This document provides an overview of the CRISPR-Cas immune system in bacteria and its applications. It discusses:
1. The history and components of the CRISPR-Cas system, including Cas proteins, CRISPR RNA, and protospacer adjacent motifs.
2. The three main types of CRISPR-Cas systems and their mechanisms of targeting DNA or RNA.
3. How engineered versions of Cas9 can be used for targeted genome editing and modulation of gene expression in mammalian cells.
4. Applications of CRISPR-Cas in microbiology such as genetic engineering of bacteria, gene repression/activation using deactivated Cas9, and developing sequence-specific antimicrobials.
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.
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 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.
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
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 (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.
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.
The next generation of crispr–cas technologies and Applicationsiqraakbar8
The prokaryote-derived CRISPR–Cas genome editing systems have transformed our ability to manipulate, detect, image and annotate specific DNA and RNA sequences in living cells of diverse species. The ease of use and robustness of this technology have revolutionized genome editing for research ranging from fundamental science to translational medicine. Initial successes have inspired efforts to discover new systems for targeting and manipulating nucleic acids, including those from Cas9, Cas12, Cascade and Cas13 orthologues.
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.
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 (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.
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.
The next generation of crispr–cas technologies and Applicationsiqraakbar8
The prokaryote-derived CRISPR–Cas genome editing systems have transformed our ability to manipulate, detect, image and annotate specific DNA and RNA sequences in living cells of diverse species. The ease of use and robustness of this technology have revolutionized genome editing for research ranging from fundamental science to translational medicine. Initial successes have inspired efforts to discover new systems for targeting and manipulating nucleic acids, including those from Cas9, Cas12, Cascade and Cas13 orthologues.
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.
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.
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.
An Introduction to Crispr Genome Editing
Crispr cas: A new tool of genome editing
CRISPRs (Clustered Regularly Interspaced Short Palindromic Repeats) are part of an adaptive defense mechanism in bacteria and archaea. Use of the CRISPR/Cas9 system for genome editing has been a major technological breakthrough, making genome modification in cells or organisms fast, more efficient, and much more robust than previous genome editing methods. Single guide RNAs (sgRNAs) or guide RNAs (gRNAs) direct and activate the Cas9 endonuclease at a specific genomic sequence. Cas9 then cleaves the target DNA, making it available for repair by the non-homologous end joining (NHEJ) system or for creating an insertion site for exogenous donor DNA by homologous recombination.
CRISPR is one of the mind blowing discovery which completely change the science of microorganisms. It is am efficient tool for genome editing and make the scientist enable to treat disease. The vast application of CRISPR technology covered almost all every aspect of life ranging from individual life to commercial aspect.
Purpose:
The purpose of this webinar is to develop creative scientific thinking in youngster and make them familiar with the miricals of science discovery.
Introduction, History, components, cas9 protein structure and function,gRNA variants, Cas9 nuclease variants, CRISPR in bacteria as the immune system, mechanism, steps of working, Applications, and pros and cons.
A brief information about the SCOP protein database used in bioinformatics.
The Structural Classification of Proteins (SCOP) database is a comprehensive and authoritative resource for the structural and evolutionary relationships of proteins. It provides a detailed and curated classification of protein structures, grouping them into families, superfamilies, and folds based on their structural and sequence similarities.
What is greenhouse gasses and how many gasses are there to affect the Earth.moosaasad1975
What are greenhouse gasses how they affect the earth and its environment what is the future of the environment and earth how the weather and the climate effects.
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.
Richard's entangled aventures in wonderlandRichard Gill
Since the loophole-free Bell experiments of 2020 and the Nobel prizes in physics of 2022, critics of Bell's work have retreated to the fortress of super-determinism. Now, super-determinism is a derogatory word - it just means "determinism". Palmer, Hance and Hossenfelder argue that quantum mechanics and determinism are not incompatible, using a sophisticated mathematical construction based on a subtle thinning of allowed states and measurements in quantum mechanics, such that what is left appears to make Bell's argument fail, without altering the empirical predictions of quantum mechanics. I think however that it is a smoke screen, and the slogan "lost in math" comes to my mind. I will discuss some other recent disproofs of Bell's theorem using the language of causality based on causal graphs. Causal thinking is also central to law and justice. I will mention surprising connections to my work on serial killer nurse cases, in particular the Dutch case of Lucia de Berk and the current UK case of Lucy Letby.
