205 PRODUCTION OF Cas9-EXPRESSING CATTLE USING DNA TRANSPOSON

2017 ◽  
Vol 29 (1) ◽  
pp. 211
Author(s):  
S.-E. Hahn ◽  
S.-Y. Yum ◽  
S.-J. Lee ◽  
C.-I. Lee ◽  
H.-S. Kim ◽  
...  
Keyword(s):  
Primary Culture ◽  
Genome Editing ◽  
Gene Editing ◽  
Primary Cells ◽  
Veterinary Science ◽  
Valuable Resource ◽  
Piggybac Transposon ◽  
Transgenic Cattle ◽  
A Genome ◽  
First Time

A genome-editing technology, CRISPR/Cas9 system is proved to be a powerful tool for knockout and knock-in in various species. When 2 components [Cas9 and single guide (sg) RNA] are delivered into cells or embryos, the events of gene editing occur. Because Cas9 is essential for every gene editing based on the CRISPR/Cas9 system, some studies reported that efficiency of gene editing would be increased as Cas9 was integrated into cells or animals. Accordingly, if the Cas9-expressing cattle is born, it would be broadly used for gene editing in cattle. For this study, the Cas9 and RFP genes were cloned into the PiggyBac transposon system. PiggyBac-Cas9-RFP and transposase were microinjected into 1436 IVF embryos and 241 blastocysts were formed. Blastocysts with RFP expression accounted for 14.1% of total formed blastocysts. Five blastocysts were selected and transferred into 5 recipient cow (1 embryo per recipient). After gestation periods, 4 transgenic cattle were delivered without any veterinary assistance. From transgenic cattle, ear skin tissue was collected for primary culture. On those primary cells, sgRNA in DNA form for various genes such as PRNP, RB1, and BLG were transfected with 2 μg of sgRNA per 5 × 105 cells using electroporation. As expected, every group of each sgRNA delivered was confirmed to be mutated by T7E1 assay. The data demonstrated that, for the first time, transgenic cattle with Cas9 expression were born, grown up to date (age = 5 months) and will be a valuable resource for genome editing in cattle. This work was supported by BK21 PLUS Program for Creative Veterinary Science and Seoul Milk Coop (SNU 550–20160004).

scholarly journals Progress and Challenges in the Improvement of Ornamental Plants by Genome Editing

Plants ◽  
2020 ◽  
Vol 9 (6) ◽  
pp. 687 ◽  
Author(s):  
Chang Ho Ahn ◽  
Mummadireddy Ramya ◽  
Hye Ryun An ◽  
Pil Man Park ◽  
Yae-Jin Kim ◽  
...  
Keyword(s):  
Genome Editing ◽  
Gene Editing ◽  
Genetically Modified Organisms ◽  
Ornamental Plants ◽  
Safety Evaluation ◽  
Evaluation Process ◽  
Vase Life ◽  
Transcription Activator ◽  
A Genome ◽  
Dna Specificity

Biotechnological approaches have been used to modify the floral color, size, and fragrance of ornamental plants, as well as to increase disease resistance and vase life. Together with the advancement of whole genome sequencing technologies, new plant breeding techniques have rapidly emerged in recent years. Compared to the early versions of gene editing tools, such as meganucleases (MNs), zinc fingers (ZFNs), and transcription activator-like effector nucleases (TALENs), clustered regularly interspaced short palindromic repeat (CRISPR) is capable of altering a genome more efficiently and with higher accuracy. Most recently, new CRISPR systems, including base editors and prime editors, confer reduced off-target activity with improved DNA specificity and an expanded targeting scope. However, there are still controversial issues worldwide for the recognition of genome-edited plants, including whether genome-edited plants are genetically modified organisms and require a safety evaluation process. In the current review, we briefly summarize the current progress in gene editing systems and also introduce successful/representative cases of the CRISPR system application for the improvement of ornamental plants with desirable traits. Furthermore, potential challenges and future prospects in the use of genome-editing tools for ornamental plants are also discussed.

