scholarly journals CRISPR-Cas9 Gene Editing in Lizards Through Microinjection of Unfertilized Oocytes

2019 ◽  
Author(s):  
Ashley M. Rasys ◽  
Sungdae Park ◽  
Rebecca E. Ball ◽  
Aaron J. Alcala ◽  
James D. Lauderdale ◽  
...  
Keyword(s):  
Reproductive Biology ◽  
Gene Function ◽  
Gene Editing ◽  
Targeted Mutagenesis ◽  
Efficient Production ◽  
Direct Manipulation ◽  
Induced Mutations ◽  
New Approach ◽  
Evolution And Development

AbstractCRISPR-Cas9 mediated gene editing has enabled the direct manipulation of gene function in many species. However, the reproductive biology of reptiles presents unique barriers for the use of this technology, and there are currently no reptiles with effective methods for targeted mutagenesis. Here we present a new approach that enables the efficient production of CRISPR-Cas9 induced mutations in Anolis lizards, an important model for studies of reptile evolution and development.

scholarly journals Highly Efficient and Heritable Targeted Mutagenesis in Wheat via the Agrobacterium tumefaciens-Mediated CRISPR/Cas9 System

2019 ◽  
Vol 20 (17) ◽  
pp. 4257 ◽  
Author(s):  
Shujuan Zhang ◽  
Rongzhi Zhang ◽  
Jie Gao ◽  
Tiantian Gu ◽  
Guoqi Song ◽  
...  
Keyword(s):  
Gene Editing ◽  
Genome Engineering ◽  
Targeted Mutagenesis ◽  
Mutation Rates ◽  
Next Generation ◽  
Induced Mutations ◽  
D Genome ◽  
Agrobacterium Mediated Transformation ◽  
Biallelic Mutations ◽  
New Mutations

The CRISPR/Cas9 system has been successfully used in hexaploid wheat. Although it has been reported that the induced mutations can be passed to the next generation, gene editing and transmission patterns in later generations still need to be studied. In this study, we demonstrated that the CRISPR/Cas9 system could achieve efficient mutagenesis in five wheat genes via Agrobacterium-mediated transformation of an sgRNA targeting the D genome, an sgRNA targeting both the A and B homologues and three tri-genome guides targeting the editing of all three homologues. High mutation rates and putative homozygous or biallelic mutations were observed in the T0 plants. The targeted mutations could be stably inherited by the next generation, and the editing efficiency of each mutant line increased significantly across generations. The editing types and inheritance of targeted mutagenesis were similar, which were not related to the targeted subgenome number. The presence of Cas9/sgRNA could cause new mutations in subsequent generations, while mutated lines without Cas9/sgRNA could retain the mutation type. Additionally, off-target mutations were not found in sequences that were highly homologous to the selected sgRNA sequences. Overall, the results suggested that CRISPR/Cas9-induced gene editing via Agrobacterium-mediated transformation plays important roles in wheat genome engineering.

scholarly journals Latest Advances of Virology Research Using CRISPR/Cas9-Based Gene-Editing Technology and Its Application to Vaccine Development

Viruses ◽  
2021 ◽  
Vol 13 (5) ◽  
pp. 779
Author(s):  
Man Teng ◽  
Yongxiu Yao ◽  
Venugopal Nair ◽  
Jun Luo
Keyword(s):  
Biological Sciences ◽  
Gene Therapy ◽  
Genetic Engineering ◽  
Gene Function ◽  
Viral Genome ◽  
Gene Editing ◽  
Vaccine Development ◽  
Future Prospects ◽  
Dna Viruses ◽  
Viral Genomes

In recent years, the CRISPR/Cas9-based gene-editing techniques have been well developed and applied widely in several aspects of research in the biological sciences, in many species, including humans, animals, plants, and even in viruses. Modification of the viral genome is crucial for revealing gene function, virus pathogenesis, gene therapy, genetic engineering, and vaccine development. Herein, we have provided a brief review of the different technologies for the modification of the viral genomes. Particularly, we have focused on the recently developed CRISPR/Cas9-based gene-editing system, detailing its origin, functional principles, and touching on its latest achievements in virology research and applications in vaccine development, especially in large DNA viruses of humans and animals. Future prospects of CRISPR/Cas9-based gene-editing technology in virology research, including the potential shortcomings, are also discussed.

