This document provides an overview of CRISPR/Cas9 genome editing. It discusses the history and limitations of prior genome engineering techniques like recombinant DNA and zinc finger nucleases. It then explains how CRISPR/Cas9 works as a RNA-guided DNA endonuclease and how this allows it to efficiently and specifically edit genomes. The document outlines several applications of CRISPR/Cas9 like generating knockout animals and cell lines. It also notes some concerns about using the technique for human genome editing.
CRISPR (clustered regularly interspaced short palindromic repeats) is a family of DNA sequences found within the genomes of prokaryotic organisms such as bacteria and archaea. These sequences are derived from DNA fragments of bacteriophages that have previously infected the prokaryote and are used to detect and destroy DNA from similar phages during subsequent infections. Hence these sequences play a key role in the antiviral defense system of prokaryotes.
Cas9 (CRISPR-associated protein 9) is an enzyme that uses CRISPR sequences as a guide to recognize and cleave specific strands of DNA that are complementary to the CRISPR sequence. Cas9 enzymes together with CRISPR sequences form the basis of a technology known as CRISPR-Cas9 that can be used to edit genes within organisms.This editing process has a wide variety of applications including basic biological research, development of biotechnology products, and treatment of diseases.
The CRISPR-Cas system is a prokaryotic immune system that confers resistance to foreign genetic elements such as those present within plasmids and phages that provides a form of acquired immunity. RNA harboring the spacer sequence helps Cas (CRISPR-associated) proteins recognize and cut foreign pathogenic DNA. Other RNA-guided Cas proteins cut foreign RNA. CRISPR are found in approximately 50% of sequenced bacterial genomes and nearly 90% of sequenced archaea.
CRISPR-Cas9 is a genome editing tool that is creating a buzz in the science world. It is faster, cheaper and more accurate than previous techniques of editing DNA and has a wide range of potential applications.
i explained about basics of genome engineering and crispr system.
CRISPR will change the world and it is just the beginning, are you ready to meet the future? you think its great and beautiful or.....?
please give your feedback to my email
pooyanaghshbandi@yahoo.com
i am starting to write a critical and fantastic review article about CRISPR, if you are interested to join please contact me.
CRISPR (clustered regularly interspaced short palindromic repeats) is a family of DNA sequences found within the genomes of prokaryotic organisms such as bacteria and archaea. These sequences are derived from DNA fragments of bacteriophages that have previously infected the prokaryote and are used to detect and destroy DNA from similar phages during subsequent infections. Hence these sequences play a key role in the antiviral defense system of prokaryotes.
Cas9 (CRISPR-associated protein 9) is an enzyme that uses CRISPR sequences as a guide to recognize and cleave specific strands of DNA that are complementary to the CRISPR sequence. Cas9 enzymes together with CRISPR sequences form the basis of a technology known as CRISPR-Cas9 that can be used to edit genes within organisms.This editing process has a wide variety of applications including basic biological research, development of biotechnology products, and treatment of diseases.
The CRISPR-Cas system is a prokaryotic immune system that confers resistance to foreign genetic elements such as those present within plasmids and phages that provides a form of acquired immunity. RNA harboring the spacer sequence helps Cas (CRISPR-associated) proteins recognize and cut foreign pathogenic DNA. Other RNA-guided Cas proteins cut foreign RNA. CRISPR are found in approximately 50% of sequenced bacterial genomes and nearly 90% of sequenced archaea.
CRISPR-Cas9 is a genome editing tool that is creating a buzz in the science world. It is faster, cheaper and more accurate than previous techniques of editing DNA and has a wide range of potential applications.
i explained about basics of genome engineering and crispr system.
CRISPR will change the world and it is just the beginning, are you ready to meet the future? you think its great and beautiful or.....?
please give your feedback to my email
pooyanaghshbandi@yahoo.com
i am starting to write a critical and fantastic review article about CRISPR, if you are interested to join please contact me.
CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)Akshay Deshmukh
clustered regularly interspaced short palindromic repeats is a family of DNA sequences found in the genomes of prokaryotic organisms such as bacteria. Now CRISPR use as genome editing tool in different Plant Breeder to manipulate the DNA of the crop
A simple version of the CRISPR/Cas system, CRISPR/Cas9, has been modified to edit genomes. By delivering the Cas9 nuclease complexed with a synthetic guide RNA (gRNA) into a cell, the cell's genome can be cut at a desired location, allowing existing genes to be removed and/or new ones added.
Genome editing with the CRISPR-Cas9 system has become one of the major tools in modern biotechnology. This slide share discusses the fundamentals in a simple, easy to understand format.
Have you considered that protein over-expression or inefficient mRNA knockdown may be masking physiological effects in your assays? Increasingly scientists are moving to endogenous gene-editing to characterise the function of their genes of interest.
Dr Chris Thorne from Cambridge Biotech Horizon Discovery discusses the ground breaking gene-editing technology CRISPR. The simplicity of experimental design has led to rapid adoption of the technology across the scientific community. However, challenges remain.
This Slidedeck focuses specifically on implementing CRISPR experiments, and explore a number of key considerations crucial to maximising chances of targeting success, whether your goal is to generate a knock-out or a knock-in. Chris also takes a look at some of the alternative uses of CRISPR, including sgRNA genome wide synthetic lethality screens.