Richard's aventures in two entangled wonderlandsRichard Gill
Since the loophole-free Bell experiments of 2020 and the Nobel prizes in physics of 2022, critics of Bell's work have retreated to the fortress of super-determinism. Now, super-determinism is a derogatory word - it just means "determinism". Palmer, Hance and Hossenfelder argue that quantum mechanics and determinism are not incompatible, using a sophisticated mathematical construction based on a subtle thinning of allowed states and measurements in quantum mechanics, such that what is left appears to make Bell's argument fail, without altering the empirical predictions of quantum mechanics. I think however that it is a smoke screen, and the slogan "lost in math" comes to my mind. I will discuss some other recent disproofs of Bell's theorem using the language of causality based on causal graphs. Causal thinking is also central to law and justice. I will mention surprising connections to my work on serial killer nurse cases, in particular the Dutch case of Lucia de Berk and the current UK case of Lucy Letby.
Observation of Io’s Resurfacing via Plume Deposition Using Ground-based Adapt...Sérgio Sacani
Since volcanic activity was first discovered on Io from Voyager images in 1979, changes
on Io’s surface have been monitored from both spacecraft and ground-based telescopes.
Here, we present the highest spatial resolution images of Io ever obtained from a groundbased telescope. These images, acquired by the SHARK-VIS instrument on the Large
Binocular Telescope, show evidence of a major resurfacing event on Io’s trailing hemisphere. When compared to the most recent spacecraft images, the SHARK-VIS images
show that a plume deposit from a powerful eruption at Pillan Patera has covered part
of the long-lived Pele plume deposit. Although this type of resurfacing event may be common on Io, few have been detected due to the rarity of spacecraft visits and the previously low spatial resolution available from Earth-based telescopes. The SHARK-VIS instrument ushers in a new era of high resolution imaging of Io’s surface using adaptive
optics at visible wavelengths.
Comparing Evolved Extractive Text Summary Scores of Bidirectional Encoder Rep...University of Maribor
Slides from:
11th International Conference on Electrical, Electronics and Computer Engineering (IcETRAN), Niš, 3-6 June 2024
Track: Artificial Intelligence
https://www.etran.rs/2024/en/home-english/
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.
(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.
2. CRISPR - cas : A New tool for Genetic
Manipulations from Bacterial Immunity
Systems
2
M. Raveendra Reddy
Ph D, UAS, GKVK, BANGLORE
Viral SS DNA
RNA Guide
CRISPR Cascade
subunit
3. Content…
1. Introduction
2. History
3. Components of CRISPR
4. Different Cas proteins and their function
5. Types of CRISPR cas system
6. Variants of Cas9
7. Targeted Genome editing in Mammalian
cells
8. Applications in Microbiology
9. Conclusion
4. CRISPR
• It represents a family of DNA repeats in most archaeal (~90%) and
bacterial (~40%) genomes provides acquired immunity against
viruses and phages.
CRISPRs (Clustered Regularly Interspaced Short Palindromic
Repeats) are DNA loci containing short repetitions of base sequences
which separated by short "spacer DNA" from previous exposures to
a virus or plasmid.
5. 5
The size of CRISPR repeats and spacers varies between 23 to 47
base pairs (bp) and 21 to 72 bp, respectively.
Generally, CRISPR repeat sequences are highly conserved within
a given CRISPR locus.
6. The spacer region of each CRISPR RNA base pairs with complementary
nucleic acids, driving cleavage or degradation by the Cas proteins (cas 1 and
Cas 2) within minutes of invasion.
7. History
Yoshizumi Ishino in 1987, from Osaka University who accidentally
cloned part of a CRISPR together with the iap gene in E.coli
Francis Mojica (1995) studied CRISPR in the archaeal
organism Haloferax mediteranii and its function at the University of
Alicante, Spain.
The wide spread dispersal of CRISPR among prokaryotes was
presented at the Genomes 2000 meeting at the Institute Pasteur in Paris
by Jansen (named SPacer Interspersed Direct Repeat (SPIDR)) and
published by Mojica in the same year.
