scholarly journals sgRNA design for DLT gene editing using CRISPR-Cas9 and in-silico mutation prediction in Rice cv. Hawara Bunar

2021 ◽  
Vol 948 (1) ◽  
pp. 012083
Author(s):  
I Halim ◽  
M H Fendiyanto ◽  
Miftahudin
Keyword(s):  
Reference Gene ◽  
Gene Editing ◽  
Amino Acid Sequences ◽  
Protein Domain ◽  
Pcr Product ◽  
Protospacer Adjacent Motif ◽  
Family Protein ◽  
Exon 1 ◽  
Sgrna Design ◽  
Mutation Prediction

Abstract The DWARF AND LOW TILLERRING (DLT) gene is a transcription factor for a gene involved in Brassinosteroid (BR) biosynthesis. Manipulating BR biosynthesis will affect the height and tiller number of rice. CRISPR-Cas9 is an accurate tool to edit a gene sequence. The accuracy of site editing of the CRISPR-Cas9-mediated target gene editing is determined by the 20 nucleotide sequences in the sgRNA and the binding site known as the Protospacer Adjacent Motif (PAM). The study aimed to design sgRNA and predict the DLT gene mutation sites in rice cv. Hawara Bunar. The exon 1 of the DLT gene was amplified using a primer pair designed from the reference gene. The PCR product was then sequenced, and the sequence was used to design sgRNA. The study has designed sgRNA located on the targeted sequence that corresponds to the Gras family protein domain of the exon1 DLT gene. The mutation sites were predicted to be at the domain site through the alignment of the nucleotide and amino acid sequences of the DLT gene and the reference gene. It is predicted that mutations in the target site that corresponds to the protein domain will change the protein structure and its function.

Organization and expression of the chicken N-myc gene

1990 ◽  
Vol 10 (5) ◽  
pp. 2017-2026
Author(s):  
S Sawai ◽  
K Kato ◽  
Y Wakamatsu ◽  
H Kondoh
Keyword(s):  
Genomic Sequence ◽  
Amino Acid Sequences ◽  
Coding Regions ◽  
Noncoding Exon ◽  
Exon 1 ◽  
Myc Gene ◽  
The Central Nervous System ◽  
Flanking Region ◽  
High Level

We cloned the chicken N-myc gene and analyzed its structure and expression. We found that it consisted of three exons with coding regions in exons 2 and 3. Comparison to mammalian N-myc genomic sequence indicated that nucleotide sequences of the 5'-flanking region, noncoding exon 1, and introns were not conserved, but coding and 3' noncoding sequences showed significant homology to mammalian N-myc. Alignment of deduced amino acid sequences of chicken and mammalian N-myc proteins revealed nine conserved domains interrupted by different lengths of nonhomologous sequences. Two of the domains were specific to N-myc proteins, and the other seven were common to c-myc proteins. Northern blot (immunoblot) and in situ hybridization analyses of 3.5-day-old chicken embryos revealed that high-level expression of the N-myc gene was confirmed to certain tissues, e.g., the central nervous system, neural crest derivatives, and mesenchyme of limb buds. In the beak and limb primordia, N-myc expression in the mesenchyme was higher toward the distal end, suggesting possible involvement in positional assignment of the tissue within the rudimentary structures.

CRISPR/Cas9-induced β-carotene Hydroxylase Mutation in Dunaliella Salina CCAP19/18

2021 ◽  
Author(s):  
Lina Hu ◽  
Shu ying FENG ◽  
Gaofeng Liang ◽  
Jingxia Du ◽  
Aifang Li ◽  
...  
Keyword(s):  
High Performance ◽  
Dunaliella Salina ◽  
Gene Editing ◽  
Expression System ◽  
Transformation Method ◽  
Practical Significance ◽  
Exon 1 ◽  
Targeted Gene Editing ◽  
Foreign Genes ◽  
Β Carotene