The slides aim to support those researchers either planning to or already using CRISPR gene-editing in their lab. Horizon Discovery have also recently launched a program aimed specifically at academic cell biologists to promote the adoption of CRISPR by offering FREE CRISPR Reagents for knock-out cell line generation - more information available here. http://www.horizondiscovery.com/what-we-do/discovery-toolbox/genassist-crispr--raav-genome-editing-tools
Crispr-Cas9 system works on the concept of bacterial defence mechanism. The idea of which was replicated in eukaryotic cell in in- vitro condition by the researchers.
a brief description on the new emerging genome editing technology CRISPR-Cas9. this technique is making its place stronger and stronger day by day. and impossible things can be possible by this technique. and some main and famous names who discovered this technique.
The CRISPR (clustered regularly interspaced short palindromic repeats)–Cas9 (CRISPR-associated nuclease 9), a genome editing system adapted from the bacterial immune mechanism that is poised to transform genetic engineering by providing a simple, efficient and economical method to precisely manipulate the genome of any organism. Compared with zinc finger nucleases (ZFN) and transcription activator-like effector nucleases (TALEN), CRISPR/Cas9 is simpler with higher specificity and less toxicity. This RNA-guided nuclease (RGN)-based approach has been effectively used to induce targeted mutations(knock in or knock out) in multiple genes simultaneously, create conditional alleles, and generate endogenously tagged proteins.It has a wide variety of applications such as gene therapy, gene expression regulation, genome wide functional screening, virus resistance, transgenic animal production, site specific DNA integration etc. In the future CRISPR/Cas9 technology will play a significant role in innovating the life science research and industrial fields.
CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)Akshay Deshmukh
clustered regularly interspaced short palindromic repeats is a family of DNA sequences found in the genomes of prokaryotic organisms such as bacteria. Now CRISPR use as genome editing tool in different Plant Breeder to manipulate the DNA of the crop
A simple version of the CRISPR/Cas system, CRISPR/Cas9, has been modified to edit genomes. By delivering the Cas9 nuclease complexed with a synthetic guide RNA (gRNA) into a cell, the cell's genome can be cut at a desired location, allowing existing genes to be removed and/or new ones added.
Genome editing with the CRISPR-Cas9 system has become one of the major tools in modern biotechnology. This slide share discusses the fundamentals in a simple, easy to understand format.
Have you considered that protein over-expression or inefficient mRNA knockdown may be masking physiological effects in your assays? Increasingly scientists are moving to endogenous gene-editing to characterise the function of their genes of interest.
Dr Chris Thorne from Cambridge Biotech Horizon Discovery discusses the ground breaking gene-editing technology CRISPR. The simplicity of experimental design has led to rapid adoption of the technology across the scientific community. However, challenges remain.
This Slidedeck focuses specifically on implementing CRISPR experiments, and explore a number of key considerations crucial to maximising chances of targeting success, whether your goal is to generate a knock-out or a knock-in. Chris also takes a look at some of the alternative uses of CRISPR, including sgRNA genome wide synthetic lethality screens.
The slides aim to support those researchers either planning to or already using CRISPR gene-editing in their lab. Horizon Discovery have also recently launched a program aimed specifically at academic cell biologists to promote the adoption of CRISPR by offering FREE CRISPR Reagents for knock-out cell line generation - more information available here. http://www.horizondiscovery.com/what-we-do/discovery-toolbox/genassist-crispr--raav-genome-editing-tools
Crispr-Cas9 system works on the concept of bacterial defence mechanism. The idea of which was replicated in eukaryotic cell in in- vitro condition by the researchers.
a brief description on the new emerging genome editing technology CRISPR-Cas9. this technique is making its place stronger and stronger day by day. and impossible things can be possible by this technique. and some main and famous names who discovered this technique.
The CRISPR (clustered regularly interspaced short palindromic repeats)–Cas9 (CRISPR-associated nuclease 9), a genome editing system adapted from the bacterial immune mechanism that is poised to transform genetic engineering by providing a simple, efficient and economical method to precisely manipulate the genome of any organism. Compared with zinc finger nucleases (ZFN) and transcription activator-like effector nucleases (TALEN), CRISPR/Cas9 is simpler with higher specificity and less toxicity. This RNA-guided nuclease (RGN)-based approach has been effectively used to induce targeted mutations(knock in or knock out) in multiple genes simultaneously, create conditional alleles, and generate endogenously tagged proteins.It has a wide variety of applications such as gene therapy, gene expression regulation, genome wide functional screening, virus resistance, transgenic animal production, site specific DNA integration etc. In the future CRISPR/Cas9 technology will play a significant role in innovating the life science research and industrial fields.
An Introduction to Crispr Genome EditingChris Thorne
In this short presentation, I make a case for doing genome editing vs some of the approaches that have gone before, describe some of the tools available, and the focus on CRISPR-Cas9, what it is, where it's come from and how it works.
This presentation highlights the basics and application of genome editing strategies in plants, strategies to reduce off-target mutation, identification of mutant analysis etc.
