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Article ; Online: CRISPR-Cas9 Mutagenesis in

Blitz, Ira L / Nakayama, Takuya

Cold Spring Harbor protocols

2022 Volume 2022, Issue 3

Abstract: CRISPR-Cas9 mutagenesis is being widely used to create targeted loss-of-function mutations in the diploid ... ...

Abstract	CRISPR-Cas9 mutagenesis is being widely used to create targeted loss-of-function mutations in the diploid frog
MeSH term(s)	Animals ; CRISPR-Cas Systems/genetics ; Chromosomes, Human, Y ; Gene Editing/methods ; Humans ; Male ; Mosaicism ; Mutagenesis ; Phenotype ; Xenopus/genetics
Language	English
Publishing date	2022-03-01
Publishing country	United States
Document type	Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't
ISSN	1559-6095
ISSN (online)	1559-6095
DOI	10.1101/pdb.prot106971
Database	MEDical Literature Analysis and Retrieval System OnLINE

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Article ; Online: Primordial Germ Cell Transplantation for CRISPR/Cas9-based Leapfrogging in Xenopus.

Blitz, Ira L

Journal of visualized experiments : JoVE

2018 , Issue 132

Abstract: The creation of mutant lines by genome editing is accelerating genetic analysis in many organisms. CRISPR/Cas9 methods have been adapted for use in the African clawed frog, Xenopus, a longstanding model organism for biomedical research. Traditional ... ...

Abstract	The creation of mutant lines by genome editing is accelerating genetic analysis in many organisms. CRISPR/Cas9 methods have been adapted for use in the African clawed frog, Xenopus, a longstanding model organism for biomedical research. Traditional breeding schemes for creating homozygous mutant lines with CRISPR/Cas9-targeted mutagenesis have several time-consuming and laborious steps. To facilitate the creation of mutant embryos, particularly to overcome the obstacles associated with knocking out genes that are essential for embryogenesis, a new method called leapfrogging was developed. This technique leverages the robustness of Xenopus embryos to "cut and paste" embryological methods. Leapfrogging utilizes the transfer of primordial germ cells (PGCs) from efficiently-mutagenized donor embryos into PGC-ablated wildtype siblings. This method allows for the efficient mutation of essential genes by creating chimeric animals with wildtype somatic cells that carry a mutant germline. When two F0 animals carrying "leapfrog transplants" (i.e., mutant germ cells) are intercrossed, they produce homozygous, or compound heterozygous, null F1 embryos, thus saving a full generation time to obtain phenotypic data. Leapfrogging also provides a new approach for analyzing maternal effect genes, which are refractory to F0 phenotypic analysis following CRISPR/Cas9 mutagenesis. This manuscript details the method of leapfrogging, with special emphasis on how to successfully perform PGC transplantation.
MeSH term(s)	Animals ; CRISPR-Cas Systems/genetics ; Cell Transplantation ; Gene Targeting/methods ; Germ Cells/transplantation ; Mutation ; Xenopus laevis
Language	English
Publishing date	2018-02-01
Publishing country	United States
Document type	Journal Article ; Research Support, N.I.H., Extramural ; Video-Audio Media
ZDB-ID	2259946-0
ISSN	1940-087X ; 1940-087X
ISSN (online)	1940-087X
ISSN	1940-087X
DOI	10.3791/56035
Database	MEDical Literature Analysis and Retrieval System OnLINE

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Article ; Online: Control of zygotic genome activation in Xenopus.

Blitz, Ira L / Cho, Ken W Y

Current topics in developmental biology

2021 Volume 145, Page(s) 167–204

Abstract: The fertilized frog egg contains all the materials needed to initiate development of a new organism, including stored RNAs and proteins deposited during oogenesis, thus the earliest stages of development do not require transcription. The onset of ... ...

