CRISPR-Cas is a natural defense system in bacteria that uses CRISPR sequences and Cas proteins to target and degrade foreign DNA such as from viruses. It has been adapted for genome editing in other organisms using a Cas9 protein guided by a synthetic single guide RNA to introduce targeted double-strand breaks. This system allows for precise genome modifications and has applications in biomedical research, disease treatment, and engineering of plants and other organisms. However, off-target effects and delivery methods require further optimization.
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-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.
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.
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.
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.
CRISPR is easily the best gene editing tool to date. For decades, scientists have been looking for a way to to perform precise changes to genetic sequences. In the past several years, researchers were able to exploit the immune systems of bacteria to edit the genome of other living cells. CRISPR is reported to have higher targeting efficiencies when compared to TALENs and Zinc Fingers. It is efficient, easy to use and cheap; making it a scalable genetic engineering tool that is highly desirable in various industry-wide applications.
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.
Bacteriophage vectors
Bacteriophage
WHY BACTERIOPHAGE AS A VECTOR?
M13 phage
Genome of m13 phage
Life cycle and dna replication of m13
CONSTRUCTION M13 AS PHAGE VECTOR
M13 MP 2 vector
M13MP7 VECTOR
Selection of recombinants
Lambda replacement vectors
LAMBDA EMBL 4 VECTOR
P1 PHAGE
GENOME OF P1 PHAGE
P1 PHAGE AS VECTOR
P1 phage vector system
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-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.
CRISPR is easily the best gene editing tool to date. For decades, scientists have been looking for a way to to perform precise changes to genetic sequences. In the past several years, researchers were able to exploit the immune systems of bacteria to edit the genome of other living cells. CRISPR is reported to have higher targeting efficiencies when compared to TALENs and Zinc Fingers. It is efficient, easy to use and cheap; making it a scalable genetic engineering tool that is highly desirable in various industry-wide applications.
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.
Bacteriophage vectors
Bacteriophage
WHY BACTERIOPHAGE AS A VECTOR?
M13 phage
Genome of m13 phage
Life cycle and dna replication of m13
CONSTRUCTION M13 AS PHAGE VECTOR
M13 MP 2 vector
M13MP7 VECTOR
Selection of recombinants
Lambda replacement vectors
LAMBDA EMBL 4 VECTOR
P1 PHAGE
GENOME OF P1 PHAGE
P1 PHAGE AS VECTOR
P1 phage vector system
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.
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 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.
The purpose of this paper is to provide a study on some of the genome editing tools, namely CRISPR/CAS9 and RNAi (RNA interference). Both of these tool have been the latest innovations/discovery in the field of biology which has been used in altering genes in humans and other living organisms alike. This paper will discuss the working of each tool, their pros and cons, and at last, be compared with each other.
CRISPR : CLUSTERED REGULARLY INTERSPACED SHORT PALINDROMIC REPEAT
It 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.
It forms the basis of a genome editing technology known as CRISPR-Cas9 that allows permanent modifications of genes within organisms.
CRISPR-Cas system consist of two key molecules that introduce a change into the DNA sequence 1. Cas 9 - act as molecular scissors 2. gRNA – guides Cas9 to the right part of the genome gRNA = crispr rRNA + tracrRNA
Prezi Link: https://prezi.com/q8lkxnmwk25-/untitled-prezi/?utm_campaign=share&utm_medium=copy
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.
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.
This presentation explores a brief idea about the structural and functional attributes of nucleotides, the structure and function of genetic materials along with the impact of UV rays and pH upon them.
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/
Earliest Galaxies in the JADES Origins Field: Luminosity Function and Cosmic ...Sérgio Sacani
We characterize the earliest galaxy population in the JADES Origins Field (JOF), the deepest
imaging field observed with JWST. We make use of the ancillary Hubble optical images (5 filters
spanning 0.4−0.9µm) and novel JWST images with 14 filters spanning 0.8−5µm, including 7 mediumband filters, and reaching total exposure times of up to 46 hours per filter. We combine all our data
at > 2.3µm to construct an ultradeep image, reaching as deep as ≈ 31.4 AB mag in the stack and
30.3-31.0 AB mag (5σ, r = 0.1” circular aperture) in individual filters. We measure photometric
redshifts and use robust selection criteria to identify a sample of eight galaxy candidates at redshifts
z = 11.5 − 15. These objects show compact half-light radii of R1/2 ∼ 50 − 200pc, stellar masses of
M⋆ ∼ 107−108M⊙, and star-formation rates of SFR ∼ 0.1−1 M⊙ yr−1
. Our search finds no candidates
at 15 < z < 20, placing upper limits at these redshifts. We develop a forward modeling approach to
infer the properties of the evolving luminosity function without binning in redshift or luminosity that
marginalizes over the photometric redshift uncertainty of our candidate galaxies and incorporates the
impact of non-detections. We find a z = 12 luminosity function in good agreement with prior results,
and that the luminosity function normalization and UV luminosity density decline by a factor of ∼ 2.5
from z = 12 to z = 14. We discuss the possible implications of our results in the context of theoretical
models for evolution of the dark matter halo mass function.
