scholarly journals CRISPR-Cas9: Role in Processing of Modular Metabolic Engineered Bio-Based Products

2021 ◽  
Author(s):  
Vishnu Sharma ◽  
Tarun Kumar Kumawat ◽  
Anjali Pandit ◽  
Bhoomika Sharma ◽  
Pooja Agarwal ◽  
...  
Keyword(s):  
Immune System ◽  
Genome Editing ◽  
Metabolic Product ◽  
Modifying Genes ◽  
Genome Modification ◽  
Foreign Dna ◽  
Genome Level

Biogenetic engineering is a significant technology to sensibly manage microbial metabolic product factories. Genome modification methods for efficiently controlling and modifying genes at the genome level have progressed in biogenetic engineering during the last decade. CRISPR is genome editing technology that allows for the modification of organisms’ genomes. CRISPR and its related RNA-guided endonuclease are versatile advanced immune system frameworks for defending against foreign DNA and RNAs. CRISPR is efficient, accessible, and trustworthy genomic modification tool in unparalleled resolution. At present, CRISPR-Cas9 method is expanded to industrially manipulate cells. Metabolically modified organisms are quickly becoming interested in the production of different bio-based components. Here, chapter explore about the control productivity of targeted biomolecules in divergent cells based on the use of different CRISPR-related Cas9.

scholarly journals Programming Native CRISPR Arrays for the Generation of Targeted Immunity

mBio ◽  
2016 ◽  
Vol 7 (3) ◽  
Author(s):  
Alexander P. Hynes ◽  
Simon J. Labrie ◽  
Sylvain Moineau
Keyword(s):  
Immune System ◽  
Nucleic Acid ◽  
Genome Editing ◽  
Dna Sequence ◽  
Adaptive Immune System ◽  
Natural Process ◽  
The Public ◽  
Adaptive Immune ◽  
Public Consciousness ◽  
Industrial Settings

ABSTRACT The adaptive immune system of prokaryotes, called CRISPR-Cas (clustered regularly interspaced short palindromic repeats and CRISPR-associated genes), results in specific cleavage of invading nucleic acid sequences recognized by the cell’s “memory” of past encounters. Here, we exploited the properties of native CRISPR-Cas systems to program the natural “memorization” process, efficiently generating immunity not only to a bacteriophage or plasmid but to any specifically chosen DNA sequence. IMPORTANCE CRISPR-Cas systems have entered the public consciousness as genome editing tools due to their readily programmable nature. In industrial settings, natural CRISPR-Cas immunity is already exploited to generate strains resistant to potentially disruptive viruses. However, the natural process by which bacteria acquire new target specificities (adaptation) is difficult to study and manipulate. The target against which immunity is conferred is selected stochastically. By biasing the immunization process, we offer a means to generate customized immunity, as well as provide a new tool to study adaptation.

scholarly journals Mechanism of Foreign DNA Selection in a Bacterial Adaptive Immune System

2012 ◽  
Vol 46 (5) ◽  
pp. 606-615 ◽  
Author(s):  
Dipali G. Sashital ◽  
Blake Wiedenheft ◽  
Jennifer A. Doudna
Keyword(s):  
Immune System ◽  
Adaptive Immune System ◽  
Adaptive Immune ◽  
Foreign Dna

scholarly journals Sequential Genome Editing and Induced Excision of the Transgene in N. tabacum BY2 Cells

2020 ◽  
Vol 11 ◽  
Author(s):  
Maor Sheva ◽  
Uri Hanania ◽  
Tami Ariel ◽  
Albina Turbovski ◽  
Vishal Kumar Rameshchandra Rathod ◽  
...  
Keyword(s):  
Host Cell ◽  
Genome Editing ◽  
Cell Lines ◽  
Recombinant Proteins ◽  
Plant Cells ◽  
Second Step ◽  
Selectable Markers ◽  
Additional Gene ◽  
Bright Yellow ◽  
Foreign Dna