Abstract	The fertilized frog egg contains all the materials needed to initiate development of a new organism, including stored RNAs and proteins deposited during oogenesis, thus the earliest stages of development do not require transcription. The onset of transcription from the zygotic genome marks the first genetic switch activating the gene regulatory network that programs embryonic development. Zygotic genome activation occurs after an initial phase of transcriptional quiescence that continues until the midblastula stage, a period called the midblastula transition, which was first identified in Xenopus. Activation of transcription is programmed by maternally supplied factors and is regulated at multiple levels. A similar switch exists in most animals and is of great interest both to developmental biologists and to those interested in understanding nuclear reprogramming. Here we review in detail our knowledge on this major switch in transcription in Xenopus and place recent discoveries in the context of a decades old problem.
MeSH term(s)	Animals ; Genome/genetics ; Oogenesis ; Xenopus laevis/embryology ; Xenopus laevis/genetics ; Zygote/cytology ; Zygote/metabolism
Language	English
Publishing date	2021-04-19
Publishing country	United States
Document type	Journal Article ; Research Support, N.I.H., Extramural ; Research Support, U.S. Gov't, Non-P.H.S. ; Review
ISSN	1557-8933 ; 0070-2153
ISSN (online)	1557-8933
ISSN	0070-2153
DOI	10.1016/bs.ctdb.2021.03.003
Database	MEDical Literature Analysis and Retrieval System OnLINE

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Article: Primordial germ cell transplantation for crispr/cas9-based leapfrogging in Xenopus

Blitz, Ira L

Journal of visualized experiments. 2018 Feb. 01, , no. 132

2018

Abstract	The creation of mutant lines by genome editing is accelerating genetic analysis in many organisms. CRISPR/Cas9 methods have been adapted for use in the African clawed frog, Xenopus, a longstanding model organism for biomedical research. Traditional breeding schemes for creating homozygous mutant lines with CRISPR/Cas9-targeted mutagenesis have several time-consuming and laborious steps. To facilitate the creation of mutant embryos, particularly to overcome the obstacles associated with knocking out genes that are essential for embryogenesis, a new method called leapfrogging was developed. This technique leverages the robustness of Xenopus embryos to "cut and paste" embryological methods. Leapfrogging utilizes the transfer of primordial germ cells (PGCs) from efficiently-mutagenized donor embryos into PGC-ablated wildtype siblings. This method allows for the efficient mutation of essential genes by creating chimeric animals with wildtype somatic cells that carry a mutant germline. When two F0 animals carrying "leapfrog transplants" (i.e., mutant germ cells) are intercrossed, they produce homozygous, or compound heterozygous, null F1 embryos, thus saving a full generation time to obtain phenotypic data. Leapfrogging also provides a new approach for analyzing maternal effect genes, which are refractory to F0 phenotypic analysis following CRISPR/Cas9 mutagenesis. This manuscript details the method of leapfrogging, with special emphasis on how to successfully perform PGC transplantation.
Keywords	Xenopus laevis ; animals ; biomedical research ; breeding ; cell transplantation ; embryogenesis ; essential genes ; gene editing ; genetic analysis ; germ cells ; heterozygosity ; homozygosity ; maternal effect ; models ; mutagenesis ; mutants ; phenotype ; siblings ; somatic cells
Language	English
Dates of publication	2018-0201
Size	p. e56035.
Publishing place	Journal of Visualized Experiments
Document type	Article
ZDB-ID	2259946-0
ISSN	1940-087X
ISSN	1940-087X
DOI	10.3791/56035
Database	NAL-Catalogue (AGRICOLA)

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Article ; Online: Histone deacetylase 1 maintains lineage integrity through histone acetylome refinement during early embryogenesis.

Zhou, Jeff Jiajing / Cho, Jin Sun / Han, Han / Blitz, Ira L / Wang, Wenqi / Cho, Ken W Y

eLife

2023 Volume 12

Abstract: Histone acetylation is a pivotal epigenetic modification that controls chromatin structure and regulates gene expression. It plays an essential role in modulating zygotic transcription and cell lineage specification of developing embryos. While the ... ...