Earliest Galaxies in the JADES Origins Field: Luminosity Function and Cosmic ...
Crispr cas
1. DONE BY:
SHRUTHI K (18308019)
1st M.Sc MICROBIOLOGY
PONDICHERRY UNIVERSITY
2. WHAT IS CRISPR CAS ?
Clustered Regularly Insterspaced Short Palindromic Repeats
(CRISPR) are short segments of prokaryotic DNA that contain
repititive base sequences. Spacer- DNA of viral proteins
The protein Cas9 (or "CRISPR-associated") is an enzyme that
acts like a pair of molecular scissors, capable of cutting strands
of DNA. They help in gene modification.
They are the natural defense modification in bacteria and archaea
to foil attacks by viruses and other foreign bodies.
3.
4. KEY COMPONENTS OF CRISPR CAS
crRNA : contains DNA that locates the correct section of host
DNA along with region that binds to tracrRNA as hairpin loop
forming an active complex. TracrRNA binds to crRNA forms an
active complex. (stem loop) – need to stimulate cas9.
sgRNA: chimera of crRNA and tracrRNA (modification of
crRNA )
Cas9 protein: protein whose active form is abvle to modify DNA.
Many variants exist with differing function. (nicks, double strand
breaks, DNA binding)
5. CRISPR-Cas IN BACTERIA
After insertion of exogenous DNA from viruses or plasmids, a Cas
complex recognizes foreign DNA and integrates a novel repeat-spacer
unit at the leader end of the CRISPR locus.
Type I systems. A Cas protein cleaves at the base of the stem–loop short
crRNA guides. The Cascade– crRNA complex scans the target DNA for a
matching sequence (known as protospacer), which is flanked by a
protospacer-adjacent motif (PAM, in green). Annealing of the crRNA to
the target strand forms an R-loop; the Cas3 nuclease is recruited and
cleaves the target downstream of the PAM (red arrowhead) and also
degrades the opposite strand.
6. Type II systems. These systems encode another small RNA
known as trans-encoded crRNA which has regions
complementary to crRNA. The tracrRNA dsRNA is cleaved by
RNase III and cleaves both strands of the protospacer/crRNA R-
loop. A PAM is located downstream of the target sequence.
7.
8. GENOME EDITING WITH CRISPR
tracrRNA might be required for target
DNAbinding and/or to stimulate the
nuclease activity of Cas9 downstream
of target recognition.
Cas9, which can site-specifically
cleave double-stranded DNA, resulting
in the activation of the doublestrand
break (DSB) repair machinery. DSBs
can be repaired by the cellular Non-
Homologous End Joining (NHEJ)
pathway
DOUDNA.J,
CHARPENTIER.E
The new frontier of genome
engineering with CRISPR-
Cas9, Science mag.org
9. Cas9, which can site-specifically cleave double-stranded DNA, resulting
in the activation of the doublestrand break (DSB) repair machinery. DSBs
can be repaired by the cellular Non-Homologous End Joining (NHEJ)
pathway.
Alternatively, if a donor template with homology to the targeted locus is
supplied, the DSB may be repaired by the homology-directed repair
(HDR) pathway allowing for precise replacement mutations to be made.
The tracrRNA and crRNA are complementary to each other and form a
stem loop structure. The loop is linked artificially by adding GAAA
nucleotides.
10. If we want to cut a specific part of DNA and insert certain
nucleotides, we create a guide RNA containing
corresponding bit of RNA.
Once the DNA to be cut is in place in the cas 9 complex to
form a DNA-RNA hybrid, the rev and hnh endonucleases
snip the dsDNA at the specific place.
The third component apart from guideRNA and cas9 is host
RNA which contains the set of nucleotides that we want to
insert in the break to repair it.
11.
12.
13. Jennifer doudna and emmanuelle charpentier- crispr in 2012
in bacterial cells in vitro. UC files patent.
2013- Feng Zhang, DNA editing in cell invivo in mouse.
Broad files for patent.
UC claimed “interference proceeding”
Patent office ruled Broad’s patent different.
2019 Feb – UC granted patent for basic use of CRISPR in all
kinds of cells.
Patent worth- 100 million
Other than cas9, other cas12, casX also by Broad institute
patented.
(rooting for UC) Broad can file challenging patent.
Final outcome tough to predict.
15. APPLICATIONS
GENOME EDITING OF IMPORTANT MODEL ORGANISMS AND
CELL TYPES:
Groundbreaking research was begun by Jinek et al. [2012] when they
investigated the mature crRNA that forms a pair with the transactivating
crRNA (tracrRNA)
Reengineered dual-RNA:Cas9 specificity by changing the nucleotides of the
crRNAs to make single and multiple nucleotide changes. They used two
crRNAs to simultaneously create the desired mutations in Streptococcus
pneumoniae.