While plant cells in suspension are becoming a popular platform for expressing biotherapeutic proteins, the need to pre-engineer these cells to better comply with their role as host cell lines is emerging. Heterologous DNA and selectable markers are used for transformation and genome editing designated to produce improved host cell lines for overexpression of recombinant proteins. The removal of these heterologous DNA and selectable markers, no longer needed, can be beneficial since they limit additional gene stacking in subsequent transformations and may pose excessive metabolic burden on the cell machinery. In this study we developed an innovative stepwise methodology in which the CRISPR-Cas9 is used sequentially to target genome editing, followed by its own excision. The first step included a stable insertion of a CRISPR-Cas9 cassette, targeted to knockout the β(1,2)-xylosyltranferase (XylT) and the α(1,3)-fucosyltransferase (FucT) genes in Nicotiana tabacum L. cv Bright Yellow 2 (BY2) cell suspension. The second step included the excision of the inserted cassette of 14.3 kbp by induction of specific sgRNA designed to target the T-DNA boundaries. The genome editing step and the transgene removal step are achieved in one transformation run. This mechanism enables CRISPR genome editing and subsequently eliminating the introduced transgenes thus freeing the cells from foreign DNA no longer needed.

scholarly journals A Crisper Look at Genome Editing: RNA-guided Genome Modification

2013 ◽  
Vol 21 (4) ◽  
pp. 720-722 ◽  
Author(s):  
Mara Damian ◽  
Matthew H Porteus
Keyword(s):  
Genome Editing ◽  
Genome Modification

scholarly journals Detection of High Titers of Antibody against Helicobacter Cysteine-Rich Proteins A, B, C, and E in Helicobacter pylori-Infected Individuals

2003 ◽  
Vol 10 (4) ◽  
pp. 542-545 ◽  
Author(s):  
Peer R. E. Mittl ◽  
Lucas Lüthy ◽  
Christoph Reinhardt ◽  
Hellen Joller
Keyword(s):  
Helicobacter Pylori ◽  
Immune System ◽  
Control Group ◽  
Clear Correlation ◽  
Weak Correlation ◽  
The Family ◽  
Genome Level ◽  
Immunological Assays ◽  
H Pylori

ABSTRACT The family of Helicobacter cysteine-rich proteins (Hcp) constitutes one of the largest protein families that are specific for proteobacteria from the delta/epsilon subgroup. Most of the proteins belonging to this family have so far only been recognized on the genome level. To investigate the expression of Hcp proteins in vivo we analyzed titers of antibody against HcpA (HP0211), HcpB (HP0336), HcpC (HP1098), and HcpE (HP0235) in sera from 30 Helicobacter pylori-positive individuals and in a control group of six H. pylori-negative individuals. Significantly higher titers of antibody were observed for H. pylori-positive individuals (P < 0.00005). The highest and lowest titers were observed for HcpC (Δ mean = 1.06) and HcpB (Δ mean = 0.333), respectively. There is a clear correlation among anti-HcpA, -HcpC, and -HcpE immunoglobulin G titers in H. pylori-positive individuals (correlation > 0.7), but there is only a weak correlation for HcpB (correlation < 0.4). These results confirm that Hcp proteins are expressed by H. pylori under natural environmental conditions and that these proteins are recognized by the immune system of the host. The observed correlations are in agreement with the expected distribution of Hcp proteins among H. pylori strains. HcpA, HcpC, and HcpE are present in the genomes of strains 26695 and J99, whereas HcpB is absent from most strains. Since Hcp proteins are specific for H. pylori, immunological assays including Hcp proteins might be of value to detect H. pylori infection and perhaps to distinguish among different groups of H. pylori-positive patients.

scholarly journals CRISPR Cas9 Tip of an Iceberg: It’s Potential in Genome Editing and Many More

2021 ◽  
Author(s):  
Jalal Ahmad ◽  
Nayyer Siddique
Keyword(s):  
Genome Editing ◽  
Genetic Diseases ◽  
Adaptive Immune System ◽  
Biological Research ◽  
Adaptive Immune ◽  
Scientific World ◽  
A Genome ◽  
Foreign Dna ◽  
Immense Potential ◽  
Potential Use

