scholarly journals Resources for genome editing in livestock: Cas9-expressing chickens and pigs

2020 ◽  
Author(s):  
Denise Bartsch ◽  
Hicham Sid ◽  
Beate Rieblinger ◽  
Romina Hellmich ◽  
Antonina Schlickenrieder ◽  
...  
Keyword(s):  
Genome Editing ◽  
Target Genes ◽  
Germ Line ◽  
Genetically Modified ◽  
Agricultural Science ◽  
Genetic Traits ◽  
Organ Specific ◽  
Species Comparisons ◽  
Transgenic Chickens

AbstractGenetically modified animals continue to provide important insights in biomedical sciences. Research has focused mostly on genetically modified mice so far, but other species like pigs resemble more closely the human physiology. In addition, cross-species comparisons with phylogenetically distant species such as chickens provide powerful insights into fundamental biological and biomedical processes. One of the most versatile genetic methods applicable across species is CRISPR/Cas9. Here, we report for the first time the generation of Cas9 transgenic chickens and pigs that allow in vivo genome editing in these two important agricultural species. We demonstrated that Cas9 is constitutively expressed in all organs of both species and that the animals are healthy and fertile. In addition, we confirmed the functionality of Cas9 for a number of different target genes and for a variety of cell types. Taken together, these transgenic animal species expressing Cas9 provide an unprecedented tool for agricultural and biomedical research, and will facilitate organ specific reverse genetics as well as cross-species comparisons.Significance statementGenome engineering of animals is crucial for translational medicine and the study of genetic traits. Here, we generated transgenic chickens and pigs that ubiquitously express the Cas9 endonuclease, providing the basis for in vivo genome editing. We demonstrated the functionality of this system by successful genome editing in chicken and porcine cells and tissues. These animals facilitate organ specific in vivo genome editing in both species without laborious germ line modifications, which will reduce the number of animals needed for genetic studies. They also provide a new tool for functional genomics, developmental biology and numerous other applications in biomedical and agricultural science.

scholarly journals Cas9-expressing chickens and pigs as resources for genome editing in livestock

2021 ◽  
Vol 118 (10) ◽  
pp. e2022562118
Author(s):  
Beate Rieblinger ◽  
Hicham Sid ◽  
Denise Duda ◽  
Tarik Bozoglu ◽  
Romina Klinger ◽  
...  
Keyword(s):  
Genome Editing ◽  
Reverse Genetics ◽  
Target Genes ◽  
Genetically Modified ◽  
Transgenic Animals ◽  
Cell Types ◽  
Species Comparisons ◽  
Health And Disease ◽  
Transgenic Chickens

Genetically modified animals continue to provide important insights into the molecular basis of health and disease. Research has focused mostly on genetically modified mice, although other species like pigs resemble the human physiology more closely. In addition, cross-species comparisons with phylogenetically distant species such as chickens provide powerful insights into fundamental biological and biomedical processes. One of the most versatile genetic methods applicable across species is CRISPR-Cas9. Here, we report the generation of transgenic chickens and pigs that constitutively express Cas9 in all organs. These animals are healthy and fertile. Functionality of Cas9 was confirmed in both species for a number of different target genes, for a variety of cell types and in vivo by targeted gene disruption in lymphocytes and the developing brain, and by precise excision of a 12.7-kb DNA fragment in the heart. The Cas9 transgenic animals will provide a powerful resource for in vivo genome editing for both agricultural and translational biomedical research, and will facilitate reverse genetics as well as cross-species comparisons.

scholarly journals Efficient genome editing in primary cells and in vivo using viral-derived “Nanoblades” loaded with Cas9/sgRNA ribonucleoproteins

2017 ◽  
Author(s):  
Philippe E. Mangeot ◽  
Valérie Risson ◽  
Floriane Fusil ◽  
Aline Marnef ◽  
Emilie Laurent ◽  
...  
Keyword(s):  
Stem Cells ◽  
Genome Editing ◽  
Target Genes ◽  
Bone Marrow Cells ◽  
Leukemia Virus ◽  
Target Cells ◽  
Mouse Bone Marrow ◽  
Primary Cells ◽  
Hematopoietic Stem

