-tips allow for a rapid, stand-alone four-step genotyping workflow by simply binding the DNA contained in the primary lysate to the cellulose filter, washing it in water and eluting it directly into the buffer for the downstream application (e.g. PCR). This drastically cuts down processing time to maximum 30 seconds per sample, with the potential for parallelizing and automation. We show the ease and sensitivity of our procedure by genotyping genetically modified medaka (Oryzias latipes) and zebrafish (Danio rerio) embryos (targeted by CRISPR/Cas9 knock-out and knock-in) in a 96-well plate format. The robust isolation and detection of multiple alleles of various abundancies in a mosaic genetic background allows phenotype-genotype correlation already in the injected generation, demonstrating the reliability and sensitivity of the protocol. Our method is applicable across kingdoms to samples ranging from cells to tissues i. e. plant seedlings, adult flies, mouse cell culture and tissue as well as adult fish fin-clips.
MeSH term(s)	Animals ; Animals, Genetically Modified/genetics ; CRISPR-Cas Systems ; Embryo, Nonmammalian/cytology ; Embryo, Nonmammalian/metabolism ; Gene Editing/methods ; Gene Knockout Techniques/methods ; Genome ; Oryzias/genetics ; Zebrafish/genetics
Language	English
Publishing date	2019-03-05
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	2267670-3
ISSN	1932-6203 ; 1932-6203
ISSN (online)	1932-6203
ISSN	1932-6203
DOI	10.1371/journal.pone.0213317
Database	MEDical Literature Analysis and Retrieval System OnLINE

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Article: Natural genetic variation quantitatively regulates heart rate and dimension.

Gierten, Jakob / Welz, Bettina / Fitzgerald, Tomas / Thumberger, Thomas / Hummel, Oliver / Leger, Adrien / Weber, Philipp / Hassel, David / Hübner, Norbert / Birney, Ewan / Wittbrodt, Joachim

bioRxiv : the preprint server for biology

2023

Abstract: The polygenic contribution to heart development and function along the health-disease continuum remains unresolved. To gain insight into the genetic basis of quantitative cardiac phenotypes, we utilize highly inbred Japanese rice fish models, ...

Abstract	The polygenic contribution to heart development and function along the health-disease continuum remains unresolved. To gain insight into the genetic basis of quantitative cardiac phenotypes, we utilize highly inbred Japanese rice fish models,
Language	English
Publishing date	2023-11-02
Publishing country	United States
Document type	Preprint
DOI	10.1101/2023.09.01.555906
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Article ; Online: CRISPR-based knockout and base editing confirm the role of MYRF in heart development and congenital heart disease.

Doering, Lino / Cornean, Alex / Thumberger, Thomas / Benjaminsen, Joergen / Wittbrodt, Beate / Kellner, Tanja / Hammouda, Omar T / Gorenflo, Matthias / Wittbrodt, Joachim / Gierten, Jakob

Disease models & mechanisms

2023 Volume 16, Issue 8

Abstract: High-throughput DNA sequencing studies increasingly associate DNA variants with congenital heart disease (CHD). However, functional modeling is a crucial prerequisite for translating genomic data into clinical care. We used CRISPR-Cas9-mediated targeting ...

Abstract	High-throughput DNA sequencing studies increasingly associate DNA variants with congenital heart disease (CHD). However, functional modeling is a crucial prerequisite for translating genomic data into clinical care. We used CRISPR-Cas9-mediated targeting of 12 candidate genes in the vertebrate model medaka (Oryzias latipes), five of which displayed a novel cardiovascular phenotype spectrum in F0 (crispants): mapre2, smg7, cdc42bpab, ankrd11 and myrf, encoding a transcription factor recently linked to cardiac-urogenital syndrome. Our myrf mutant line showed particularly prominent embryonic cardiac defects recapitulating phenotypes of pediatric patients, including hypoplastic ventricle. Mimicking human mutations, we edited three sites to generate specific myrf single-nucleotide variants via cytosine and adenine base editors. The Glu749Lys missense mutation in the conserved intramolecular chaperon autocleavage domain fully recapitulated the characteristic myrf mutant phenotype with high penetrance, underlining the crucial function of this protein domain. The efficiency and scalability of base editing to model specific point mutations accelerate gene validation studies and the generation of human-relevant disease models.
MeSH term(s)	Humans ; Child ; Gene Editing ; Mutation/genetics ; Point Mutation ; Transcription Factors/metabolism ; Heart Defects, Congenital/genetics ; CRISPR-Cas Systems/genetics
Chemical Substances	Transcription Factors
Language	English
Publishing date	2023-08-16
Publishing country	England
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	2451104-3
ISSN	1754-8411 ; 1754-8403
ISSN (online)	1754-8411
ISSN	1754-8403
DOI	10.1242/dmm.049811
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