Abstract	Histone acetylation is a pivotal epigenetic modification that controls chromatin structure and regulates gene expression. It plays an essential role in modulating zygotic transcription and cell lineage specification of developing embryos. While the outcomes of many inductive signals have been described to require enzymatic activities of histone acetyltransferases and deacetylases (HDACs), the mechanisms by which HDACs confine the utilization of the zygotic genome remain to be elucidated. Here, we show that histone deacetylase 1 (Hdac1) progressively binds to the zygotic genome from mid-blastula and onward. The recruitment of Hdac1 to the genome at blastula is instructed maternally.
MeSH term(s)	Histones/metabolism ; Histone Deacetylase 1/genetics ; Histone Deacetylase 1/metabolism ; Chromatin/metabolism ; Blastocyst/metabolism ; Histone Deacetylases/genetics ; Histone Deacetylases/metabolism ; Embryonic Development/genetics ; Acetylation ; Histone Deacetylase 2/metabolism
Chemical Substances	Histones ; Histone Deacetylase 1 (EC 3.5.1.98) ; Chromatin ; Histone Deacetylases (EC 3.5.1.98) ; Histone Deacetylase 2 (EC 3.5.1.98)
Language	English
Publishing date	2023-03-27
Publishing country	England
Document type	Journal Article ; Research Support, Non-U.S. Gov't ; Research Support, N.I.H., Extramural ; Research Support, U.S. Gov't, Non-P.H.S.
ZDB-ID	2687154-3
ISSN	2050-084X ; 2050-084X
ISSN (online)	2050-084X
ISSN	2050-084X
DOI	10.7554/eLife.79380
Database	MEDical Literature Analysis and Retrieval System OnLINE

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Article ; Online: DNase-seq to Study Chromatin Accessibility in Early

Cho, Jin Sun / Blitz, Ira L / Cho, Ken W Y

Cold Spring Harbor protocols

2019 Volume 2019, Issue 4

Abstract: Transcriptional regulatory elements are typically found in relatively nucleosome-free genomic regions, often referred to as "open chromatin." Deoxyribonuclease I (DNase I) can digest nucleosome-depleted DNA (presumably bound by transcription factors), ... ...

Abstract	Transcriptional regulatory elements are typically found in relatively nucleosome-free genomic regions, often referred to as "open chromatin." Deoxyribonuclease I (DNase I) can digest nucleosome-depleted DNA (presumably bound by transcription factors), but DNA in nucleosomes or higher-order chromatin fibers is less accessible to the nuclease. The DNase-seq method uses high-throughput sequencing to permit the interrogation of DNase hypersensitive sites (DHSs) across the entire genome and does not require prior knowledge of histone modifications, transcription factor binding sites, or high quality antibodies to identify potentially active regions of chromatin. Here, discontinuous iodixanol gradients are used as a gentle preparation of the nuclei from
MeSH term(s)	Animals ; Chromatin/metabolism ; Deoxyribonuclease I/metabolism ; Embryo, Nonmammalian/metabolism ; Regulatory Sequences, Nucleic Acid ; Transcription, Genetic ; Xenopus/embryology
Chemical Substances	Chromatin ; Deoxyribonuclease I (EC 3.1.21.1)
Language	English
Publishing date	2019-04-01
Publishing country	United States
Document type	Journal Article
ISSN	1559-6095
ISSN (online)	1559-6095
DOI	10.1101/pdb.prot098335
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Article ; Online: Histone deacetylase 1 maintains lineage integrity through histone acetylome refinement during early embryogenesis

Jeff Jiajing Zhou / Jin Sun Cho / Han Han / Ira L Blitz / Wenqi Wang / Ken WY Cho