Genome Editing with CRISPR

2016 ◽  
Vol 41 (3) ◽  
pp. 1-3
Author(s):  
Nicanor Pier Giorgio Austriaco ◽  
Keyword(s):  
Genetic Engineering ◽  
Genome Editing ◽  
Gene Editing ◽  
Life Sciences ◽  
Great Promise ◽  
Bacterial Cells ◽  
Kitchen Table ◽  
A Genome ◽  
Do It Yourself

There has been much discussion regarding the proper use of the powerful CRISPR technologies that can be used to edit the genome. CRISPR is a technique borrowed from bacterial cells that will allow scientists to quickly and precisely change the DNA of nearly any organism, including humans. Unlike other gene-editing technologies, CRISPR is cheap, quick, and easy to use. In fact, do-it-yourself CRISPR genome editing kits are available online for less than $200, which will enable anyone, including so-called biohackers, to do genetic engineering at the kitchen table. In only three years—CRISPR as a genome-editing tool was first described in 2012—it is already universally acknowledged that this technology will revolutionize the life sciences. But CRISPR’s great promise has also sparked a great ethical and societal debate on its legitimate uses, most significantly on whether it should be used to alter the genomes of our children and grandchildren.

scholarly journals Optimization of CRISPR/Cas System for Improving Genome Editing Efficiency in Plasmodium falciparum

2021 ◽  
Vol 11 ◽  
Author(s):  
Yuemeng Zhao ◽  
Fei Wang ◽  
Changhong Wang ◽  
Xiaobai Zhang ◽  
Cizhong Jiang ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Genome Editing ◽  
Gene Editing ◽  
Molecular Mechanisms ◽  
Related Gene ◽  
Malaria Parasites ◽  
Human Malaria Parasite ◽  
Genome Feature ◽  
First Time ◽  
Transgenic Strains

Studies of molecular mechanisms and related gene functions have long been restricted by limited genome editing technologies in malaria parasites. Recently, a simple and effective genome editing technology, the CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated) system, has greatly facilitated these studies in many organisms, including malaria parasites. However, due to the special genome feature of malaria parasites, the manipulation and gene editing efficacy of the CRISPR/Cas system in this pathogen need to be improved, particularly in the human malaria parasite, Plasmodium falciparum. Herein, based on the CRISPR/Cas9 system, we developed an integrating strategy to generate a Cas9i system, which significantly shortened the time for generation of transgenic strains in P. falciparum. Moreover, with this Cas9i system, we have successfully achieved multiplexed genome editing (mutating or tagging) by a single-round transfection in P. falciparum. In addition, we for the first time adapted AsCpf1 (Acidaminococcus sp. Cpf1), an alternative to Cas9, into P. falciparum parasites and examined it for gene editing. These optimizations of the CRISPR/Cas system will further facilitate the mechanistic research of malaria parasites and contribute to eliminating malaria in the future.

scholarly journals CRISPR-Cas9: A Genome Editing Tool in Crop Plants: A Review

2021 ◽  
Author(s):  
Varsha Kumari ◽  
Priyanka Kumawat ◽  
Sharanabasappa Yeri ◽  
Shyam Singh Rajput
Keyword(s):  
Genome Editing ◽  
Gene Editing ◽  
Crop Improvement ◽  
End Joining ◽  
Homology Directed Repair ◽  
Ligase Iv ◽  
A Genome ◽  
Target Dna ◽  
Non Homologous End Joining ◽  
Dna Strand

Clustered regularly interspaced short palindromic repeats/CRISPR associated nuclease 9 (CRISPR-Cas9) system is a rapid technology for gene editing. CRISPR-Cas9 is an RNA guided gene editing tool where Cas9 acts as endonuclease by cutting the target DNA strand. Double Stranded Breaks (DBS) can be repaired by non-homologous end joining (NHEJ) and homology-directed repair (HDR). The NHEJ employs DNA ligase IV to rejoin the broken ends which cause insertion or deletion mutations, whereas HDR repairs the DSBs based on a homologous complementary template and results in perfect repair of broken ends. CRISPR-Cas9 impart diverse advantageous features in contrast with the conventional methods. In this review article, we have discussed CRISPR-Cas9 based genome editing along with its mechanism of action and role in crop improvement.

scholarly journals In vivo genome editing using the Cpf1 ortholog derived from Eubacterium eligens

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Woo-Chan Ahn ◽  
Kwang-Hyun Park ◽  
In Seon Bak ◽  
Hyung-Nam Song ◽  
Yan An ◽  
...  
Keyword(s):  
Genome Editing ◽  
Knockout Mice ◽  
Dna Cleavage ◽  
Gene Editing ◽  
Essential Gene ◽  
Bacterial Species ◽  
A Genome ◽  
Target Dna ◽  
Metal Cofactor

Abstract Cpf1 is an RNA-guided endonuclease that can be programmed to cleave DNA targets. Specific features, such as containing a short crRNA, creating a staggered cleavage pattern and having a low off-target rate, render Cpf1 a promising gene-editing tool. Here, we present a new Cpf1 ortholog, EeCpf1, as a genome-editing tool; this ortholog is derived from the gut bacterial species Eubacterium eligens. EeCpf1 exhibits a higher cleavage activity with the Mn2+ metal cofactor and efficiently cuts the target DNA with an engineered, nucleotide extended crRNA at the 5′ target site. When mouse blastocysts were injected with multitargeting crRNAs against the IL2R-γ gene, an essential gene for immunodeficient mouse model production, EeCpf1 efficiently generated IL2R-γ knockout mice. For the first time, these results demonstrate that EeCpf1 can be used as an in vivo gene-editing tool for the production of knockout mice. The utilization of engineered crRNA with multiple target sites will help to explore the in vivo DNA cleavage activities of Cpf1 orthologs from other species that have not been demonstrated.