ONLY THE LAST QUESTION IS THE POINT OF POST. THE OTHER PAGES ARE B.pdfamzonknr
ONLY THE LAST QUESTION IS THE POINT OF POST. THE OTHER PAGES ARE
BACKGROUND CONTEXT Lab: Differential Expression Differential gene expression provides
the ability for a cell or organism to respond to a constantly changing external environment. The
specific constellation of proteins required for optimal function and growth varies with cellular
age and environmental context. Thus, protein production is carefully regulated by multiple
mechanisms that modulate both transcriptional and translational pathways. Control of
transcription initiation by RNA polymerase is a predominant mechanism for regulating
expression of specific proteins, presumably because it provides maximal conservation of energy
for the cell. We can often observe the consequence of differential transcription due to the
presence or absence of particular proteins or the growth in particular environments. Control can
also occur at translation; the mRNA is synthesized, but only in certain circumstances is it
translated. Control can also occur at the level of protein function; the protein is inactive, or
activity is not observed due to the lack of the substrate. In this lab we will observe differential
expression of two different genes encoded on plasmids. We will analyze transcriptional activity,
translational activity, and protein function. Plasmids are extra-chromosomal DNA. Bacteria often
have plasmids and will replicate the plasmid and pass it to daughter cells (vertical transmission)
and to neighboring cells (horizontal). Plasmids are a mechanism of gene diversity. In order to
stably retain the plasmid, there needs to be some type of metabolic reason for the bacteria to
maintain the plasmid. In other words, the plasmid confers an advantage. Plasmids contain: 1. Ori:
the plasmid may present is low or high copy number. 2. Lab generated plasmids typically also
contain a selectable marker (antibiotic resistance), 3. Additional gene for ease of visual screening
4. Multiple cloning site
pUC19 is one of a series of plasmid cloning vectors created by Joachim Messing and co-workers.
The designation "pUC" is derived from the classical "p" prefix (denoting "plasmid") and the
abbreviation for the University of California, where early work on the plasmid series had been
conducted. It is a circular double stranded DNA and has 2686 base pairs. pUC19 is one of the
most widely used vector molecules as the recombinants, or the cells into which foreign DNA has
been introduced, can be easily distinguished from the non-recombinants based on color
differences of colonies on growth media. pUC18 is similar to pUC19, but the MCS region is
reversed. - pUC 19 has an origin of replication and is maintained at a high copy number. -
pUC19 encodes for an ampicillin resistance gene (amopR), via a -lactamase enzyme that
functions by degrading ampicillin and reducing its toxicity to the host. - It has an N-terminal
fragment of -galactosidase (lacZ) gene of E. coli which allows for visual screening of
recombinant.
ONLY THE LAST QUESTION IS THE POINT OF POST. THE OTHER PAGES ARE BAC.pdfamzonknr
ONLY THE LAST QUESTION IS THE POINT OF POST. THE OTHER PAGES ARE
BACKGROUND CONTEXT Lab: Differential Expression Differential gene expression provides
the ability for a cell or organism to respond to a constantly changing external environment. The
specific constellation of proteins required for optimal function and growth varies with cellular
age and environmental context. Thus, protein production is carefully regulated by multiple
mechanisms that modulate both transcriptional and translational pathways. Control of
transcription initiation by RNA polymerase is a predominant mechanism for regulating
expression of specific proteins, presumably because it provides maximal conservation of energy
for the cell. We can often observe the consequence of differential transcription due to the
presence or absence of particular proteins or the growth in particular environments. Control can
also occur at translation; the mRNA is synthesized, but only in certain circumstances is it
translated. Control can also occur at the level of protein function; the protein is inactive, or
activity is not observed due to the lack of the substrate. In this lab we will observe differential
expression of two different genes encoded on plasmids. We will analyze transcriptional activity,
translational activity, and protein function. Plasmids are extra-chromosomal DNA. Bacteria often
have plasmids and will replicate the plasmid and pass it to daughter cells (vertical transmission)
and to neighboring cells (horizontal). Plasmids are a mechanism of gene diversity. In order to
stably retain the plasmid, there needs to be some type of metabolic reason for the bacteria to
maintain the plasmid. In other words, the plasmid confers an advantage. Plasmids contain: 1. Ori:
the plasmid may present is low or high copy number. 2. Lab generated plasmids typically also
contain a selectable marker (antibiotic resistance), 3. Additional gene for ease of visual screening
4. Multiple cloning site
pUC19 is one of a series of plasmid cloning vectors created by Joachim Messing and co-workers.
The designation "pUC" is derived from the classical "p" prefix (denoting "plasmid") and the
abbreviation for the University of California, where early work on the plasmid series had been
conducted. It is a circular double stranded DNA and has 2686 base pairs. pUC19 is one of the
most widely used vector molecules as the recombinants, or the cells into which foreign DNA has
been introduced, can be easily distinguished from the non-recombinants based on color
differences of colonies on growth media. pUC18 is similar to pUC19, but the MCS region is
reversed. - pUC 19 has an origin of replication and is maintained at a high copy number. -
pUC19 encodes for an ampicillin resistance gene (amopR), via a -lactamase enzyme that
functions by degrading ampicillin and reducing its toxicity to the host. - It has an N-terminal
fragment of -galactosidase (lacZ) gene of E. coli which allows for visual screening of
recombinant.
A concise and well fabricated presentation the current techniques used for plant genome editing including CRISPER/cas9 system, TALENS, TELES, ZINC FINGER NUCLEASES(ZFN), HEJ (homologous endjoing) and many other high throughout techniques along references.
Making genome edits in mammalian cellsChris Thorne
Looking at the kind of modifications that can be made in mammalian cells, and how at Horizon moving to a haploid model system has significantly improved efficiency of both editing and validation
Genome editing tools form the basis for personalized medicine, especially for therapies requiring change in genome. Currently there are four contenders to this – Meganucleases, ZNF Nucleases, TALENs and CRISPRs. Although, the technologies are many, there are very few commercial providers of this technology. This is attributed to the fact that select few possess the intellectual property rights of turning these technologies to valid form of therapy; for example, ZFN patent with Sangamo BioSciences and TALENs with Cellectis, Transposagen and Life Technologies.