Gene silencing or gene editing: the pros and cons.

2021 ◽  
pp. 47-53
Author(s):  
Huw D. Jones
Keyword(s):  
Gene Expression ◽  
Gene Silencing ◽  
Gene Function ◽  
Gene Editing ◽  
Mutation Breeding ◽  
Gene Transcript ◽  
Commercial Activities ◽  
Targeted Mutation ◽  
Pros And Cons ◽  
Or Gene

Abstract Research into plant genetics often requires the suppression or complete knockout of gene expression to scientifically validate gene function. In addition, the phenotypes obtained from gene suppression can occasionally have commercial value for plant breeders. Until recently, the methodological choices to achieve these goals fell into two broad types: either some form of RNA-based gene silencing; or the screening of large numbers of natural or induced random genomic mutations. The more recent invention of gene editing as a tool for targeted mutation potentially gives researchers and plant breeders another route to block gene function. RNAi is widely used in animal and plant research and functions to silence gene expression by degrading the target gene transcript. Although RNAi offers unique advantages over genomic mutations, it often leads to the formation of a genetically modified organism (GMO), which for commercial activities has major regulatory and acceptance issues in some regions of the world. Traditional methods of generating genomic mutations are more laborious and uncertain to achieve the desired goals but possess a distinct advantage of not being governed by GMO regulations. Gene editing (GE) technologies have some of the advantages of both RNAi and classical mutation breeding in that they can be designed to give simple knockouts or to modulate gene expression more subtly. GE also has a more complex regulatory position, with some countries treating it as another conventional breeding method whilst the EU defines GE as a technique of genetic modification and applies the normal GMO authorization procedures. This chapter explores the pros and cons of RNAi alongside other methods of modulating gene function.

scholarly journals Proxies of CRISPR/Cas9 Activity To Aid in the Identification of Mutagenized Arabidopsis Plants

2020 ◽  
Vol 10 (6) ◽  
pp. 2033-2042 ◽  
Author(s):  
Renyu Li ◽  
Charles Vavrik ◽  
Cristian H. Danna
Keyword(s):  
Arabidopsis Thaliana ◽  
Gene Function ◽  
Good Predictor ◽  
Gene Editing ◽  
Selection Strategy ◽  
Directed Mutagenesis ◽  
Loss Of Function ◽  
Editing Event ◽  
Endogenous Gene ◽  
Non Coding Rnas

CRISPR/Cas9 has become the preferred gene-editing technology to obtain loss-of-function mutants in plants, and hence a valuable tool to study gene function. This is mainly due to the easy reprogramming of Cas9 specificity using customizable small non-coding RNAs, and to the possibility of editing several independent genes simultaneously. Despite these advances, the identification of CRISPR-edited plants remains time and resource-intensive. Here, based on the premise that one editing event in one locus is a good predictor of editing event/s in other locus/loci, we developed a CRISPR co-editing selection strategy that greatly facilitates the identification of CRISPR-mutagenized Arabidopsis thaliana plants. This strategy is based on targeting the gene/s of interest simultaneously with a proxy of CRISPR-Cas9-directed mutagenesis. The proxy is an endogenous gene whose loss-of-function produces an easy-to-detect visible phenotype that is unrelated to the expected phenotype of the gene/s under study. We tested this strategy via assessing the frequency of co-editing of three functionally unrelated proxy genes. We found that each proxy predicted the occurrence of mutations in each surrogate gene with efficiencies ranging from 68 to 100%. The selection strategy laid out here provides a framework to facilitate the identification of multiplex edited plants, thus aiding in the study of gene function when functional redundancy hinders the effort to define gene-function-phenotype links.

scholarly journals Highly Efficient Targeted Gene Editing in Upland Cotton Using the CRISPR/Cas9 System

2018 ◽  
Vol 19 (10) ◽  
pp. 3000 ◽  
Author(s):  
Shouhong Zhu ◽  
Xiuli Yu ◽  
Yanjun Li ◽  
Yuqiang Sun ◽  
Qianhao Zhu ◽  
...  
Keyword(s):  
Gene Editing ◽  
Crop Improvement ◽  
Targeted Mutagenesis ◽  
Cotton Fibers ◽  
Efficient Tool ◽  
Gene Encoding ◽  
Editing Event ◽  
Highly Efficient ◽  
Target Sites ◽  
Targeted Gene Editing

The clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) gene editing system has been shown to be able to induce highly efficient mutagenesis in the targeted DNA of many plants, including cotton, and has become an important tool for investigation of gene function and crop improvement. Here, we developed a simple and easy to operate CRISPR/Cas9 system and demonstrated its high editing efficiency in cotton by targeting-ALARP, a gene encoding alanine-rich protein that is preferentially expressed in cotton fibers. Based on sequence analysis of the target site in the 10 transgenic cottons containing CRISPR/Cas9, we found that the mutation frequencies of GhALARP-A and GhALARP-D target sites were 71.4–100% and 92.9–100%, respectively. The most common editing event was deletion, but deletion together with large insertion was also observed. Mosaic mutation editing events were detected in most transgenic plants. No off-target mutation event was detected in any the 15 predicted sites analyzed. This study provided mutants for further study of the function of GhALARP in cotton fiber development. Our results further demonstrated the feasibility of use of CRISPR/Cas9 as a targeted mutagenesis tool in cotton, and provided an efficient tool for targeted mutagenesis and functional genomics in cotton.

Direct Manipulation of Feature Models in Web-Based Collaborative Design

2004 ◽  
Author(s):  
Rafael Bidarra ◽  
Andre´ van Bunnik ◽  
Willem F. Bronsvoort
Keyword(s):  
Collaborative Design ◽  
Feature Model ◽  
Feature Models ◽  
Direct Manipulation ◽  
Model Data ◽  
Web Based ◽  
New Approach ◽  
Client Server ◽  
Collaborative Modeling ◽  
Interactive Feature

Providing advanced 3D interactive facilities to users of a client-server collaborative modeling system presents a great challenge when thin clients are involved, mainly due to their lack of both a full-fledged CAD model and adequate modeling and solving functionalities. This paper presents a new approach that provides a convenient representation of feature model data suitable for direct manipulation of feature models at such clients. In particular, feature handles are proposed to support interactive feature editing. This approach combines all advantages of a thin client approach with the sort of 3D direct manipulation facilities usually only found in powerful standalone CAD systems.

scholarly journals A new view of transcriptome complexity and regulation through the lens of local splicing variations

eLife ◽  
2016 ◽  
Vol 5 ◽  
Author(s):  
Jorge Vaquero-Garcia ◽  
Alejandro Barrera ◽  
Matthew R Gazzara ◽  
Juan González-Vallinas ◽  
Nicholas F Lahens ◽  
...  
Keyword(s):  
Genetic Variation ◽  
Alternative Splicing ◽  
Gene Function ◽  
Specific Protein ◽  
Mrna Splicing ◽  
New Approach ◽  
Genome Wide ◽  
Mouse Tissues ◽  
Splice Variation ◽  
Novel Isoforms

Alternative splicing (AS) can critically affect gene function and disease, yet mapping splicing variations remains a challenge. Here, we propose a new approach to define and quantify mRNA splicing in units of local splicing variations (LSVs). LSVs capture previously defined types of alternative splicing as well as more complex transcript variations. Building the first genome wide map of LSVs from twelve mouse tissues, we find complex LSVs constitute over 30% of tissue dependent transcript variations and affect specific protein families. We show the prevalence of complex LSVs is conserved in humans and identify hundreds of LSVs that are specific to brain subregions or altered in Alzheimer's patients. Amongst those are novel isoforms in the Camk2 family and a novel poison exon in Ptbp1, a key splice factor in neurogenesis. We anticipate the approach presented here will advance the ability to relate tissue-specific splice variation to genetic variation, phenotype, and disease.

Analysis of MTH1 gene function in mice with targeted mutagenesis

2001 ◽  
Vol 477 (1-2) ◽  
pp. 71-78 ◽  
Author(s):  
Teruhisa Tsuzuki ◽  
Akinori Egashira ◽  
Shinobu Kura
Keyword(s):  
Gene Function ◽  
Targeted Mutagenesis
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