Abstract Dunaliella salina (D. salina) has been exploited as a novel expression system for the field of genetic engineering. However, owing to the low or inconsistent expression of target proteins, it has been greatly restricted to practical production of recombinant proteins. Since the accurate gene editing function of CRISPR/Cas system, β-carotene hydroxylase gene was chosen as an example to explore D. salina application with the purpose of improving expression level of foreign genes. In this paper, based on pKSE401 backbone, three CRISPR/Cas9 binary vectors were constructed to targeting exon 1 and 3 of the β-carotene hydroxylase of D. salina CCAP19/18 (Dschyb). D. salina mutants were obtained by salt gradient transformation method, and the expression of Dschyb gene were identified through real-time fluorescent quantitative PCR. Moreover, carotenoids content was analyzed by high-performance liquid chromatography at different time points after high intensity treatment. Compared with wild type strains, the β-carotene levels of mutants showed a significant increase, nearly up to 1.4 μg/ml, and the levels of zeaxanthin decreased to various degrees in mutants. All the results provide a compelling evidence for targeted gene editing in D. salina. This study gave a first successful gene editing of D. salina which has a very important practical significance for increasing carotene yield and meeting realistic industry demand. Furthermore, it provides an approach to overcome the current obstacles of D. salina, and then gives a strong tool to facilitates the development and application of D. salina system.

scholarly journals Dizygotic Twinning Is Not Linked to Variation at the α -Inhibin Locus on Human Chromosome 2*

2000 ◽  
Vol 85 (9) ◽  
pp. 3391-3395
Author(s):  
Grant W. Montgomery ◽  
David L. Duffy ◽  
Jeff Hall ◽  
Barbara R. Haddon ◽  
Masataka Kudo ◽  
...  
Keyword(s):  
Multiple Pregnancy ◽  
Racial Groups ◽  
Chromosome 2 ◽  
Lod Score ◽  
Α Subunit ◽  
Small Peak ◽  
Pcr Product ◽  
Exon 1 ◽  
Log Odds ◽  
Complete Exclusion

Abstract Natural multiple pregnancy in women leading to dizygotic (DZ) twins is familial and varies across racial groups, suggesting a genetic predisposition. Mothers of DZ twins have a higher incidence of spontaneous multiple ovulation and elevated FSH concentrations. FSH release is controlled by feedback of inhibin peptides from the ovary, and immunization against inhibin α-subunit results in an increased ovulation rate in animals. The inhibin α-subunit is therefore a candidate gene for mutations that may increase the frequency of DZ twinning. Restriction digests of a PCR product from exon 1 with the enzyme SpeI detects a C/T polymorphism at bp 128 with two alleles of 447 and 323/124 bp. The polymorphism was typed in 1125 individuals from 326 pedigrees with 717 mothers of spontaneous DZ twins. The α-inhibin locus mapped within 3 centimorgans of D2S164, and linkage with DZ twinning was excluded [decimal log odds ratio (LOD) score, −2.81 at θ = 0]. There was complete exclusion of linkage (LOD, less than −2) of a gene conferring relative risk 1.8 (λs, >1.8) across the chromosome, except at the p-terminus region and a small peak (maximum LOD score, 0.6) in the region of D2S151-D2S326. Analysis using either recessive or dominant models excluded linkage with DZ twinning in this population (LOD score, less than −2.5) across chromosome 2. We conclude that dizygotic twinning is not linked to variation in the α-inhibin locus. The results also suggest that mutations in other candidates on chromosome 2, including the receptor for FSH and the βB-inhibin subunit (INHBB) cannot be major contributors to risk for DZ twinning.

Characterization of cytoplasmic fibril structures found in gliding cells of Saprospira sp.