In 2007 the first experimental evidence that CRISPR was an adaptive
immune system in Streptocous thermophilus was published (Hsu et al,
2014. Cell. 157 (6): 1262–78. )
8. A 2010 study provided direct evidence that CRISPR-Cas cuts
both strands of phage and plasmid DNA in S. thermophilus
( Garneau J.E, 2010. Nature. 468 (7320): 67–71)
Jennifer Doudna and Emmanuelle Charpentier
(2012) engineered Cas9 endonuclease into a more manageable two-
component system by fusing the two RNA molecules into a "single-
guide RNA" . (Science. 337 (6096): 816–21)
Jennifer Doudna Emmanuelle Charpentier
9. Components of CRISPR
1. Protospacer adjacent motif (PAM)
2. CRISPR-RNA (crRNA)
3. trans-activating crRNA (tracrRNA)
4. Cas proteins
10. Different Cas proteins and their function
Protein Distribution Process Function
Cas1 Universal Spacer
acquisition
DNAse, not sequence specfic, can
bind RNA; present in all Types
Cas2 Universal Spacer
acquisition
specific to U-rich regions; present
in all Types
Cas3 Type I
signature
Target
interference
DNA helicase, endonuclease
Cas4 Type I, II Spacer
acquisition
RecB-like nuclease with
exonuclease activity homologous
to RecB
Cas5 Type I crRNA
expression
RAMP protein, endoribonuclease
involved in crRNA biogenesis;
part of CASCADE 10
11. Cas6 Type I, III crRNA
expression
RAMP protein, endoribonuclease involved
in crRNA biogenesis; part of CASCADE
Cas7 Type I crRNA
expression
RAMP protein, endoribonuclease involved in
crRNA biogenesis; part of CASCADE
Cas8 Type I crRNA
expression
Large protein with McrA/HNH-nuclease
domain and RuvC-like nuclease; part of
CASCADE
Cas9 Type II
signature
Target
interference
Large multidomain protein with McrA-HNH
nuclease domain and RuvC-like nuclease
domain; necessary for interference and
target cleavage
Cas10 Type III
signature
crRNA
expression
and
interference
HD nuclease domain, palm domain, Zn
ribbon; some homologies with CASCADE
elements
12. Types of CRISPR CAS system
There are three types of CRISPR-Cas systems, which
vary in their specific target and mechanism of action.
Type I systems cleave and degrade DNA,
Type II systems cleave DNA ,
Type III systems cleave DNA or RNA .
13. Type I and II systems require two principal factors to
effectively target DNA:
(i) complementarity between the CRISPR RNA spacer and
the target “protospacer” sequence.
(ii) a protospacer-adjacent motif (PAM) specific to each
CRISPR-Cas system flanking the protospacer.
14. Different CRISPR-Cas system in Bacterial Adaptive
Immunity
Class 1- type I (CRISPR-Cas3) and type III
(CRISPR-Cas10)
uses several Cas proteins and the
crRNA
Class 2- type II (CRISPR-Cas9) and type
V (CRISPR-Cpf1)
employ a large single-component
Cas-9 protein in conjunction with
crRNA and tracerRNA.
Zetsche et al., (2015)
functioning of type II CRISPR
system
15. Includes CRISPR associated
complex for antiviral defense
(cascade) and cas3
CRISPR array is transcribed as
crRNA and processed to liberate
short mature crRNA
Cas6 endoribonuclease cleaves
the repeat sequence eight
nucleotides upstream of the spacer
sequence and liberates a small
crRNA containing a full spacer
flanked by partial repeats.
Type 1 CRISPR-Cas Systems
16. Efficient immunity requires
interaction between the first
eight nucleotides of the target
and the complementary
sequence of the crRNA guide at
the 5l end of the DNA:RNA
duplex.
A second requirement for
type I immunity is the presence
the protospacer-adjacent motif
(PAM).
17. For the type I-E system of E.
coli the PAM is an AWG
trinucleotide, and it is recognized
by CasA, a member of the
Cascade complex.
If both conditions are met, the
Cas3 ssDNA nuclease is
recruited by Cascade to cleave
the displaced DNA strand
within the target sequence and
degrade it with 3l →5l
18. -defined by presence of the RNA-
guided endonuclease Cas9
- the simplest of all CRISPR-Cas
systems
-type II CRISPR loci produce a
small trans-encoded crRNA
(tracrRNA) with a region of
complementarity to the repeat
sequence
gRNA = crRNA + tracrRNA
Type II CRISPR-Cas Systems
19. -The Cas9 nuclease binds the tracrRNA, which anneals to the
repeat sequences of the precursor crRNA.
20. Binding of Cas9 to the PAM favors unwinding of the target
sequence immediately upstream of the motif, allowing the crRNA
to probe for a matching sequence.
Productive annealing results
in formation of a
crRNA:target R-loop that
triggers cleavage by Cas9.
The enzyme has two
nuclease domains HNH and
RuvC - cuts one target DNA
strand.