scholarly journals Interrogating Mitochondrial Biology and Disease Using CRISPR/Cas9 Gene Editing

Genes ◽  
2021 ◽  
Vol 12 (10) ◽  
pp. 1604
Author(s):  
Jia Xin Tang ◽  
Angela Pyle ◽  
Robert W. Taylor ◽  
Monika Oláhová
Keyword(s):  
Genome Editing ◽  
Large Scale ◽  
Gene Editing ◽  
Genetic Manipulation ◽  
Mitochondrial Diseases ◽  
Biological Research ◽  
Genetic Changes ◽  
Mitochondrial Proteome ◽  
A Genome ◽  
Indispensable Tool

Mitochondrial disease originates from genetic changes that impact human bodily functions by disrupting the mitochondrial oxidative phosphorylation system. MitoCarta is a curated and published inventory that sheds light on the mitochondrial proteome, but the function of some mitochondrially-localised proteins remains poorly characterised. Consequently, various gene editing systems have been employed to uncover the involvement of these proteins in mitochondrial biology and disease. CRISPR/Cas9 is an efficient, versatile, and highly accurate genome editing tool that was first introduced over a decade ago and has since become an indispensable tool for targeted genetic manipulation in biological research. The broad spectrum of CRISPR/Cas9 applications serves as an attractive and tractable system to study genes and pathways that are essential for the regulation and maintenance of mitochondrial health. It has opened possibilities of generating reliable cell and animal models of human disease, and with further exploitation of the technology, large-scale genomic screenings have uncovered a wealth of fundamental mechanistic insights. In this review, we describe the applications of CRISPR/Cas9 system as a genome editing tool to uncover new insights into pathomechanisms of mitochondrial diseases and/or biological processes involved in mitochondrial function.

scholarly journals Establishment of a Genome Editing Tool Using CRISPR-Cas9 in Chlorella vulgaris UTEX395

2021 ◽  
Vol 22 (2) ◽  
pp. 480
Author(s):  
Jongrae Kim ◽  
Kwang Suk Chang ◽  
Sangmuk Lee ◽  
EonSeon Jin
Keyword(s):  
Genome Editing ◽  
Chlorella Vulgaris ◽  
Negative Selection ◽  
Screening Strategy ◽  
Feed Additive ◽  
Cell Factory ◽  
Dna Transformation ◽  
Research Groups ◽  
A Genome ◽  
Food And Feed

To date, Chlorella vulgaris is the most used species of microalgae in the food and feed additive industries, and also considered as a feasible cell factory for bioproducts. However, the lack of an efficient genetic engineering tool makes it difficult to improve the physiological characteristics of this species. Therefore, the development of new strategic approaches such as genome editing is trying to overcome this hurdle in many research groups. In this study, the possibility of editing the genome of C. vulgaris UTEX395 using clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (Cas9) has been proven to target nitrate reductase (NR) and adenine phosphoribosyltransferase (APT). Genome-edited mutants, nr and apt, were generated by a DNA-mediated and/or ribonucleoprotein (RNP)-mediated CRISPR-Cas9 system, and isolated based on the negative selection against potassium chlorate or 2-fluoroadenine in place of antibiotics. The null mutation of edited genes was demonstrated by the expression level of the correspondent proteins or the mutation of transcripts, and through growth analysis under specific nutrient conditions. In conclusion, this study offers relevant empirical evidence of the possibility of genome editing in C. vulgaris UTEX395 by CRISPR-Cas9 and the practical methods. Additionally, among the generated mutants, nr can provide an easier screening strategy during DNA transformation than the use of antibiotics owing to their auxotrophic characteristics. These results will be a cornerstone for further advancement of the genetics of C. vulgaris.

scholarly journals Plastid phylogenomics resolves ambiguous relationships within the orchid family and provides a solid timeframe for biogeography and macroevolution

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Maria Alejandra Serna-Sánchez ◽  
Oscar A. Pérez-Escobar ◽  
Diego Bogarín ◽  
María Fernanda Torres-Jimenez ◽  
Astrid Catalina Alvarez-Yela ◽  
...  
Keyword(s):  
Phylogenetic Relationships ◽  
Divergence Times ◽  
Molecular Clocks ◽  
Phylogenetic Placement ◽  
Plastid Genomes ◽  
Phylogenetic Reconstructions ◽  
A Genome ◽  
Tree Models ◽  
Phylogenomic Analyses ◽  
First Time