eLife, Vol

2023 Volume 12

Abstract	Histone acetylation is a pivotal epigenetic modification that controls chromatin structure and regulates gene expression. It plays an essential role in modulating zygotic transcription and cell lineage specification of developing embryos. While the outcomes of many inductive signals have been described to require enzymatic activities of histone acetyltransferases and deacetylases (HDACs), the mechanisms by which HDACs confine the utilization of the zygotic genome remain to be elucidated. Here, we show that histone deacetylase 1 (Hdac1) progressively binds to the zygotic genome from mid-blastula and onward. The recruitment of Hdac1 to the genome at blastula is instructed maternally. Cis-regulatory modules (CRMs) bound by Hdac1 possess epigenetic signatures underlying distinct functions. We highlight a dual function model of Hdac1 where Hdac1 not only represses gene expression by sustaining a histone hypoacetylation state on inactive chromatin, but also maintains gene expression through participating in dynamic histone acetylation–deacetylation cycles on active chromatin. As a result, Hdac1 maintains differential histone acetylation states of bound CRMs between different germ layers and reinforces the transcriptional program underlying cell lineage identities, both in time and space. Taken together, our study reveals a comprehensive role for Hdac1 during early vertebrate embryogenesis.
Keywords	Hdac1 ; histone acetylation ; germ layer ; epigenetics ; zygotic genome activation ; Xenopus ; Medicine ; R ; Science ; Q ; Biology (General) ; QH301-705.5
Subject code	572
Language	English
Publishing date	2023-03-01T00:00:00Z
Publisher	eLife Sciences Publications Ltd
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article ; Online: Navigating the Xenopus tropicalis genome.

Blitz, Ira L

Methods in molecular biology (Clifton, N.J.)

2012 Volume 917, Page(s) 43–65

Abstract: The frog Xenopus laevis has for more than 60 years served as a model system for the study of vertebrate embryogenesis, molecular and cell biology, and physiology. Recently, there has been great interest in the related species Xenopus tropicalis, in part ... ...

Abstract	The frog Xenopus laevis has for more than 60 years served as a model system for the study of vertebrate embryogenesis, molecular and cell biology, and physiology. Recently, there has been great interest in the related species Xenopus tropicalis, in part because it is diploid, unlike the allotetraploid X. laevis, and therefore amenable to forward genetics, adding to the strengths of the frog system. The genome sequence of X. tropicalis was published in 2010 and this resource is facilitating rapid progress in applying transcriptomic, genomic, proteomic, and systems biological approaches for which the Xenopus system is well suited. However, the availability of the primary nucleotide sequence is only the first step in using the genome. Accessing information embedded in the genome sequence requires well-annotated genes and knowledge of how to navigate the data. The current chapter provides a step-by-step guide for the novice to finding genes of interest in the current genome assembly, supplemented with detailed notes to improve understanding. Several publically available internet-based tools for examining the X. tropicalis genome are discussed, with special emphasis placed on the examination of synteny. Accurate determination of gene identity is enhanced by examination of orthology relationships with other organisms and thus synteny is a powerful tool. This chapter provides an access path into the Xenopus genome to enhance the researchers' ability to manipulate the organism.
MeSH term(s)	Animals ; Data Mining/methods ; Databases, Genetic ; Genome ; Online Systems ; Search Engine ; Sequence Analysis, DNA ; Synteny ; User-Computer Interface ; Xenopus/genetics
Language	English
Publishing date	2012
Publishing country	United States
Document type	Journal Article
ISSN	1940-6029
ISSN (online)	1940-6029
DOI	10.1007/978-1-61779-992-1_4
Database	MEDical Literature Analysis and Retrieval System OnLINE

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Article ; Online: Morpholinos Do Not Elicit an Innate Immune Response during Early Xenopus Embryogenesis.

Paraiso, Kitt D / Blitz, Ira L / Zhou, Jeff J / Cho, Ken W Y

Developmental cell

2019 Volume 49, Issue 4, Page(s) 643–650.e3

Abstract: It has recently been reported that a common side effect of translation-blocking morpholino antisense oligonucleotides is the induction of a set of innate immune response genes in Xenopus embryos and that splicing-blocking morpholinos lead to unexpected ... ...