The tracrRNA - cofactor of
Cas9 - DNA cleavage
Ruvc
HNH
21. 7
Type III CRISPR-Cas Systems
Type III CRISPR-Cas systems are possibly the most complex of all
Characterised by the presence of genes encoding Cas10 and repeat-
associated mysterious protein (RAMP) modules Csm for type III-A and
cmr for type III-B.
precursor processing is achieved by the Cas6 repeat-specific
endoribonuclease like in Type I System , but contrast to type I systems,
however, Cas6 is not part of the Cas10 complex
22. Two unique features of type III CRISPR-Cas systems are
(a) that transcription across the target is required for immunity and
(b) both DNA and RNA targets are cleaved.
In addition, only crRNA guides complementary to the coding DNA
strand—not those complementary to the template DNA strand—provide
effective immunity
In contrast to the other CRISPR-Cas, seed sequence has not identified.
23.
24.
25. Homology-directed repair (HDR) and nonhomologous end joining
(NHEJ) are DNA repair pathways exploited for targeted genome editing in
mammalian cells
Double-strand DNA breaks (DSBs) can increase recombination events in
the cleaved region up to 1,000-fold and programmable nucleases that can
cleave any sequence within the genome of interest are essential tools for
genome engineering.
Specific mutations can be introduced when cells are supplied with
exogenous DNA fragments as templates for recombination with the cleaved
site through HDR.
TARGETED GENOME EDITING IN MAMMALIAN CELLS
26. In the absence of a DNA template for HDR, DSBs are typically
repaired by the error-prone NHEJ pathway, in which insertion or
deletion mutations (indels) of various lengths are introduced into the
target locus.
These indels usually alter the gene’s open reading frame and therefore
lead to the generation of gene knockouts.
To reduce mutational effects of DSBs, biologists have looked for
programmable sequence-specific nucleases.
1. Meganucleases,
2. Zinc finger nucleases (ZFNs),
3. Transcription activator–like effector nucleases (TALENs).
27. Meganucleases:
Meganucleases are naturally occurring sequence-specific endonucleases found in
many microorganisms and plants.
They are characterized by a large DNA recognition site ranging from 12 to 40 base
pairs .
Meganucleases have not been widely adopted as genome editing tools…
…… because sequence specificity is hard to engineer, and despite the existence of
hundreds of meganucleases in nature, the probability of finding one able to cut a given
gene at a desired location is extremely slim.
28. Both ZFNs and TALENs are engineered nucleases in which a modular
DNA-binding domain is linked to the nuclease domain of the restriction
enzyme FokI.
Zinc Finger Nucleses
In ZFNs, the DNA-binding domain is a tandem array of different Cys2
His2 zinc fingers, each of which recognizes a three- base-pair sequence in
the target DNA.
29. In TALENs, the DNA-binding
domain is derived from DNA-binding
proteins found in Xanthomonas
bacteria and contains tandem array of
15-19 nucleotides.
Each module binds a single base
pair and consists of 34 amino acid
residues, with different amino acid
sequences recognizing different base
pairs
Trancription Activator Like Effector Nucleases (TALENS)
30. In both ZFNs and TALENs, sequence specificity is conferred by the
programmable DNA- binding domain.
In addition, the assembly of TALEN genes encoding large numbers of
TALE modules presents a challenge for molecular cloning as well as for
efficient viral delivery to target cells.
Difficulties in predicting and modulating ZFN and TALEN interaction
with the target DNA, as well as the laborious process of protein design,
limit the utility of these nucleases as tools for genome editing.
31. CRISPR/Cas 9 engineering tool
DNA cleavage is based on RNA/DNA pattern and not anymore on
Protein/DNA
Change require only in the first 20 nucleotides of the gRNA
(former crRNA)
Possibility of targeting multiple DNA sequences at once
Much more easier to target DNA sequence
13
32. Some limitations: off-target
Off-target: tolerance of Cas9 to mismatches in the RNA
guide sequence.
Limited by PAM motif
Depend of mismatch locations, lengths, compositions
Difficult to predict
14
33. Mutations in the RuvC (D10A) and HNH (H840A) domains abolish
cleavage but do not impair binding of Cas9 to its targets.
The catalytically dead version of Cas9 (dCas9) can be exploited for
multiple uses in molecular biology as a sequence-specific, RNA-
guided DNA-binding protein.
Most important applications of dCas9 is modulation of transcription .
Binding of dCas9 to promoter sequences or open reading frames can
prevent transcription initiation or elongation, respectively.
Repression also can be enhanced.
