This document provides an overview of CRISPR/Cas9 genome editing. It begins with definitions of key genetic concepts like genomes, genes, and chromosomes. It then discusses the history and timeline of discoveries around CRISPR dating back to 1987, including its identification in bacteria and role in adaptive immunity. The document explains the basic principles and mechanism of CRISPR/Cas9, how it was harnessed for genome editing in 2012-2013, and current and potential applications in research and medicine. It concludes by noting some ethical concerns around genome editing technologies.
Leveraging Programmable CRISPR-Associated Transposases for Next-Generation Ge...InsideScientific
Dr. Sam Sternberg discusses a novel CRISPR-Cas9 system using programmable, RNA-guided transposase, and highlights its implications for kilobase-scale genome engineering in cell and gene therapies.
The utility of programmable, RNA-guided CRISPR-Cas systems in genome engineering continues to evolve. Nature has afforded scientists novel and diverse gene editing functionality, from nuclease-dependent CRISPR-Cas9 to second-generation base and prime editors that do not produce double-strand breaks.
In this webinar, Dr. Sam Sternberg describes a new CRISPR-Cas9 paradigm relying on nuclease-deficient bacterial transposons that catalyze RNA-guided integration of mobile genetic elements into the genome. The discovery of a fully programmable, RNA-guided transposase lays the foundation for kilobase-scale genome engineering with broad applications for developing cell and gene therapies.
Key Topics Include:
- The basics of first- and second-generation CRISPR-Cas technologies from a scientist at the forefront of their development
- Mechanisms, accommodation, and cell type diversity of CRISPR-Cas programmable transposition
- How transposase factor coordination enables highly specific, genome-wide DNA integration to target sites
- Implications of programmable transposases that obviate the need for DNA double-strand breaks and homologous recombination
Leveraging Programmable CRISPR-Associated Transposases for Next-Generation Ge...InsideScientific
Dr. Sam Sternberg discusses a novel CRISPR-Cas9 system using programmable, RNA-guided transposase, and highlights its implications for kilobase-scale genome engineering in cell and gene therapies.
The utility of programmable, RNA-guided CRISPR-Cas systems in genome engineering continues to evolve. Nature has afforded scientists novel and diverse gene editing functionality, from nuclease-dependent CRISPR-Cas9 to second-generation base and prime editors that do not produce double-strand breaks.
In this webinar, Dr. Sam Sternberg describes a new CRISPR-Cas9 paradigm relying on nuclease-deficient bacterial transposons that catalyze RNA-guided integration of mobile genetic elements into the genome. The discovery of a fully programmable, RNA-guided transposase lays the foundation for kilobase-scale genome engineering with broad applications for developing cell and gene therapies.
Key Topics Include:
- The basics of first- and second-generation CRISPR-Cas technologies from a scientist at the forefront of their development
- Mechanisms, accommodation, and cell type diversity of CRISPR-Cas programmable transposition
- How transposase factor coordination enables highly specific, genome-wide DNA integration to target sites
- Implications of programmable transposases that obviate the need for DNA double-strand breaks and homologous recombination
Presentation from the 2014 Waterloo iGEM team at the Giant Jamboree in Boston. Read more about Staphylocide, our microbe engineered to silence antiobiotic resistance, on our 2014 wiki: http://2014.igem.org/Team:Waterloo.
This presentation is also available on the iGEM website: http://2014.igem.org/files/presentation/Waterloo_Championship.pdf
Phylogenomic methods for comparative evolutionary biology - University Colleg...Joe Parker
Invited research seminar given to MSc students at University College Dublin on 24th October 2013.
I introduce the discipline of phylogenomics - comparative phylogenetic analyses of DNA sequences across genomes - and some of the applications and recent breakthroughs in the field.
As an in-depth case study I explain the methods and significance of our 2013 Nature paper on adaptive genotypic molecular convergence in echolocating mammals.
I then highlight some of the avenues of study on the frontiers of current research.
Genome: The entire chromosomal genetic material of an organism.
Sequencing a genome: Determining the identity and order of nucleotides in the genetic material – usually DNA, sometimes RNA, of an organism.
This pdf is about the Schizophrenia.