AbstractProgrammable nucleases have enabled rapid and accessible genome engineering in eukaryotic cells and living organisms. However, their delivery into target cells can be technically challenging when working with primary cells or in vivo. Using engineered murine leukemia virus-like particles loaded with Cas9/sgRNA ribonucleoproteins (“Nanoblades”), we were able to induce efficient genome-editing in cell lines and primary cells including human induced pluripotent stem cells, human hematopoietic stem cells and mouse bone-marrow cells. Transgene-free Nanoblades were also capable of in vivo genome-editing in mouse embryos and in the liver of injected mice. Nanoblades can be complexed with donor DNA for “all-in-one” homology-directed repair or programmed with modified Cas9 variants to mediate transcriptional up-regulation of target genes. Nanoblades preparation process is simple, relatively inexpensive and can be easily implemented in any laboratory equipped for cellular biology.

scholarly journals Tagged Mutagenesis by Efficient Minos-Based Germ Line Transposition

2009 ◽  
Vol 30 (1) ◽  
pp. 68-77 ◽  
Author(s):  
Ton de Wit ◽  
Sylvia Dekker ◽  
Alex Maas ◽  
Guido Breedveld ◽  
Tobias A. Knoch ◽  
...  
Keyword(s):  
Target Genes ◽  
Germ Line ◽  
Rapid Screening ◽  
Developmental Defects ◽  
Disease Phenotypes ◽  
Gene Transposition ◽  
Functional Screen ◽  
Mouse Mutants ◽  
Early Mouse

ABSTRACT Germ line gene transposition technology has been used to generate “libraries” of flies and worms carrying genomewide mutations. Phenotypic screening and DNA sequencing of such libraries provide functional information resulting from insertional events in target genes. There is also a great need to have a fast and efficient way to generate mouse mutants in vivo to model developmental defects and human diseases. Here we describe an optimized mammalian germ line transposition system active during early mouse spermatogenesis using the Minos transposon. Transposon-positive progeny carry on average more than 2 new transpositions, and 45 to 100% of the progeny carry an insertion in a gene. The optimized Minos-based system was tested in a small rapid dominant functional screen to identify mutated genes likely to cause measurable cardiovascular “disease” phenotypes in progeny/embryos. Importantly this system allows rapid screening for modifier genes.

scholarly journals Self-Association of Gata1 Enhances Transcriptional Activity In Vivo in Zebra Fish Embryos

2003 ◽  
Vol 23 (22) ◽  
pp. 8295-8305 ◽  
Author(s):  
Keizo Nishikawa ◽  
Makoto Kobayashi ◽  
Atsuko Masumi ◽  
Susan E. Lyons ◽  
Brant M. Weinstein ◽  
...  
Keyword(s):  
Transcriptional Activity ◽  
Molecular Mechanisms ◽  
Target Genes ◽  
Germ Line ◽  
Zebra Fish ◽  
Wild Type ◽  
Lysine Residues ◽  
Self Association ◽  
Gata Motif

ABSTRACT Gata1 is a prototype transcription factor that regulates hematopoiesis, yet the molecular mechanisms by which Gata1 transactivates its target genes in vivo remain unclear. We previously showed, in transgenic zebra fish, that Gata1 autoregulates its own expression. In this study, we characterized the molecular mechanisms for this autoregulation by using mutations in the Gata1 protein which impair autoregulation. Of the tested mutations, replacement of six lysine residues with alanine (Gata1KA6), which inhibited self-association activity of Gata1, reduced the Gata1-dependent induction of reporter gene expression driven by the zebra fish gata1 hematopoietic regulatory domain (gata1 HRD). Furthermore, overexpression of wild-type Gata1 but not Gata1KA6 rescued the expression of Gata1 downstream genes in vlad tepes, a germ line gata1 mutant fish. Interestingly, both GATA sites in the double GATA motif in gata1 HRD were critical for the promoter activity and for binding of the self-associated Gata1 complex, whereas only the 3′-GATA site was required for Gata1 monomer binding. These results thus provide the first in vivo evidence that the ability of Gata1 to self-associate critically contributes to the autoregulation of the gata1 gene.