EXPLOITING THE RNA-GUIDED DNA-BINDING ACTIVITY OF CAS9
36. Advantages of dCas9 modulation of gene expression (CRISPRi)
over RNAi technology
key distinctions between dCas9 and RNAi are that
(a) dCas9 downregulation of transcripts acts at the DNA level, in
contrast to posttranscriptional regulation by RNAi,
(b) dCas9 offers the possibility of upregulating gene expression,
allowing for discovery of gain-of-function phenotypes that are not
possible to obtain with RNAi, and
(c) dCas9 can act on miRNAs and any other genomic region.
37. Streptococcus pyogenes Cas9 :
-the standard cas9 used in researches
- PAM seq : 5’ NGG 3’
Staphylococcus aureus Cas9 :
Smaller than S. pyogenes Cas9
- PAM seq : 5’ NNGRRT 3’
Another important use of
dCas9 is visualization of specific
DNA loci in living cells by fusing it
with fluorescent proteins
Chen et al. fused dCas9 to
an enhanced green fluorescent
protein (eGFP) to interrogate the
spatiotemporal dynamics of DNA
sequences in living cells.
38.
39. CAS9-BASED APPLICATIONS IN MICROBIOLOGY
1. Genetic Engineering
Bikard et al (2012) programmed the human pathogen
Streptococcus pneumoniae with CRISPR sequences that target capsule
genes (virulence factor).
CRISPR interference can prevent transformation of non
encapsulated, avirulent pneumococci into capsulated, virulent strains during
infection in mice.
Cell Host Microbe. 2012 Aug 16;12(2):177-86.
40. Result demonstrate that CRISPR interference can prevent the
emergence of virulence in vivo and that strong selective pressure for
virulence or antibiotic resistance can lead to CRISPR loss in bacterial
pathogens.
This approach has been successfully applied for genetic manipulation
in numerous bacterial species, including E. coli , Streptococcus
pneumoniae , Lactobacillus reuteri , Clostridium beijerinckii and
Streptomyces coelicolor as well as bacteriophages.
41. 2. dCas9-Based Gene Repression and Activation
Bikard et al. (2013) fused catalytically dead cas9 (dCas9) to
the ω subunit of RNA polymerase (RNAP) and expressed this fusion
(dCas9-ω) in an E. coli mutant lacking the gene for ω.
dCas9-ω occupies the place of ω in the RNAP complex, where it can
mediate the recruitment of RNAP to specific promoters using the
appropriate guide crRNA. This method enhances transcription.
Nucleic Acids Res. 41:7429–37
42. Sequence-Specific Antimicrobials
Cas9 chromosomal cleavage leads to toxicity to cell. This property is used for the
development of sequence specific antimicrobials by RNA guided nuclease.
In human microbiota, could selectively kill bacteria with a specific genetic
sequence. In contrast to conventional antibiotics, the Cas9 antimicrobial could be
programmed with a guide RNA matching a specific virulence gene sequence to kill
the bacterial pathogen harboring this gene but spare the rest of the microbiota.
43. Phagemids : (pDB114)
Phages can be used to deliver cas9-containing plasmids harboring
CRISPR RNA sequences that direct Cas9 to unique chromosomal loci to
achieve sequence-specific killing. (Staphylococcus aureus)
a) Only target cells with the cognate sequence are killed, whereas
nontarget cells survive the phagemid treatment.
44. Significant challenges must be overcome before Phagemid can be
developed as a suitable drug.
1. The most important obstacle is the method of delivery, given that
phagemids have the same narrow host. This substantially limits the
number of species that can receive the Cas9 plasmid; only those
expressing the appropriate phage receptor can be treated.
2. Phagemids are also immunogenic, and their large structure limits their
access to certain infection sites.
3. Furthermore, phagemids deliver Cas9 in the form of plasmid DNA,
which could be destroyed and neutralized by bacterial restriction
enzymes.
46. Comparision between sgRNA and crRNA-tracrRNA
hetero duplex
Advantages
• Flexible targeting
• Sequence specific
• Efficient
• Precise cleavage
• Affordable
• Quick
• Multiplex guides
Limitation
Cas9 is a large protein
PAM – dependent design limitations
Off –target cleavage
47. Cas9-based tools are a formidable asset for studies in basic
science, biotechnology, and medicine. Cas9-based technologies
are spreading at a remarkable speed that speaks to their simplicity
and efficiency. One outstanding objective is the precise
determination of off-target effects and their consequences.
Although current data seem to indicate that these are not a
major obstacle, a better characterization of Cas9 cleavage is
required before the technology is used in humans.
Ethical considerations are a second outstanding issue
Conclusion