Abstract	It has recently been reported that a common side effect of translation-blocking morpholino antisense oligonucleotides is the induction of a set of innate immune response genes in Xenopus embryos and that splicing-blocking morpholinos lead to unexpected off-target mis-splicing events. Here, we present an analysis of all publicly available Xenopus RNA sequencing (RNA-seq) data in a reexamination of the effects of translation-blocking morpholinos on the innate immune response. Our analysis does not support the authors' general conclusion, which was based on a limited number of RNA-seq datasets. Moreover, the strong induction of an immune response appears to be specific to the tbxt/tbxt2 morpholinos. The more comprehensive study presented here indicates that using morpholinos for targeted gene knockdowns remains of considerable value for the rapid identification of gene function.
MeSH term(s)	Animals ; Embryonic Development/drug effects ; Gene Knockdown Techniques ; Immunity, Innate/immunology ; Immunity, Innate/physiology ; Morpholinos/immunology ; Morpholinos/metabolism ; Oligonucleotides, Antisense/genetics ; RNA Splicing ; T-Box Domain Proteins/genetics ; T-Box Domain Proteins/metabolism ; Transcriptome/genetics ; Xenopus/embryology ; Xenopus/genetics ; Xenopus Proteins/genetics ; Xenopus Proteins/metabolism ; Xenopus laevis/embryology ; Xenopus laevis/genetics
Chemical Substances	Morpholinos ; Oligonucleotides, Antisense ; T-Box Domain Proteins ; TBXT protein, Xenopus ; Xenopus Proteins
Language	English
Publishing date	2019-05-04
Publishing country	United States
Document type	Journal Article ; Research Support, N.I.H., Extramural ; Research Support, U.S. Gov't, Non-P.H.S. ; Review
ZDB-ID	2054967-2
ISSN	1878-1551 ; 1534-5807
ISSN (online)	1878-1551
ISSN	1534-5807
DOI	10.1016/j.devcel.2019.04.019
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Article ; Online: Leapfrogging: primordial germ cell transplantation permits recovery of CRISPR/Cas9-induced mutations in essential genes.

Blitz, Ira L / Fish, Margaret B / Cho, Ken W Y

Development (Cambridge, England)

2016 Volume 143, Issue 15, Page(s) 2868–2875

Abstract: CRISPR/Cas9 genome editing is revolutionizing genetic loss-of-function analysis but technical limitations remain that slow progress when creating mutant lines. First, in conventional genetic breeding schemes, mosaic founder animals carrying mutant ... ...

Abstract	CRISPR/Cas9 genome editing is revolutionizing genetic loss-of-function analysis but technical limitations remain that slow progress when creating mutant lines. First, in conventional genetic breeding schemes, mosaic founder animals carrying mutant alleles are outcrossed to produce F1 heterozygotes. Phenotypic analysis occurs in the F2 generation following F1 intercrosses. Thus, mutant analyses will require multi-generational studies. Second, when targeting essential genes, efficient mutagenesis of founders is often lethal, preventing the acquisition of mature animals. Reducing mutagenesis levels may improve founder survival, but results in lower, more variable rates of germline transmission. Therefore, an efficient approach to study lethal mutations would be useful. To overcome these shortfalls, we introduce 'leapfrogging', a method combining efficient CRISPR mutagenesis with transplantation of mutated primordial germ cells into a wild-type host. Tested using Xenopus tropicalis, we show that founders containing transplants transmit mutant alleles with high efficiency. F1 offspring from intercrosses between F0 animals that carry embryonic lethal alleles recapitulate loss-of-function phenotypes, circumventing an entire generation of breeding. We anticipate that leapfrogging will be transferable to other species.
MeSH term(s)	Animals ; Anura ; Blastula/cytology ; Blastula/metabolism ; CRISPR-Cas Systems/genetics ; CRISPR-Cas Systems/physiology ; Embryo, Nonmammalian ; Female ; Germ Cells/cytology ; Germ Cells/metabolism ; Male ; Mutagenesis ; Mutation/genetics ; Transcription Activator-Like Effector Nucleases/genetics ; Transcription Activator-Like Effector Nucleases/metabolism ; Xenopus
Chemical Substances	Transcription Activator-Like Effector Nucleases (EC 3.1.-)
Language	English
Publishing date	2016-07-06
Publishing country	England
Document type	Journal Article ; Research Support, N.I.H., Extramural
ZDB-ID	90607-4
ISSN	1477-9129 ; 0950-1991
ISSN (online)	1477-9129
ISSN	0950-1991
DOI	10.1242/dev.138057
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