Hwang et al. [2013] used a CRISPR-Cas9 system in vivo for targeted
genome editing in zebrafish embryos.
Mali et al. [2013] engineered a (sgRNAs) and a human codon-optimized
Cas9 for targeted genome editing.
16. CRISPR-Cas9 AS SMART ANTIMICROBIALAGENTS AGAINST
MDR PATHOGENS:
In order to test the multiplexity of CRISPR-Cas9, they designed two
sgRNAs to target the superantigen enterotoxin sek gene and a region of the
mecA gene.
They observed that target strains had been killed with comparable efficiency
CRISPR-Cas9 FOR THE ERADICATION OF HUMAN
VIRUSES:
The hepatitis viruses include hepatitis A, hepatitis B, and hepatitis C, three
distinct viruses that cause acute and chronic infections in human [Zhang et
al., 2014; Zeng, 2014]. CRISPR-Cas9 has been developed to eliminate HBV
through the targeting of P1 and XCp sites.
Used to eliminate HPV, HSV, HIV
17. CRISPR-cas9 IN THE NEUROSCIENCES:
Recently CRISPR‐Cas9 technology has been used for creating a new in vitro
and in vivo animal model for testing and characterizing the nervous system
and neurological diseases.
CRISPR‐Cas9 interruptions in and around the DISC1 gene - knocked‐down
levels of the protein expression. Further study is required to see whether or
not DISC1 can be corrected in vivo to overcome the schizophrenia.
[Srikanth et al.,2015]
CRISPR‐Cas9 have been applied in iPSC‐based disease models to explore
the mechanism of epilepsy caused by SCN1A loss‐of‐function mutations
[Liu et al., 2016]
Wang et al. [2015] have made mutations in CHD8 gene, replicating autism
spectrum disorders (ASDs).
18. CRISPR-cas9 IN HUMAN DISEASE THERAPY:
CRISPR‐Cas9 has been revolutionized for combating the cardiovascular
diseases. Mutation in the PCSK9 gene results in a reduction in (LDL‐C),
which can improve the heart health and reduce the cardiovascular disease.
Ding et al. [2014] have used CRISPR‐Cas9 for in vivo mutation of
the PCSK9 gene in mouse liver.
Yang et al. [2016] have developed an adeno‐associated virus delivery system
for CRISPR‐Cas9 toward the correction of OTC gene in newborn mice –
hyperammonemia.
CRISPR‐Cas9 has been used to correct the mutated dmd (Duche nne
muscular dystrophy) gene in the mdx mice germ line and found 2–100%
corrections efficiency Long et al., 2014; Nelson et al., 2016].
19. CRISPR-cas9 IN CANCER IMMUNOTHERAPY:
The generation of CAR-T (chimeric antigen receptor) cells is one of the most
eye-catching applications of CRISPR-Cas9 technology in cancer
immunotherapy.
Ren et al used the CRISPR/Cas9 system to disrupt multiple genomic sites for
constructing CAR-T cells with defective TCR and HLA class I expression,
which shows potent antitumour activity.
CRISPR-Cas9-mediated genome editing can also be applied to eliminate
genes that encode inhibitory T cell surface receptors, such as programmed
cell death protein 1 (PD-1)
Zhang et al successfully generated lymphocyte activating gene-3 (LAG-3)
knockout CAR-T cells using CRISPR-Cas9.
20. Off target effects
Unwanted deletions and insertions
Production of gRNA – posttranslational modicfication processes
Efficient delivery system – adeno and lentivirus.
21. REFERENCES
Jennifer A. Doudna Emmanuelle Charpentier, The new frontier of
genome engineering with CRISPR-Cas9, Science 28 Nov 2014:
Vol. 346, Issue 6213, DOI: 10.1126/science.1258096
Philippe Horvath and Rodolphe Barrangou, CRISPR/Cas, the
Immune System of Bacteria and Archaea, Science 327 (5962), 167-
170. DOI: 10.1126/science.1179555
Chun-Hao Huang, Ko-Chuan Lee, and Jennifer A. Doudna ,
Applications of CRISPR-Cas Enzymes in Cancer Therapeutics and
Detection , Cell Press Reviews.
Martin Jinek, Krzysztof Chylinski, Ines Fonfara, Michael Hauer,
Jennifer A. Doudna and Emmanuelle Charpentier, A
Programmable Dual-RNA-Guided DNA Endonuclease in
Adaptive Bacterial Immunity, Science 337 (6096), 816-821.
DOI: 10.1126/science.1225829originally published online June 28,
2012
Editor's Notes
Genome editing, types of nucleases, why
The predicted tracrRNA:crRNA secondary structure includes base pairing between the 22 nucleotides at the 3′ terminus of the crRNA and a segment near the 5′ end of the mature tracrRNA
Unknown viral dna- broken by cas1 and genes stored in spacers
Homing rna- where the pam sequence is in grna for editing