Lab: Differential Expression Differential gene expression provides the ability for a cell or
organism to respond to a constantly changing external environment. The specific constellation of
proteins required for optimal function and growth varies with cellular age and environmental
context. Thus, protein production is carefully regulated by multiple mechanisms that modulate
both transcriptional and translational pathways. Control of transcription initiation by RNA
polymerase is a predominant mechanism for regulating expression of specific proteins,
presumably because it provides maximal conservation of energy for the cell. We can often
observe the consequence of differential transcription due to the presence or absence of particular
proteins or the growth in particular environments. Control can also occur at translation; the
mRNA is synthesized, but only in certain circumstances is it translated. Control can also occur at
the level of protein function; the protein is inactive, or activity is not observed due to the lack of
the substrate. In this lab we will observe differential expression of two different genes encoded
on plasmids. We will analyze transcriptional activity, translational activity, and protein function.
Plasmids are extra-chromosomal DNA. Bacteria often have plasmids and will replicate the
plasmid and pass it to daughter cells (vertical transmission) and to neighboring cells (horizontal).
Plasmids are a mechanism of gene diversity. In order to stably retain the plasmid, there needs to
be some type of metabolic reason for the bacteria to maintain the plasmid. In other words, the
plasmid confers an advantage. Plasmids contain: 1. Ori: the plasmid may present is low or high
copy number. 2. Lab generated plasmids typically also contain a selectable marker (antibiotic
resistance), 3. Additional gene for ease of visual screening 4. Multiple cloning site
pUC19 is one of a series of plasmid cloning vectors created by Joachim Messing and co-workers.
The designation "pUC" is derived from the classical "p" prefix (denoting "plasmid") and the
abbreviation for the University of California, where early work on the plasmid series had been
conducted. It is a circular double stranded DNA and has 2686 base pairs. pUC19 is one of the
most widely used vector molecules as the recombinants, or the cells into which foreign DNA has
been introduced, can be easily distinguished from the non-recombinants based on color
differences of colonies on growth media. pUC18 is similar to pUC19, but the MCS region is
reversed. - pUC 19 has an origin of replication and is maintained at a high copy number. -
pUC19 encodes for an ampicillin resistance gene (amopR), via a -lactamase enzyme that
functions by degrading ampicillin and reducing its toxicity to the host. - It has an N-terminal
fragment of -galactosidase (lacZ) gene of E. coli which allows for visual screening of
recombinant plasmids. The transformed cells containing the plasmid with the gene of interest ca.
Lab: Differential Expression Differential gene expression provides the ability for a cell or
organism to respond to a constantly changing external environment. The specific constellation of
proteins required for optimal function and growth varies with cellular age and environmental
context. Thus, protein production is carefully regulated by multiple mechanisms that modulate
both transcriptional and translational pathways. Control of transcription initiation by RNA
polymerase is a predominant mechanism for regulating expression of specific proteins,
presumably because it provides maximal conservation of energy for the cell. We can often
observe the consequence of differential transcription due to the presence or absence of particular
proteins or the growth in particular environments. Control can also occur at translation; the
mRNA is synthesized, but only in certain circumstances is it translated. Control can also occur at
the level of protein function; the protein is inactive, or activity is not observed due to the lack of
the substrate. In this lab we will observe differential expression of two different genes encoded
on plasmids. We will analyze transcriptional activity, translational activity, and protein function.
Plasmids are extra-chromosomal DNA. Bacteria often have plasmids and will replicate the
plasmid and pass it to daughter cells (vertical transmission) and to neighboring cells (horizontal).
Plasmids are a mechanism of gene diversity. In order to stably retain the plasmid, there needs to
be some type of metabolic reason for the bacteria to maintain the plasmid. In other words, the
plasmid confers an advantage. Plasmids contain: 1. Ori: the plasmid may present is low or high
copy number. 2. Lab generated plasmids typically also contain a selectable marker (antibiotic
resistance), 3. Additional gene for ease of visual screening 4. Multiple cloning site
pUC19 is one of a series of plasmid cloning vectors created by Joachim Messing and co-workers.
The designation "pUC" is derived from the classical "p" prefix (denoting "plasmid") and the
abbreviation for the University of California, where early work on the plasmid series had been
conducted. It is a circular double stranded DNA and has 2686 base pairs. pUC19 is one of the
most widely used vector molecules as the recombinants, or the cells into which foreign DNA has
been introduced, can be easily distinguished from the non-recombinants based on color
differences of colonies on growth media. pUC18 is similar to pUC19, but the MCS region is
reversed. - pUC 19 has an origin of replication and is maintained at a high copy number. -
pUC19 encodes for an ampicillin resistance gene (amopR), via a -lactamase enzyme that
functions by degrading ampicillin and reducing its toxicity to the host. - It has an N-terminal
fragment of -galactosidase (lacZ) gene of E. coli which allows for visual screening of
recombinant plasmids. The transformed cells containing the plasmid with the gene of interest ca.
Recent advances in CRISPR-CAS9 technology: an alternative to transgenic breedingJyoti Prakash Sahoo
These are the part of the Bacterial immune system which detects and recognize the foreign DNA and cleaves it.
THE CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) loci
Cas (CRISPR- associated) proteins can target and cleave invading DNA in a sequence – specific manner.
CRISPR array is composed of a series of repeats interspaced by spacer sequences acquired from invading genomes.
Slide 1: Title Slide
Extrachromosomal Inheritance
Slide 2: Introduction to Extrachromosomal Inheritance
Definition: Extrachromosomal inheritance refers to the transmission of genetic material that is not found within the nucleus.
Key Components: Involves genes located in mitochondria, chloroplasts, and plasmids.
Slide 3: Mitochondrial Inheritance
Mitochondria: Organelles responsible for energy production.