AbstractRecent phylogenomic analyses based on the maternally inherited plastid organelle have enlightened evolutionary relationships between the subfamilies of Orchidaceae and most of the tribes. However, uncertainty remains within several subtribes and genera for which phylogenetic relationships have not ever been tested in a phylogenomic context. To address these knowledge-gaps, we here provide the most extensively sampled analysis of the orchid family to date, based on 78 plastid coding genes representing 264 species, 117 genera, 18 tribes and 28 subtribes. Divergence times are also provided as inferred from strict and relaxed molecular clocks and birth–death tree models. Our taxon sampling includes 51 newly sequenced plastid genomes produced by a genome skimming approach. We focus our sampling efforts on previously unplaced clades within tribes Cymbidieae and Epidendreae. Our results confirmed phylogenetic relationships in Orchidaceae as recovered in previous studies, most of which were recovered with maximum support (209 of the 262 tree branches). We provide for the first time a clear phylogenetic placement for Codonorchideae within subfamily Orchidoideae, and Podochilieae and Collabieae within subfamily Epidendroideae. We also identify relationships that have been persistently problematic across multiple studies, regardless of the different details of sampling and genomic datasets used for phylogenetic reconstructions. Our study provides an expanded, robust temporal phylogenomic framework of the Orchidaceae that paves the way for biogeographical and macroevolutionary studies.

scholarly journals Simplified All-In-One CRISPR-Cas9 Construction for Efficient Genome Editing in Cryptococcus Species

2021 ◽  
Vol 7 (7) ◽  
pp. 505
Author(s):  
Ping Zhang ◽  
Yu Wang ◽  
Chenxi Li ◽  
Xiaoyu Ma ◽  
Lan Ma ◽  
...  
Keyword(s):  
Genome Editing ◽  
Fungal Pathogens ◽  
Gene Editing ◽  
Recombination Frequency ◽  
Target Sequence ◽  
Widespread Application ◽  
Genetic Studies ◽  
Efficient Gene ◽  
Cryptococcus Species ◽  
Overlap Pcr

Cryptococcus neoformans and Cryptococcus deneoformans are opportunistic fungal pathogens found worldwide that are utilized to reveal mechanisms of fungal pathogenesis. However, their low homologous recombination frequency has greatly encumbered genetic studies. In preliminary work, we described a ‘suicide’ CRISPR-Cas9 system for use in the efficient gene editing of C. deneoformans, but this has not yet been used in the C. neoformans strain. The procedures involved in constructing vectors are time-consuming, whether they involve restriction enzyme-based cloning of donor DNA or the introduction of a target sequence into the gRNA expression cassette via overlap PCR, as are sophisticated, thus impeding their widespread application. Here, we report the optimized and simplified construction method for all-in-one CRISPR-Cas9 vectors that can be used in C. neoformans and C. deneoformans strains respectively, named pNK003 (Genbank: MW938321) and pRH003 (Genbank: KX977486). Taking several gene manipulations as examples, we also demonstrate the accuracy and efficiency of the new simplified all-in-one CRISPR-Cas9 genome editing tools in both Serotype A and Serotype D strains, as well as their ability to eliminate Cas9 and gDNA cassettes after gene editing. We anticipate that the availability of new vectors that can simplify and streamline the technical steps for all-in-one CRISPR-Cas9 construction could accelerate genetic studies of the Cryptococcus species.

scholarly journals riboCIRC: a comprehensive database of translatable circRNAs

2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Huihui Li ◽  
Mingzhe Xie ◽  
Yan Wang ◽  
Ludong Yang ◽  
Zhi Xie ◽  
...  
Keyword(s):  
De Novo ◽  
Species Conservation ◽  
Structure And Function ◽  
Research Community ◽  
Genome Browser ◽  
Valuable Resource ◽  
Sequence Structure ◽  
A Genome ◽  
Context Specific ◽  
And Function

AbstractriboCIRC is a translatome data-oriented circRNA database specifically designed for hosting, exploring, analyzing, and visualizing translatable circRNAs from multi-species. The database provides a comprehensive repository of computationally predicted ribosome-associated circRNAs; a manually curated collection of experimentally verified translated circRNAs; an evaluation of cross-species conservation of translatable circRNAs; a systematic de novo annotation of putative circRNA-encoded peptides, including sequence, structure, and function; and a genome browser to visualize the context-specific occupant footprints of circRNAs. It represents a valuable resource for the circRNA research community and is publicly available at http://www.ribocirc.com.

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