2005 ◽  
Vol 51 (10) ◽  
pp. 875-880 ◽  
Author(s):  
Gou Furusawa ◽  
Takeshi Yoshikawa ◽  
Yoshitaka Takano ◽  
Kazuyuki Mise ◽  
Iwao Furusawa ◽  
...  
Keyword(s):  
Amino Acid ◽  
Protein Band ◽  
Amino Acid Sequences ◽  
Gliding Motility ◽  
Pcr Product ◽  
Nutrient Agar Medium ◽  
Sds Page ◽  
Tail Sheath ◽  
A Subunit ◽  
Triton X

The cytoplasmic fibril structures of Saprospira sp. strain SS98-5 grown on a low-nutrient agar medium were purified from cell lysates treated with Triton X-100 and were observed by electron microscopy to be about 7 nm in width and 200–300 nm in length. SDS–PAGE of the fibril structures exhibited a single protein band with a molecular mass of 61 kDa. A Saprospira cytoplasmic fibril protein (SCFP), which is a subunit of the fibril structures, was digested with trypsin to oligopeptides and analyzed for amino acid sequences. A partial nucleotide sequence of the SCFP gene was determined after PCR using primers designated from the amino acid sequences of the oligopeptides. SCFP gene including DNA fragments were detected by Southern hybridization using the PCR product for an SCFP gene as a probe and were cloned to determine whole nucleotide sequences. The SCFP gene indicated relatively higher similarity to conserved hypothetical phage tail sheath proteins. A Western immunoblotting analysis showed that SCFP was significantly expressed in gliding cells as compared with nongliding cells. The above findings with the previously reported results suggest that the cytoplasmic fibril structures are possibly related to the gliding motility of Saprospira sp. strain SS98-5.Key words: Saprospira, gliding motility, Saprospira cytoplasmic fibril protein (SCFP).

scholarly journals Squash vein yellowing virus Identified in Watermelon (Citrullus lanatus) in Indiana

Plant Disease ◽  
2007 ◽  
Vol 91 (8) ◽  
pp. 1056-1056 ◽  
Author(s):  
D. S. Egel ◽  
S. Adkins
Keyword(s):  
Citrullus Lanatus ◽  
Amino Acid Sequences ◽  
Rt Pcr ◽  
Strain Vector ◽  
First Report ◽  
Pcr Product ◽  
Field Samples ◽  
Health Progress ◽  
Vine Decline ◽  
Yellowing Virus

During September 2006, moderate vine decline symptoms including vine collapse and wilt and root rot were observed on numerous watermelon plants growing in a commercial field in Sullivan County, Indiana. No symptoms were observed on the fruit. Six plants displaying typical vine decline symptoms were collected and assayed for potyvirus infection and subsequently for Squash vein yellowing virus (SqVYV) and Papaya ringspot virus type W (PRSV-W). SqVYV is a whitefly-transmitted member of the Potyviridae, recently shown to cause watermelon vine decline in Florida (1,4). Plants infected with SqVYV in Florida are also frequently infected with PRSV-W, although SqVYV is sufficient for watermelon vine decline. The six field samples harbored one or more potyviruses as determined by ELISA (Agdia, Elkhart, IN). Mechanical inoculation of squash (Cucurbita pepo) and watermelon with sap from three of the field samples induced mosaic symptoms in both that are typical of potyviruses. Vein yellowing in squash and plant death in watermelon typical of SqVYV (1) later developed in plants inoculated with one field sample. A coat protein gene fragment was amplified by reverse transcription (RT)-PCR with SqVYV primers (1) from total RNA of five of the six field samples and also from the symptomatic, inoculated plants. Nucleotide and deduced amino acid sequences of a 957-bp region of the RT-PCR product (primer sequences deleted prior to analysis) were 100% identical to SqVYV (GenBank accession No. DQ812125). PRSV-W also was identified in two of the five SqVYV-infected field samples by ELISA (Agdia) and by sequence analysis of a 3′ genome fragment amplified by RT-PCR with previously described degenerate potyvirus primers (3). No evidence for infection by other potyviruses was obtained. To our knowledge, this is the first report of SqVYV in Indiana and the first report of the virus anywhere outside of Florida. The whitefly (Bemisia tabaci, B strain) vector of SqVYV is relatively uncommon in Indiana and the cold winter temperatures make it unlikely that any SqVYV-infected watermelon vines or whiteflies will overseason, necessitating reintroductions of virus and vector each season. We feel that the moderate and restricted occurrence of SqVYV in Indiana observed in September 2006 should pose little or no threat to commercial watermelon production in Indiana and should not cause growers to alter their growing practices. The occurrence of SqVYV in Indiana does not appear to explain the similar symptoms of mature watermelon vine decline (MWVD) that has been observed in Indiana since the 1980s. In contrast with the insect vectored SqVYV, MWVD seems to be caused by a soilborne biological agent (2). References: (1) S. Adkins et al. Phytopathology 97:145, 2007. (2) D. S. Egel et al. Online publication. doi:10.1094/PHP-2000-1227-01-HN. Plant Health Progress, 2000. (3) A. Gibbs and A. Mackenzie. J. Virol. Methods 63:9, 1997. (4) P. Roberts et al. Citrus Veg. Mag. December 12, 2004.