Mitochondrial DNA (mtDNA): Circular DNA molecule found in mitochondria.
Inheritance Pattern: Maternally inherited, meaning it is passed from mothers to all their offspring.
Diseases: Examples include Leber’s hereditary optic neuropathy (LHON) and mitochondrial myopathy.
Slide 4: Chloroplast Inheritance
Chloroplasts: Organelles responsible for photosynthesis in plants.
Chloroplast DNA (cpDNA): Circular DNA molecule found in chloroplasts.
Inheritance Pattern: Often maternally inherited in most plants, but can vary in some species.
Examples: Variegation in plants, where leaf color patterns are determined by chloroplast DNA.
Slide 5: Plasmid Inheritance
Plasmids: Small, circular DNA molecules found in bacteria and some eukaryotes.
Features: Can carry antibiotic resistance genes and can be transferred between cells through processes like conjugation.
Significance: Important in biotechnology for gene cloning and genetic engineering.
Slide 6: Mechanisms of Extrachromosomal Inheritance
Non-Mendelian Patterns: Do not follow Mendel’s laws of inheritance.
Cytoplasmic Segregation: During cell division, organelles like mitochondria and chloroplasts are randomly distributed to daughter cells.
Heteroplasmy: Presence of more than one type of organellar genome within a cell, leading to variation in expression.
Slide 7: Examples of Extrachromosomal Inheritance
Four O’clock Plant (Mirabilis jalapa): Shows variegated leaves due to different cpDNA in leaf cells.
Petite Mutants in Yeast: Result from mutations in mitochondrial DNA affecting respiration.
Slide 8: Importance of Extrachromosomal Inheritance
Evolution: Provides insight into the evolution of eukaryotic cells.
Medicine: Understanding mitochondrial inheritance helps in diagnosing and treating mitochondrial diseases.
Agriculture: Chloroplast inheritance can be used in plant breeding and genetic modification.
Slide 9: Recent Research and Advances
Gene Editing: Techniques like CRISPR-Cas9 are being used to edit mitochondrial and chloroplast DNA.
Therapies: Development of mitochondrial replacement therapy (MRT) for preventing mitochondrial diseases.
Slide 10: Conclusion
Summary: Extrachromosomal inheritance involves the transmission of genetic material outside the nucleus and plays a crucial role in genetics, medicine, and biotechnology.
Future Directions: Continued research and technological advancements hold promise for new treatments and applications.
Slide 11: Questions and Discussion
Invite Audience: Open the floor for any questions or further discussion on the topic.
Multi-source connectivity as the driver of solar wind variability in the heli...Sérgio Sacani
The ambient solar wind that flls the heliosphere originates from multiple
sources in the solar corona and is highly structured. It is often described
as high-speed, relatively homogeneous, plasma streams from coronal
holes and slow-speed, highly variable, streams whose source regions are
under debate. A key goal of ESA/NASA’s Solar Orbiter mission is to identify
solar wind sources and understand what drives the complexity seen in the
heliosphere. By combining magnetic feld modelling and spectroscopic
techniques with high-resolution observations and measurements, we show
that the solar wind variability detected in situ by Solar Orbiter in March
2022 is driven by spatio-temporal changes in the magnetic connectivity to
multiple sources in the solar atmosphere. The magnetic feld footpoints
connected to the spacecraft moved from the boundaries of a coronal hole
to one active region (12961) and then across to another region (12957). This
is refected in the in situ measurements, which show the transition from fast
to highly Alfvénic then to slow solar wind that is disrupted by the arrival of
a coronal mass ejection. Our results describe solar wind variability at 0.5 au
but are applicable to near-Earth observatories.
Cancer cell metabolism: special Reference to Lactate PathwayAADYARAJPANDEY1
Normal Cell Metabolism:
Cellular respiration describes the series of steps that cells use to break down sugar and other chemicals to get the energy we need to function.
Energy is stored in the bonds of glucose and when glucose is broken down, much of that energy is released.
Cell utilize energy in the form of ATP.
The first step of respiration is called glycolysis. In a series of steps, glycolysis breaks glucose into two smaller molecules - a chemical called pyruvate. A small amount of ATP is formed during this process.
Most healthy cells continue the breakdown in a second process, called the Kreb's cycle. The Kreb's cycle allows cells to “burn” the pyruvates made in glycolysis to get more ATP.
The last step in the breakdown of glucose is called oxidative phosphorylation (Ox-Phos).
It takes place in specialized cell structures called mitochondria. This process produces a large amount of ATP. Importantly, cells need oxygen to complete oxidative phosphorylation.
If a cell completes only glycolysis, only 2 molecules of ATP are made per glucose. However, if the cell completes the entire respiration process (glycolysis - Kreb's - oxidative phosphorylation), about 36 molecules of ATP are created, giving it much more energy to use.
IN CANCER CELL:
Unlike healthy cells that "burn" the entire molecule of sugar to capture a large amount of energy as ATP, cancer cells are wasteful.
Cancer cells only partially break down sugar molecules. They overuse the first step of respiration, glycolysis. They frequently do not complete the second step, oxidative phosphorylation.
This results in only 2 molecules of ATP per each glucose molecule instead of the 36 or so ATPs healthy cells gain. As a result, cancer cells need to use a lot more sugar molecules to get enough energy to survive.
Unlike healthy cells that "burn" the entire molecule of sugar to capture a large amount of energy as ATP, cancer cells are wasteful.
Cancer cells only partially break down sugar molecules. They overuse the first step of respiration, glycolysis. They frequently do not complete the second step, oxidative phosphorylation.