scholarly journals Maize Transformation: From Plant Material to the Release of Genetically Modified and Edited Varieties

2021 ◽  
Vol 12 ◽  
Author(s):  
Juliana Erika de Carvalho Teixeira Yassitepe ◽  
Viviane Cristina Heinzen da Silva ◽  
José Hernandes-Lopes ◽  
Ricardo Augusto Dante ◽  
Isabel Rodrigues Gerhardt ◽  
...  
Keyword(s):  
Genome Editing ◽  
Genetically Modified ◽  
Abiotic Stress Tolerance ◽  
Plant Biotechnology ◽  
Current Status ◽  
High Yield ◽  
Genetically Modified Maize ◽  
Maize Transformation ◽  
Maize Varieties

Over the past decades, advances in plant biotechnology have allowed the development of genetically modified maize varieties that have significantly impacted agricultural management and improved the grain yield worldwide. To date, genetically modified varieties represent 30% of the world’s maize cultivated area and incorporate traits such as herbicide, insect and disease resistance, abiotic stress tolerance, high yield, and improved nutritional quality. Maize transformation, which is a prerequisite for genetically modified maize development, is no longer a major bottleneck. Protocols using morphogenic regulators have evolved significantly towards increasing transformation frequency and genotype independence. Emerging technologies using either stable or transient expression and tissue culture-independent methods, such as direct genome editing using RNA-guided endonuclease system as an in vivo desired-target mutator, simultaneous double haploid production and editing/haploid-inducer-mediated genome editing, and pollen transformation, are expected to lead significant progress in maize biotechnology. This review summarises the significant advances in maize transformation protocols, technologies, and applications and discusses the current status, including a pipeline for trait development and regulatory issues related to current and future genetically modified and genetically edited maize varieties.

25 GENOME EDITING OF SOMATIC CELL NUCLEAR TRANSFER DERIVED ZYGOTES BY CLUSTERED REGULARLY INTERSPACED SHORT PALINDROMIC REPEATS (CRISPR)/Cas9 GUIDE RNA INJECTION

2016 ◽  
Vol 28 (2) ◽  
pp. 142
Author(s):  
K. M. Whitworth ◽  
S. L. Murphy ◽  
J. A. Benne ◽  
L. D. Spate ◽  
E. Walters ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Cell Line ◽  
Genome Editing ◽  
Genetically Modified ◽  
Donor Cell ◽  
T7 Promoter ◽  
Guide Rna ◽  
Rag2 Gene

Recent applications of the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system have greatly improved the efficiency of genome editing in pigs. However, in some cases, genetically modified pig models need an additional modification to improve their application. The objective of this experiment was to determine whether a combination of somatic cell NT (SCNT) by using a previously modified donor cell line and subsequent zygote injection with CRISPR/Cas9 guide RNA to target a second gene would result in embryos and offspring successfully containing both modifications. Fibroblast cell lines were collected from fumarylacetoacetate hydrolase deficient (FAH–/–) fetuses and used as the donor cell line. Somatic cell NT was performed by standard technique. A CRISPR guide RNA specific for recombination activating gene 2 (RAG2) was designed and in vitro transcribed from a synthesised gBlock (IDT) containing a T7 promoter sequence, the CRISPR Guide RNA (20 bp), and 85 bp of tracer RNA. The gBlock was PCR amplified with Q5 polymerase (NEB, Ipswich, MA, USA) and in vitro transcribed with the MEGAshortscript™ T7 Transcription Kit (Life Technologies, Grand Island, NY, USA). Guide RNA (20 ng μL–1) and polyadenylated Cas9 (20 ng μL–1, Sigma, St. Louis, MO, USA) were co-injected into the cytoplasm of SCNT zygotes at 14 to 16 h after fusion and activation. Injected SCNT were then cultured in vitro in PZM3 + 1.69 mM arginine medium (MU1) to Day 5. Three embryo transfers were performed surgically into recipient gilts on Day 4 or 5 of oestrus (50, 62, or 70 embryos per pig) to evaluate in vivo development. The remaining embryos were cultured in MU1 to Day 7 and analysed for the presence of modifications to the RAG2 gene. Embryos were classified as modified if they contained an INDEL as measured by both gel electrophoresis and DNA sequencing of PCR amplicons spanning the targeted exon. The RAG2 modification rate was 83.3% (n = 6), of which 50% (n = 3) of the embryos contained biallelic modifications. All control embryos contained a wild-type RAG2 gene (n = 5). Embryo transfer resulted in a 33.3% pregnancy rate (1/3). The combination of SCNT and CRISPR/Cas9 zygote injection can be a highly efficient tool to successfully create pig embryos with an additional modification. This additional technique further improves the usefulness of already created genetically modified pig models. This study was funded by the National Institutes of Health via U42 OD011140.