For more details visit on YouTube; @SELF-EXPLANATORY;
https://www.youtube.com/channel/UCAiarMZDNhe1A3Rnpr_WkzA/videos
Thanks...!
Presentation from the 2014 Waterloo iGEM team at the Giant Jamboree in Boston. Read more about Staphylocide, our microbe engineered to silence antiobiotic resistance, on our 2014 wiki: http://2014.igem.org/Team:Waterloo.
This presentation is also available on the iGEM website: http://2014.igem.org/files/presentation/Waterloo_Championship.pdf
Phylogenomic methods for comparative evolutionary biology - University Colleg...Joe Parker
Invited research seminar given to MSc students at University College Dublin on 24th October 2013.
I introduce the discipline of phylogenomics - comparative phylogenetic analyses of DNA sequences across genomes - and some of the applications and recent breakthroughs in the field.
As an in-depth case study I explain the methods and significance of our 2013 Nature paper on adaptive genotypic molecular convergence in echolocating mammals.
I then highlight some of the avenues of study on the frontiers of current research.
Genome: The entire chromosomal genetic material of an organism.
Sequencing a genome: Determining the identity and order of nucleotides in the genetic material – usually DNA, sometimes RNA, of an organism.
This pdf is about the Schizophrenia.
For more details visit on YouTube; @SELF-EXPLANATORY;
https://www.youtube.com/channel/UCAiarMZDNhe1A3Rnpr_WkzA/videos
Thanks...!
Seminar of U.V. Spectroscopy by SAMIR PANDASAMIR PANDA
Spectroscopy is a branch of science dealing the study of interaction of electromagnetic radiation with matter.
Ultraviolet-visible spectroscopy refers to absorption spectroscopy or reflect spectroscopy in the UV-VIS spectral region.
Ultraviolet-visible spectroscopy is an analytical method that can measure the amount of light received by the analyte.
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.
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.
Introduction:
RNA interference (RNAi) or Post-Transcriptional Gene Silencing (PTGS) is an important biological process for modulating eukaryotic gene expression.
It is highly conserved process of posttranscriptional gene silencing by which double stranded RNA (dsRNA) causes sequence-specific degradation of mRNA sequences.
dsRNA-induced gene silencing (RNAi) is reported in a wide range of eukaryotes ranging from worms, insects, mammals and plants.
This process mediates resistance to both endogenous parasitic and exogenous pathogenic nucleic acids, and regulates the expression of protein-coding genes.
What are small ncRNAs?
micro RNA (miRNA)
short interfering RNA (siRNA)
Properties of small non-coding RNA:
Involved in silencing mRNA transcripts.
Called “small” because they are usually only about 21-24 nucleotides long.
Synthesized by first cutting up longer precursor sequences (like the 61nt one that Lee discovered).
Silence an mRNA by base pairing with some sequence on the mRNA.
Discovery of siRNA?
The first small RNA:
In 1993 Rosalind Lee (Victor Ambros lab) was studying a non- coding gene in C. elegans, lin-4, that was involved in silencing of another gene, lin-14, at the appropriate time in the
development of the worm C. elegans.
Two small transcripts of lin-4 (22nt and 61nt) were found to be complementary to a sequence in the 3' UTR of lin-14.
Because lin-4 encoded no protein, she deduced that it must be these transcripts that are causing the silencing by RNA-RNA interactions.
Types of RNAi ( non coding RNA)
MiRNA
Length (23-25 nt)
Trans acting
Binds with target MRNA in mismatch
Translation inhibition
Si RNA
Length 21 nt.
Cis acting
Bind with target Mrna in perfect complementary sequence
Piwi-RNA
Length ; 25 to 36 nt.
Expressed in Germ Cells
Regulates trnasposomes activity
MECHANISM OF RNAI:
First the double-stranded RNA teams up with a protein complex named Dicer, which cuts the long RNA into short pieces.
Then another protein complex called RISC (RNA-induced silencing complex) discards one of the two RNA strands.
The RISC-docked, single-stranded RNA then pairs with the homologous mRNA and destroys it.