Clustered regularly interspaced short palindromic repeats or CRISPR, one of the major technological tools from nature's toolbox, has revolutionized the scientific world with its potential use in humans and plants. CRISPR Cas9 was first known as an adaptive immune system of bacteria. It is a system that cleaves foreign DNA. It has been exploited to be used as a genome editing tool for correcting genetic diseases in humans, for plants to create stress-resistant plants, and for a variety of different purposes. This review provides a basic overview of its applications in different areas of biological research. It has immense potential for a variety of researches, but it's still a mystery for science. It feels like scientists just know a tip of an iceberg.

scholarly journals Snippets of virus integrated into human genome suggests possibility of CRISPR-like adaptive immune system in human cells

2019 ◽  
Author(s):  
Wenfa Ng
Keyword(s):  
Immune Response ◽  
Immune System ◽  
Human Genome ◽  
Adaptive Immune Response ◽  
Adaptive Immune System ◽  
Human Cells ◽  
Rna Seq ◽  
Viral Dna ◽  
Adaptive Immune ◽  
Foreign Dna

Snippets of virus that infect humans have been shown to be incorporated into the human genome. Could such virus snippets provide a form of adaptive immunity similar to that offered by CRISPR to bacterial cells? To answer the question, RNA-seq could be used to provide a broad view of the RNA transcribed from DNA in the genome. Using known genome sequence of viruses that infect humans as template, reads obtained from RNA-seq would be profiled for virus snippets integrated into human genome and subsequently transcribed as part of an adaptive immune system. Subsequently, viruses corresponding to the virus snippets in human genome would be used to infect human cell lines to obtain direct evidence of how virus snippets mediate an adaptive immune response at the cellular level. Specifically, successful defence of the cell by virus snippets triggering an adaptive immune response would manifest as viable cells compared to lysed cells unable to mount an immune response. Following demonstration of cell viability under viral challenge, in vitro biochemical assays using cell lysate would interrogate the specific proteins and enzymes that mediate possible cutting of the foreign DNA or RNA. To this end, beads immobilized with virus snippets would serve as bait for binding to complementary viral DNA or RNA as well as potential endogenous endonuclease protein. Following precipitation and recovery of beads, possible endonuclease that bind to both viral DNA or RNA and virus snippets immobilized on beads would be isolated through gel electrophoresis and subsequently purified. Purified endonuclease would be assayed for activity against a variety of nucleic acids (both DNA and RNA) from various sources with and without added virus snippets. This provides important information on substrate range and specificity of the potential endonuclease. Amino acid sequencing of the purified endonuclease would help downstream bioinformatic search for candidate protein in the human genome. Finally, cryo-electron microscopy could help determine the structure of the endonuclease in complex with viral nucleic acids and virus snippets. Such structural information would provide more insights into mechanistic details describing the binding and cleavage of viral DNA or RNA in a CRISPR-like adaptive immune response in human cells. Overall, tantalizing clues have emerged that a CRISPR-like adaptive immune response may exist in human cells for defending against viral attack. Combination of cell biological, biochemical and structural tools could lend insights into the potential endonuclease that mediate double strand break of foreign DNA or RNA using virus snippets transcribed from the human genome as guide RNA. If demonstrated to be true for a variety of human viruses across different cell lines, the newly discovered viral defence mechanism in human cells hold important implications for understanding the adoption and evolution of CRISPR in eukaryotic cells.

scholarly journals Attenuation of cGAS/STING activity during mitosis

2020 ◽  
Vol 3 (9) ◽  
pp. e201900636 ◽  
Author(s):  
Brittany L Uhlorn ◽  
Eduardo R Gamez ◽  
Shuaizhi Li ◽  
Samuel K Campos
Keyword(s):  
Immune System ◽  
Innate Immune System ◽  
Cellular Stress ◽  
Innate Immune ◽  
Regulatory Mechanisms ◽  
Mitotic Chromosomes ◽  
Interphase Cells ◽  
Microbial Infections ◽  
Foreign Dna ◽  
Sting Pathway