Current and future approaches using genetically modified mice in endocrine research

2006 ◽  
Vol 291 (3) ◽  
pp. E429-E438 ◽  
Author(s):  
Rachel A. Davey ◽  
Helen E. MacLean
Keyword(s):  
Mouse Models ◽  
Target Genes ◽  
Genetically Modified ◽  
Advantages And Disadvantages ◽  
Genetically Modified Mice ◽  
The Many ◽  
Time Specific ◽  
Si Rna

Genetically modified mouse models have been used widely to advance our knowledge in the field of endocrinology and metabolism. A number of different approaches to generate genetically modified mice are now available, which provide the power to analyze the role of individual proteins in vivo. However, there are a number of points to be considered in the use and interpretation of these models. This review discusses the advantages and disadvantages involved in the generation and use of different genetically modified mouse models in endocrine research, including conventional techniques (e.g., overexpression, knockout, and knock-in models), tissue- and/or time-specific deletion of target genes [e.g., Cre- loxP and short interfering (si)RNA transgenic approaches], and gene-trap approaches to undertake functional genomics. This review also highlights the many factors that should be considered when assessing the phenotype of these mouse models, many of which are relevant to all murine physiological studies. These approaches are a powerful means by which to dissect the function of genes and are revolutionizing our understanding of endocrine physiology and metabolism.

‘Artificial spermatid’-mediated genome editing†

2019 ◽  
Vol 101 (3) ◽  
pp. 538-548 ◽  
Author(s):  
Lingbo Wang ◽  
Jinsong Li
Keyword(s):  
High Efficiency ◽  
Genetically Modified ◽  
Embryonic Stem ◽  
Differentially Methylated Region ◽  
Double Knockout ◽  
Genetic Traits ◽  
Periodic Cell ◽  
Haploid Embryos

Abstract For years, extensive efforts have been made to use mammalian sperm as the mediator to generate genetically modified animals; however, the strategy of sperm-mediated gene transfer (SMGT) is unable to produce stable and diversified modifications in descendants. Recently, haploid embryonic stem cells (haESCs) have been successfully derived from haploid embryos carrying the genome of highly specialized gametes, and can stably maintain haploidy (through periodic cell sorting based on DNA quantity) and both self-renewal and pluripotency in long-term cell culture. In particular, haESCs derived from androgenetic haploid blastocysts (AG-haESCs), carrying only the sperm genome, can support the generation of live mice (semi-cloned, SC mice) through oocyte injection. Remarkably, after removal of the imprinted control regions H19-DMR (differentially methylated region of DNA) and IG-DMR in AG-haESCs, the double knockout (DKO)-AG-haESCs can stably produce SC animals with high efficiency, and so can serve as a sperm equivalent. Importantly, DKO-AG-haESCs can be used for multiple rounds of gene modifications in vitro, followed by efficient generation of live and fertile mice with the expected genetic traits. Thus, DKO-AG-haESCs (referred to as ‘artificial spermatids’) combed with CRISPR-Cas technology can be used as the genetically tractable fertilization agent, to efficiently create genetically modified offspring, and is a versatile genetic tool for in vivo analyses of gene function.

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