THE RISC COMPLEX:
RISC is large(>500kD) RNA multi- protein Binding complex which triggers MRNA degradation in response to MRNA
Unwinding of double stranded Si RNA by ATP independent Helicase
Active component of RISC is Ago proteins( ENDONUCLEASE) which cleave target MRNA.
DICER: endonuclease (RNase Family III)
Argonaute: Central Component of the RNA-Induced Silencing Complex (RISC)
One strand of the dsRNA produced by Dicer is retained in the RISC complex in association with Argonaute
ARGONAUTE PROTEIN :
1.PAZ(PIWI/Argonaute/ Zwille)- Recognition of target MRNA
2.PIWI (p-element induced wimpy Testis)- breaks Phosphodiester bond of mRNA.)RNAse H activity.
MiRNA:
The Double-stranded RNAs are naturally produced in eukaryotic cells during development, and they have a key role in regulating gene expression .
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
• What is genome editing?
• Background
• CRISPR/Cas9
• Timeline
• Basic Principle
• CRISPR: A Revolutionary Gene Editing Tool
• Hacked CRISPR Applications
• Current Research
• Ethical Concerns
• Conclusion of CRISPR/Cas9
3. GENOME
An organism’s complete set of genetic instructions
GENES
Are made up of a sequence of DNA, which
acts as instructions to ultimately make
molecules called proteins
•HUMAN GENOME
Cells of different species have a characteristic
number of chromosomes.
•Bacterial Cell 1 circular Chromosome
•Typical bird 80 total Linear
•Humans 46 total Linear
CHROMOSOMES
pngimg.com
4.
5. What is Genome Editing?
Genome editing, or gene
editing is where DNA is altered,
deleted, or completely replaced
in the genome.
-BP’s to complete Gene
sequences.
7. The Beginning as Repeat Sequences in
Bacterial Genome -1987
• “Exotic junk DNA”
(Zimmer, 2015)
• New research shows a widespread
presence in Archeae and Bacteria (2013-2016)
• Osaka University in Japan published the
sequence of a gene that belonged to the
enteric microbe E. coli
• Protein-Gene Switches
8. lustered egularly nterspaced hort alindromic epeat
RISPR sociated protein
Family of genes associated with CRISPR
locus
(typically between 23 and 47 bp)
(typically 21-72 bp)
(Charpentier, Doudna, 2014)
9. Timeline
2005- French National Institute for Agricultural Research
•Proposed that CRISPR structures involved DNA fragmentation by CAS
Proteins.
•Protospacer Adjacent Motif (PAM )
• Potential protective function against
foreign DNA invasion.
(Bolotin et al., 2005)
2007- Danisco (Now owned by DuPont)
•Common Industry Phages vs. Streptococcus thermophilus
• Adaptive immune system
•How?
(Barrangou et al., 2007)
10. December, 2010 University of Laval
Cas9 cleaves target DNA
(Garneau et al., 2010).
March, 2011 Emmanuelle Charpentier
Discovery of trans-activating CRISPR RNA for
Cas9 system (tracrRNA)
(Deltcheva et al., 2011)
Timeline
August, 2008 Netherlands
Spacer sequences are transcribed into guide
RNAs called CRISPR RNAs (crRNAs)
(Brouns et al., 2008).