This results in only 2 molecules of ATP per each glucose molecule instead of the 36 or so ATPs healthy cells gain. As a result, cancer cells need to use a lot more sugar molecules to get enough energy to survive.
introduction to WARBERG PHENOMENA:
WARBURG EFFECT Usually, cancer cells are highly glycolytic (glucose addiction) and take up more glucose than do normal cells from outside.
Otto Heinrich Warburg (; 8 October 1883 – 1 August 1970) In 1931 was awarded the Nobel Prize in Physiology for his "discovery of the nature and mode of action of the respiratory enzyme.
WARNBURG EFFECT : cancer cells under aerobic (well-oxygenated) conditions to metabolize glucose to lactate (aerobic glycolysis) is known as the Warburg effect. Warburg made the observation that tumor slices consume glucose and secrete lactate at a higher rate than normal tissues.
(May 29th, 2024) Advancements in Intravital Microscopy- Insights for Preclini...Scintica Instrumentation
Intravital microscopy (IVM) is a powerful tool utilized to study cellular behavior over time and space in vivo. Much of our understanding of cell biology has been accomplished using various in vitro and ex vivo methods; however, these studies do not necessarily reflect the natural dynamics of biological processes. Unlike traditional cell culture or fixed tissue imaging, IVM allows for the ultra-fast high-resolution imaging of cellular processes over time and space and were studied in its natural environment. Real-time visualization of biological processes in the context of an intact organism helps maintain physiological relevance and provide insights into the progression of disease, response to treatments or developmental processes.
In this webinar we give an overview of advanced applications of the IVM system in preclinical research. IVIM technology is a provider of all-in-one intravital microscopy systems and solutions optimized for in vivo imaging of live animal models at sub-micron resolution. The system’s unique features and user-friendly software enables researchers to probe fast dynamic biological processes such as immune cell tracking, cell-cell interaction as well as vascularization and tumor metastasis with exceptional detail. This webinar will also give an overview of IVM being utilized in drug development, offering a view into the intricate interaction between drugs/nanoparticles and tissues in vivo and allows for the evaluation of therapeutic intervention in a variety of tissues and organs. This interdisciplinary collaboration continues to drive the advancements of novel therapeutic strategies.
THE IMPORTANCE OF MARTIAN ATMOSPHERE SAMPLE RETURN.Sérgio Sacani
The return of a sample of near-surface atmosphere from Mars would facilitate answers to several first-order science questions surrounding the formation and evolution of the planet. One of the important aspects of terrestrial planet formation in general is the role that primary atmospheres played in influencing the chemistry and structure of the planets and their antecedents. Studies of the martian atmosphere can be used to investigate the role of a primary atmosphere in its history. Atmosphere samples would also inform our understanding of the near-surface chemistry of the planet, and ultimately the prospects for life. High-precision isotopic analyses of constituent gases are needed to address these questions, requiring that the analyses are made on returned samples rather than in situ.
2. Contents
• History of Genome Engineering
• CRISPR/Cas9
• Applications
• Current Limitations and Future prospects
3. Recombinant DNA Technology :
aka “Genetic Engineering”
- Plasmid Vector
- Restriction Endonuclease
- DNA Ligase
Foundation of Modern Molecular Biology & Biotechnology
Paul Berg Herb Boyer Stanley Cohen
- PCR
- Sanger Sequencing Kary Mullis
Fred Sanger
- Transgenic Animal/Plants
Rudolph Jaenisch
4. • Size of DNA can manipulates in vitro :
~ Max 150kb.
More practically, less than 20kb
• Recombinant DNA can manipulated is mostly
episomal DNAs
• Random Integration of foreign DNA
Major Limitations of „Genetic
Engineering v1.0‟
5. Restriction Endonuclease
• Typical restriction endonuclease can recognize 6-8bp
• RE with 6bp will cut, on average, every 46 or 4096bp, while
8bp cutter will recognize 48, or 65536bp
• Therefore conventional RE is not suitable for genome level
manipulation.
• Human Genome : 3 billion bp.
• For specific cleavage of human genome, at least specific
recognition of more than 18bp would be required.
7. - Yeast
- E.coli (Lamda Red Recombinase System)
- Mouse Embryonic Stem Cell (Knockout/KnockIn mouse)
- Limitations
• Feasible in only a few model organisms (ES Cell)
• Time consuming
• Efficiencies
Homologous Recombination
8. ZFN & TALEN
Artificial restriction enzyme consist of
DNA recognition (Zinc Finger or TALE)
Cleavage Domain (FokI Nuclease)
Repeated Protein Modules (Zinc Figer or TALE) recognize
DNA bases
Dimerization of FokI nuclease domain induce cleavages of
target DNA
recognize long
stretches of bases suitable for genome-level cleavages
9. Left ZFN
9 nt target
Right ZFN
9 nt target
Cleavage by
Dimerization
10. • To recognize new target sequence, you
should develop new zinc-finger DNA binding
domain
- Modular assembly from previously generated
array
- Selection using Phage Display/One Hybrid
• Time consuming for the proper ZFP sets
• Failure rate is very high
• Off-target effects are very high
11. TALEN
Transcription activator-like effector nuclease
TAL effector : secreted protein by plant pathogenm
Xanthomonas sp.
Type III effector proteins which activate plant gene expression
Repeated highly conserved 33-34 amino acid sequences
(Except 33-34 amino acids)
14. Nonhomologus end joining (NHEJ)
- Natural pathway to repair double-strand break of DNA
- ZFN or TALEN induces double-stranded break of DNA then
NHEJ joins broken ends, although its repair ability can be limited.