scholarly journals CRISPR-Cas9 in agriculture: Approaches, applications, future perspectives, and associated challenges

2020 ◽  
Vol 3 (1) ◽  
pp. 6-16
Author(s):  
Prabin Adhikari ◽  
Mousami Poudel
Keyword(s):  
Dna Cleavage ◽  
Gene Editing ◽  
Adaptive Immune System ◽  
Genetic Manipulations ◽  
Adaptive Immune ◽  
Cargo Delivery ◽  
Genome Wide ◽  
Rna Fragments ◽  
Sgrna Design ◽  
Living Organisms

AbstractThe discovery of an adaptive immune system especially in archae and bacteria, CRISPR/Cas has revolutionized the field of agriculture and served as a potential gene editing tool, producing great excitement to the molecular scientists for the improved genetic manipulations. CRISPR/Cas9 is a RNA guided endonuclease which is popular among its predecessors ZFN and TALEN’s. The utilities of CRISPR from its predecessors is the use of short RNA fragments to locate target and breaking the double strands which avoids the need of protein engineering, thus allowing time efficiency measure for gene editing. It is a simple, flexible and highly efficient programmable DNA cleavage system that can be modified for widespread applications like knocking out the genes, controlling transcription, modifying epigenomes, controlling genome-wide screens, modifying genes for disease and stress tolerance and imaging chromosomes. However, gene cargo delivery system, off target cutting and issues on the safety of living organisms imposes major challenge to this system. Several attempts have been done to rectify these challenges; using sgRNA design software, cas9 nickases and other mutants. Thus, further addressing these challenges may open the avenue for CRISPR/cas9 for addressing the agriculture related problems.

scholarly journals Identification of Genes for Wheat Fungal Resistance Using Bioinformatics Techniques

2018 ◽  
pp. 1-10 ◽  
Author(s):  
Ahmed E. Nassar ◽  
Khaled H. Mousa ◽  
Ahmed A. Madbouly ◽  
Shafik D. Ibrahim ◽  
Alsamman M. Alsamman
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Fungal Pathogens ◽  
Gene Annotation ◽  
Average Length ◽  
Amino Acid Sequences ◽  
Protein Domain ◽  
Fungal Resistance ◽  
Domain Identification ◽  
Cereal Grain

For the majority of world populations, wheat (Triticum aestivum L.) would be the first essential and economic cereal grain crop. Pests and pathogens in both rich and developing countries are constantly threatening wheat production and sustainable development. Multiple gene pathways were recorded to share an association with fungal pathogens with wheat biological resistance. Our aim to use such tools in order to detect and classify fungal resistance genes in wheat through sequence alignment, protein domain identification and phylogenetic analysis. In addition the introduction for restriction fragment length polymorphism (RFLP) for such genes in the new primer database. Approximately 138 sequences of DNA were recovered from the wheat genome by aligning 3845 anti-fungal amino acids through tblastn tool. The NCBI blastn online tool used to detect sequences with functional genes, where 92 genes have been detected. The total number of nucleotides was 48385, where the smallest DNA sequence have 302 bp and the longest contains 977 bp with an average length of 525.9 bp per sequence. The wheat chromosomes 3D, and 4B have the highest number of sequences (9) followed by chromosomes 3B (7) and 3A(6), where wheat genomes A, B and D have 30, 35 and 27 genes, respectively. Five different amino acids motifs have been revealed among studied wheat amino acid sequences. The gene annotation tools used to infer studied amino acid gene annotation. Amino acid sequences belongs to lectin, kinase, tyrosine-protein kinase (STK), thaumatin, and cysteine-rich repeats representing 2, 9, 8, 19, 23 genes respectively, in addition to 31 hypothetical genes. The proteins chemical content have been assessed through 16 different amino acid chemical and physical characteristics.