12. CRISPR: A Revolutionary
Gene Editing Tool
In 2012,
•Scientists confirmed cleavage site and the PAM
sequence
•Two Lobes
•Trimmed Nucleotides
•Guide RNA could be simplified and reprogrammed
(Gasiunas et al., 2012) (Jinek et al., 2012)
13. CRISPR: A Revolutionary
Gene Editing Tool
January, 2013
CRISPR-Cas9 harnessed for genome editing
In vitro-
•Human Embryonic Kidney Cells
•Mouse cells
26. Conclusion of CRISPR/Cas9
• Knockout/Knock-in
• Gene Activation / Repression
• Research
• Potential Therapeutic Applications
• CRISPR and The World
27. Barrangou, R., Fremaux, C., Deveau, H., Richards, M., Boyaval, P., Moineau, S., . . . Horvath, P. (2007). CRISPR Provides Acquired
Resistance Against Viruses in Prokaryotes. Science, 315(5819), 1709-1712. doi:10.1126/science.1138140
Bolotin, A., Ehrlich, S. D., Quinquis, B., & Sorokin, A. (2005). Clustered regularly interspaced short palindrome repeats (CRISPRs)
have spacers of extrachromosomal origin. Microbiology, 151(8), 2551-2561. doi:10.1099/mic.0.28048-0
Brouns, S. J., Jore, M. M., Lundgren, M., Westra, E. R., Slijkhuis, R. J., Snijders, A. P., . . . Oost, J. V. (2008). Small CRISPR RNAs Guide
Antiviral Defense in Prokaryotes. Science, 321(5891), 960-964. doi:10.1126/science.1159689
Cong, L., Ran, F. A., Cox, D., Lin, S., Barretto, R., Habib, N., . . . Zhang, F. (2013). Multiplex Genome Engineering Using
CRISPR/Cas Systems. Science, 339(6121), 819-823. doi:10.1126/science.1231143
Deltcheva, E., Chylinski, K., Sharma, C. M., Gonzales, K., Chao, Y., Pirzada, Z. A., . . . Charpentier, E. (2011). CRISPR RNA maturation
by trans-encoded small RNA and host factor RNase III. Nature, 471(7340), 602-607. doi:10.1038/nature09886
Garneau, J. E., Dupuis, M., Villion, M., Romero, D. A., Barrangou, R., Boyaval, P., . . . Moineau, S. (2010). The CRISPR/Cas bacterial immune
system cleaves bacteriophage and plasmid DNA. Nature, 468(7320), 67-71. doi:10.1038/nature09523
Giorgio Austriaco, N. P. (2016). Genome Editing with CRISPR. Ethics & Medics, 41(3), 1-3.
Jinek, M., East, A., Cheng, A., Lin, S., Ma, E., & Doudna, J. (2013). RNA-programmed genome editing in human
cells. ELife, 2. doi:10.7554/elife.00471
Lomov, N. A., Borunova, V. V., & Rubtsov, M. A. (2015). CRISPR/Cas9 technology for targeted genome
editing. Biopolymers & Cell, 31(4), 243-248. doi:10.7124/bc.0008E7
Marraffini, L. A., & Sontheimer, E. J. (2008). CRISPR Interference Limits Horizontal Gene Transfer in Staphylococci by
Targeting DNA. Science, 322(5909), 1843-1845. doi:10.1126/science.1165771
Nishimasu, H., Ishitani, R., & Nureki, O. (2014). Crystal structure of Streptococcus pyogenes Cas9 in complex with guide
RNA and target DNA. doi:10.2210/pdb4oo8/pdb
Polstein, L. R., & Gersbach, C. A. (2015). A light-inducible CRISPR-Cas9 system for control of endogenous gene activation.
Nature Chemical Biology Nat Chem Biol, 11(3), 198-200. doi:10.1038/nchembio.1753
References
28. Torres-Ruiz, R., & Rodriguez-Perales, S. (2015). CRISPR-Cas9: A Revolutionary Tool for Cancer Modelling.
International Journal Of Molecular Sciences, 16(9), 22151-22168. doi:10.3390/ijms160922151
Wallace, J., Hu, R., Mosbruger, T. L., Dahlem, T. J., Stephens, W. Z., Rao, D. S., & ... O’Connell, R. M. (2016). Genome-Wide
CRISPR-Cas9 Screen Identifies MicroRNAs That Regulate Myeloid Leukemia Cell Growth. Plos ONE, 11(4), 1-11.
doi:10.1371/journal.pone.0153689
Wang, Z., Pan, Q., Gendron, P., Zhu, W., Guo, F., Cen, S., Liang, C. (2016). CRISPR/Cas9- Derived Mutations Both Inhibit HIV-1
Replication and Accelerate Viral Escape. Cell Reports. doi:10.1016/j.celrep.2016.03.042
Weijun, Z., Rongyue, L., Yann Le, D., Jian, L., Fei, G., Wainberg, M. A., & Chen, L. (2015). The CRISPR/Cas9 system inactivates
latent HIV-1 proviral DNA. Retrovirology, 12(1), 1-7. doi:10.1186/s12977-015-0150-z
References