ZF or TALE
ZF or TALE FokI
FokI
DSB
NHEJ
Indel Cause Frameshift -> knockout
15. Homology Directed Repair (HDR)
ZF or TALE
ZF or TALE FokI
FokI
DSB
Donor Template
(Mutation, Insertion..)
HDR
ssDNA Oligo or Plasmid
Precise Repair (Targeted Gene Integration, Site-specific Mutagenesis)
17. Humble Beginning as Exotic Repeat
Sequences in Bacterial Genome
- Found as „exotic junk DNA‟ with unknown function
Ishino et al., J.Bacteriol (1987)
- Widespread presence in Archeae and Bacteria
Jansen et al, Mol. Microbiol (2002)
- Named as..
lustered egularly nterspaced hort alindromic epeat
RISPR sociated protein (Cas)
Family of genes associated with CRISPR
- Sequence similarity between phage
18. CRISPR as bacterial immune system
against bacteriophagy
The research was carried at by researcher in DANISCO.Inc
(acquired by DuPont at 2011)
Science 2007
19.
20. Practical questions in Yogurt Fermentation
industry
- Phage contamination :
Most serious problem in fermentation industries
- Phage-resistant strains would emerged after phage
pandemics
- Hypothesis
36. One-Step Generation of Knock Out / Knock-In Mouse
Traditional Knock Out/In
Mouse Generations using ES Cell
Targeting Vector Construction/
ES Cell Knockout and selection
3 Months
Injection of ES Cell into Blastocyst
Generation Chimeric Mouse
2 Months
At least 6-12 Months is required to
generate Founder Mice
CRISPR/Cas9 Systems
Design and Generation of sgRNA andCc
Less than a week
(1 day except oligo synthesis)
Injection in Zygote
And Transfer to surrogates Mother
1 weeks
Germline transmission and backgross
Selection of Founder
~ 4 Month
(If you are lucky…)
Founder Mouse
Less than 3 weeks
Multiple gene : individual crossing…
37. 80-90% of Mouse has mutated alleles
60-70% of Mouse has Double Knocked when two sgRNAs are introduced
38. Knock-in Generations
Generation of floxed mouse in single step
• Injections of
cas9+sgRNA+ssODN(Single-
strand oligo donor nucleotide)
• Homology Dependent Repair
40. Advantage of CRISPR/Cas9 over TALEN or ZFN (1)
TALEN or ZFN
Artificial protein gene recognizing the target sequences are required
X 2
Synthesis of TALE gene is not trivial due to repeated nature of TALE
45. In CRISPR/Cas9 system…
All you need to synthesize this part
Cas9 is common protein component
regardless the nature of recognition site
- Very affordable
- Fast
- High-throughput friendly
46. Advantage of CRISPR/Cas9 over TALEN or ZFN (2)
- TALEN or ZFN : Artificial Restriciton Enzyme consisted with..
DNA binding domain + Nonspecific DNA cleavage domains
Dimerization of FokI cleavage domain is essential for DNA cleavages
If binding affinity of one of ZFN/TALEN pair is less than other, cleavage efficiency is lo
- Not as optimal compared with bona-fide endonuclease?
47. Cas9 is bona fide RNA-dependent DNA endonuclease by itself
- Higher catalytic efficiency
- Evolved to cleave Phage DNA after injection ASAP.
51. Case Studies
Buzzword about Cas9 became really loud, so we decided to join CRISPR bandwagon…
http://www.addgene.org
52. In January 2014, we got cas9 constructs from addgene..
$65 per clone
53. In vitro transcriptions of Cas9
Design and Generation of sgRNAs
- Order two DNA oligos..
-Annealing and amplification using PCR
-In vitro transcription using T7 RNA Polymerase
For the preparations of all of material, it tooks 2-3 Days..
54. Exon1OCT-4 Exon2 Exon3 Exon4 Exon5
TCCTAAAGCAGAAGAGGATCACCCTGGGATATAC
Knockout of Porcine Oct4
Injections in Porcine Zygotes (Parthernotes)
J.W. Kwon
56. DNA Oct4 Merge
Cas9 (100ng)
Cas9/
sgRNA
(10ng/ul)
Cas9/
sgRNA
(100ng/ul)
Immunostaining of Oct4 in Cas9/sgRNA
Knockdown of l Oct4 in porcine blastocyst
57. Application of CRISPR/Cas9
• Knockout/Knock-in Animal Generation
• Gene Knockout in Cultured Cell Line
• Gene Activation / Repression by dCas9
• Therapeutic Application?
• Others..
58. Generation of Animal Model in Lighting Speed
- Knockout/Knock-in generation Mouse :
at least 6~12 months
- Using CRISPR/Cas9..
You can get a founder in 2 Months with ~90% of efficiency
- Introduction of Disease Model Mutations
Variants discovered from GWAS / WGS projects
Validation in animal model would be possible
59. Knockout/KnockIn in „Other‟ Animals
- Knockout/Knock-in generation Mouse :
Established procedures even before ZFN/TALEN/CRISPR
- But in other animal?
Lack of embryonic stem cell and suitable genome level
targeting technology
Even in Rat, embryonic stem cell was
- Targeted genetic modification in domestic animal
60. With Little Helps from CRISPR/Cas9..
Rat August 2013Zebrafish January 2013
Xenopus
October 2013
Pig
January 2014
Rabbit January 2014
Rice fish
April 2014
Silkworm December 2013
Drosophila
September 2013
61. Virtually genomes of all living organisms can
be modified by CRISPR/Cas9
as “Programmable DNA endonculease”
AnimalPlant
Fungi
Bacteria
Mouse
Rat
Xenopus
Drosophila
Pig
Zebrafish
Rabbit
Goat
Arabidopsis
Rice
Tobacco
Wheat
Orange
66. Sus scorfa : Important model organism for
Xenotransplantation
Knockout of immune responsive related genes is
necessary
Alternative Source of Human Organs :
Xenotransplantation?