The innate immune system recognizes cytosolic DNA associated with microbial infections and cellular stress via the cGAS/STING pathway, leading to activation of phospho-IRF3 and downstream IFN-I and senescence responses. To prevent hyperactivation, cGAS/STING is presumed to be nonresponsive to chromosomal self-DNA during open mitosis, although specific regulatory mechanisms are lacking. Given a role for the Golgi in STING activation, we investigated the state of the cGAS/STING pathway in interphase cells with artificially vesiculated Golgi and in cells arrested in mitosis. We find that whereas cGAS activity is impaired through interaction with mitotic chromosomes, Golgi integrity has little effect on the enzyme’s production of cGAMP. In contrast, STING activation in response to either foreign DNA (cGAS-dependent) or exogenous cGAMP is impaired by a vesiculated Golgi. Overall, our data suggest a secondary means for cells to limit potentially harmful cGAS/STING responses during open mitosis via natural Golgi vesiculation.

CRISPR/Cas-Systems: characteristics and possibilities of use for editing bacterial genomes

Bacteriology ◽  
2020 ◽  
Vol 5 (2) ◽  
pp. 38-48
Author(s):  
I.A. Blatov ◽  
◽  
A.S. Shchurova ◽  
D.Yu. Guschin ◽  
S.D. Zvereva ◽  
...  
Keyword(s):  
Immune System ◽  
Genome Editing ◽  
Bacterial Species ◽  
Adaptive Immune System ◽  
Bacterial Genomes ◽  
Modern Biology ◽  
Adaptive Immune ◽  
History Of ◽  
Genomic Editing ◽  
Classification Structure

CRISPR-Cas is the adaptive immune system of bacteria and archaea. Since 2012, when the first opportunity to use the CRISPR/Cas system for genome editing was realized, the number of studies in this area has been growing rapidly. Today, genomic editing to modify specific regions of the genomes of various organisms is considered one of the key methodologies of modern biology. This review is devoted to the history of discovery, classification, structure, operational mechanisms of CRISPRCas systems and strategies for editing the genomes of various bacterial species using this technology. Key words: genome editing, genome, CRISPR-Cas system, bacteria

scholarly journals Efficient DNA-free genome editing of bread wheat using CRISPR/Cas9 ribonucleoprotein complexes

2017 ◽  
Vol 8 (1) ◽  
Author(s):  
Zhen Liang ◽  
Kunling Chen ◽  
Tingdong Li ◽  
Yi Zhang ◽  
Yanpeng Wang ◽  
...  
Keyword(s):  
Genome Editing ◽  
Bread Wheat ◽  
Deep Sequencing ◽  
Crop Plants ◽  
Good Prospect ◽  
Crop Breeding ◽  
Transgene Integration ◽  
Ribonucleoprotein Complexes ◽  
Wheat Embryos ◽  
Foreign Dna

Abstract Substantial efforts are being made to optimize the CRISPR/Cas9 system for precision crop breeding. The avoidance of transgene integration and reduction of off-target mutations are the most important targets for optimization. Here, we describe an efficient genome editing method for bread wheat using CRISPR/Cas9 ribonucleoproteins (RNPs). Starting from RNP preparation, the whole protocol takes only seven to nine weeks, with four to five independent mutants produced from 100 immature wheat embryos. Deep sequencing reveals that the chance of off-target mutations in wheat cells is much lower in RNP mediated genome editing than in editing with CRISPR/Cas9 DNA. Consistent with this finding, no off-target mutations are detected in the mutant plants. Because no foreign DNA is used in CRISPR/Cas9 RNP mediated genome editing, the mutants obtained are completely transgene free. This method may be widely applicable for producing genome edited crop plants and has a good prospect of being commercialized.

Sign in / Sign up

Export Citation Format

Share Document