scholarly journals PAVOOC: Designing CRISPR sgRNAs using 3D protein structures and functional domain annotation

2018 ◽  
Author(s):  
Moritz Schaefer ◽  
Dr. Djork-Arné Clevert ◽  
Dr. Bertram Weiss ◽  
Dr. Andreas Steffen
Keyword(s):  
Gene Editing ◽  
State Of The Art ◽  
Protein Structures ◽  
Design Tool ◽  
Protein Crystal ◽  
Functional Domain ◽  
Web Based ◽  
Domain Annotation ◽  
Sgrna Design ◽  
Processing Steps

AbstractSummary: sgRNAs targeting the same gene can significantly vary in terms of efficacy and specificity. PAVOOC (Prediction And Visualization of On- and Off-targets for CRISPR) is a web-based CRISPR sgRNA design tool that employs state-of-the art machine learning models to prioritize most effective candidate sgRNAs. In contrast to other tools, it maps sgRNAs to functional domains and protein structures and visualizes cut sites on corresponding protein crystal structures. Furthermore, PAVOOC supports HDR template generation for gene editing experiments and the visualization of the mutated amino acids in 3D.Availability and Implementation: PAVOOC is available under https://pavooc.me and accessible using current browsers (Chrome/Chromium recommended). The source code is hosted at github.com/moritzschaefer/pavooc under the MIT License. The backend, including data processing steps, and the frontend is implemented in Python 3 and ReactJS respectively. All components run in a simple Docker environment.Contact: [email protected]

scholarly journals First detection and molecular characterization of kobuvirus from a dog in Turkey

2019 ◽  
Vol 75 (05) ◽  
pp. 6248-2019
Author(s):  
ZEYNEP AKKUTAY-YOLDAR ◽  
TAYLAN KOÇ B. ◽  
ÇIĞDEM OĞUZOĞLU T.
Keyword(s):  
Molecular Characterization ◽  
Rectal Swab ◽  
Clinical Symptoms ◽  
Phylogenetic Analyses ◽  
Amino Acid Sequences ◽  
Coding Region ◽  
Gastrointestinal Infections ◽  
Protein Coding ◽  
Pcr Product ◽  
Domestic Carnivores

Canine kobuvirus (CaKVs) is a newly emerging virus detected in dogs in several countries. However, kobuvirus infection has not yet been described in domestic carnivores in Turkey. In this study, we tested blood and rectal swab samples to determine the presence of kobuvirus in a dog with clinical symptoms by reverse transcription-polymerase chain reaction (RT-PCR), using 3D (RNA polymerase) region primers of canine kobuviruses. To provide molecular characterization data, the Maximum Likelihood (ML) method was used for the phylogenetic analyses. The PCR product of the partial protein-coding region of the 3D protein gene from the rectal swab was amplified, purified, and sequenced. Phylogenetic analysis of amino acid sequences suggests that our CaKV strain was closely related to US-CaKVs,and placed on a monophyletic clade as a sister branch localized in the CaKV cluster. These results indicate that CaKV exists in dogs in Turkey. With a similarity of 94.2–96.1%, it is like other CaKVs. To our knowledge, this is the first report of CaKV infection of a dog by in Turkey. Further studies are needed to determine its role in dog gastrointestinal infections.

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