- porcine α1,3-galactosyltransferase (GGTA1)
- CMP-Neu5Ac hydroxylase
Expression of various human immune organizer in Pigs
67. Primary fetal fibroblast Genetic Modification Nuclear Transfer
Slow
Inefficient
Transgenesis
Gene targeting by
Homologous recombination/
AAV vector
ZFN/TALEN
(i.e. Cloning)
Low efficiency
Laborious
Abnormal development
Transfer Nuclues of
Genetically Modified cell
to Unfertillized /
enuclated oocyte
Traditional Way of Genetic Modifications in Pig
72. dCas9-mediated Endogenous Gene Activations
Cell Res. 2013
Double Mutant of Cas9
Inactive for cleavage
Tandem Transactivation Domain
Position of sgRNA
74. Therapeutic Potential of CRISPR/Cas9
CCR5 HIV receptor targeting by ZFN
http://www.sangamo.com/pipeline/sb-728.html
75. Editas genomics was found late 2013
Zhang + Church + Doudna
http://www.editas.com
76. Mutation Corrections Cataract (백내장) in Model Organism
Wu et al., Cell Stem Cell, 2013
Repair of
Dominant Negative
Heterozygote
Using WT allele
Repair of
Dominant Negative
Heterozygote
Using oligonucleotide
77. Functional Repair of CTFR by CRISPR/Cas9 in Intestinal
Stem Cell Organoids of Cystic Fibrosis Patients
Schwank et al., Cell Stem Cell 2013
Delta F508 : Most common CTFR mutation : resulting abnormal channel proteins
78. Genome Editing in Adult Mouse
- Mouse model of hereditary tyrosinemia type I
- Caused by mutation on fumarylacetoacetate hydrolase (Exon skipping)
79.
80. Correction of Mutations in Zygote stages of Human?
We have more knowledge and techniques on Human Embryo than Monkey‟s
81. Assisted Reproduction Technology is common
In 2012, 176,275 ART Cycle (In vitro fertillization) were performed and 65,179
live born infants
Over 1% of all infants born in the United States are conceived using ART
ICSI (intracytoplasmic sperm injection) was involved in 30-40% of cases
86. 1-Cell Embryo
(Zygote)
sgRNAs
Cas9
Donor DNA
Injection
3 Days
PGD-NGS Genotyping
(Fast turnaround
required)
8-Cell Embryo
Blastocyst with
Desired Modification
Without off-site mutaton
Blastocyst witout
Modification or
With off-site
mutaton
Embryo Transfer
Or
Storage in liquid N2
Potential Workflow for ‘GMO’ human?
5 Days
87. Ethical Concerns
- Regulations
- Safety
- Ethical concerns (GMO Human?)
- 생명윤리및 안전에 관한 법률
Mad Scientist aka “Frankenstein builder”
No relation with http://madscientist.wordpress.com
88. BGI invested significant resources on PGD screening
http://www.genomics.cn/en/navigation/show_navigation?nid=5687
89. They are also trying to sequence a million people‟s genome
For what?
90.
91. “Rising of „designer babies‟ industry?”
“성형외과 지고 성형산부인과 뜬다?”
Welcome To the Brave New World.
“Designer Baby” Patent issued to 23andMe.com
US. 8,620,594 B2
92. Current Pitfall of CRISPR/Cas9
- Off-target effects
-Cas9 recognition is mainly rely on ~15bp upstream of PAM
93. Although Off-target effect and toxicity of CRISPR is much lower than those of ZFN..
Fuji et al., NAR 2013
94. How to avoid off-target effects?
- Optimization of Injection conditions (less cas9/sgRNA)
- Bioinformatics : Find a sgRNA target for less off-targets
“CRISPR Design” (http://crispr.mit.edu)
95. Double-Nicking System
- Using Cas9 Nickase (Can cleave only single strand of DNA)
Ran et al., Cell 2013
- Reduces off-target mutagnesis
by 50-1,000 fold
- Efficient indel / HDR as similar
with wt Cas9
- More restriction in cleavage
site
96. Sequence requirement of Cas9
Streptococcus pyrogenes Cas9
5’-NNNNNNNNNNNN-NGG-3’
Neisseria meningitidis Cas9
5’-NNNNNNNNNNN-NNNNGATT-3’
NmCas9 can gene distruptions
In Human ES Cells
Hou, Thomson JA
PNAS 2013
Streptococcus thermophillus
5’-NNNNNNNNNNNNNN-NNAGAA-3’
Treponema denticola
5‟-NNNNNNNNNNNNNN-NAAAAC-3‟
Screening of novel Cas9? With different PAM specifity?
97. Engineering of Cas9
Now structure of Cas9-sgRNA is in our hands, it is time to engineer it
- PAM Specificity?
- Removal of nonessential part (spCas9 is too big in some vector system)
- Efficient fusion with other functional domains (Epigenetics?)
98. Roles of other Cas proteins and possible applications
We do not understand exact
functional roles
of all of Cas proteins
Some of Cas proteins may enhance
Genome engineering efficiency further
99. Delivery of Cas9/sgRNA
More efficient delivery method would be crucial for in vivo application
Viral vector?
Plasmid?
Ribonucleoprotein complex?
Delivery without transfection agent?