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CRISPR
The document summarizes key aspects of CRISPR genome editing technology. It describes how CRISPR uses Cas9 and guide RNA to precisely target and edit DNA sequences. It provides a brief history of CRISPR discovery and outlines its components and mechanism of action. The document also discusses several medical applications of CRISPR including treating Duchenne muscular dystrophy, beta-thalassemia, and testing for viruses. It concludes that CRISPR is a flexible and accurate gene editing tool being explored for various applications in agriculture, biotechnology, and medicine.
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CRISPR
- 1. Click to editMaster text styles Second level Third level Fourth level Fifth level CRISP R :A NEW BREAK THRO UGH Dr. SHERIN SHAJI Dr. VEDICA SETHI Dr. ZEENATH GHOUSKHAN
- 2. INTRODUCTION •Genome editing isa form of genetic engineering in which DNA sequences are inserted , deleted or replaced in the genome of living organisms using molecular scissors like nucleases. The four main families of nucleases is Meganucleases Zinc finger nucleases(ZFNS) Transcription activator like effector based effector nucleases (TALEN) CRISPR
- 3. CRISPR : CLUSTERED REGULARLYINTERSPACED 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
- 4. 4HISTORY: Key Events •1987-CRISPR sequences were first discovered in Escherichia coli. •2002- Coined CRISPR name, defined signature Cas genes. •2007- First experimental for CRISPR adaptive immunity. •2012- Idea of using CRISPR/Cas 9 as a genome engineering tool by Jennifer Doudna and Emmanuelle Charpentier. •2013- Demonstration of Cas 9 genome in eukaryotic cells. •2015- The CRISPR-Cas system was selected by Science as 2015 Breakthrough of the Year.
- 5. Different CRISPR-Cas system inBacterial Adaptive Immunity- •Type 1 – contain Cas3 gene which encodes large proteins with separate helicase and DNAase activity, in addition to genes encoding protien that form cascade like complexes with different compositions. •Type 2 – includes HNH type system in which Cas9, a single very large protein is sufficient to generate crRNA and cleaving the target DNA in addition to ubiquitous Cas 1 and Cas 2. •Type 3 - contain polymerase and RAMP modules in which at least some of the RAMPs seem to be involved in the processing of the spacer–repeat transcripts,
- 7. CRISPR LOCUS Spacer :the direct repeats in a CRISPR locus are seperated by short stretches of nonrepetitive DNA called spacers that are derived from invading plasmid or phage DNA. Protospacers : the nucleotide sequence of the spacers which is similar to a region in the phage genome which block phage replication. Leader sequence : is a conserved sequence associated with CRISPR locii located upstream of CRISPR. CRISPR array : composed of series of repeats interspaced by spacer sequences acquired from invading genome.
- 8. KEY COMPONENTS OF CRISPR crRNA: contains the guide RNA that locates the correct section of host DNA along with a region that binds to tracrRNA (in a hairpin loop form) forming an active complex. tracrRNA : binds to crRNA and forms an active complex. gRNA : a combined RNA consisting of tracrRNA and at least one crRNA. Cas 9 : they are RNA guided DNA endonuclease associated with CRISPR to interrograte foreign DNA.
- 9. MECHANISM OF ACTIONOF CRISPR
- 10. WHAT MAKES CRISPR SYSTEM THEIDEAL GENOME EDITING TOOL? •High potency and specificity •Broadly applicable to both invivo and exvivo application •Simple editing tools, allow unique ability to scale and optimize speed •Potential one time curative treatment for genetic disease •Ability to target multiple DNA site simultaneously which makes it different from other genome editing tools like ZFNs and TALENs •Multifunctional programability to insert, delete or repair gene
- 12. APPLICATION OF CRISPRIN MODERN MEDICINE •To understand the role that specific mutations in specific genes influence a particular trait of an organism. •To recreate known stable mutations in cell lines that can serve as models of a particular disease. •To create stable mutations in whole organisms (plants / animals) and create strains that can be used in research or commerce. •To create gene therapies in order to treat or diagnose congenital diseases, infections, or cancers especially new outbreaks like ZIKA virus •To create gene drives that can modify populations of organisms in a specific way
- 14. CRISPR TREATMENT FOR DUCHENEMUSCULAR DYSTROPHY •New gene editing enzyme CRISPR cpf1 corrected DMD in human cell •DMD mutation in dystrophin gene •CRISPR cpf1 differ from CRISPR Cas9 : much smaller than Cas9 enzyme which make it easier for the delivery to muscles • skipping a mutation region or precisely repairing a mutation in gene CRISPR cpf1 mediated genome editing corrects DMD mutation but also muscle contractility and strength
- 15. CRISPR targets pointmutation in exon 23 it creates a stop codon and serve as a model of DMD Efficacy by using a two vector system of CRISPR rather than single vectors for the guide RNA and Cas9 which will restore the dystrophin positive fibers Recovery: cell level- decrease infiltration, inflammatory cells and decrease fibrosis and reversal of necerosis.
- 16. Final outcome: •Improved gripstrength •Force generation, resistance against eccentric contractions •Decreased blood levels of creatinine kinase •Dystrophin expression on vascular smooth muscle •Important source of oxygenation during activity and cardiomyocytes- restored dystrophin.
- 18. TREATMENT FOR MYOTUBULAR MYOPATHY •Is aX-linked genetic disease affecting new born boys. •Caused by mutation in MTM1 gene encoding myotubularin, a protein involved in functioning of muscle cells. •Research conducted by University of Washington and Harvard Medical School in USA achieved a new method of treatment of MM by gene
- 19. CORRECTION OF BETA- THALASSEMIAMUTATION USING CRISPR CAS9 •Beta- Thalassemia caused by mutation in human Hb heritage. •Creation of human induced pluripotent stem cells (iPSC) from Beta Thalassemia patients could offer an approach to cure the disease. •Correction of disease causing mutation in iPSC’s restore normal function and provide a rich source of cells in transplantation. •CRISPR/Cas9 correct the HBB mutation in patient derived iPSC without leaving any residual foot print.
- 21. CRISPR-Cas13a: DIAGNOSTIC TOOL FOR ZIKAAND DENGUE VIRUS “The Sherlock” •SHERLOCK uses an RNA guide that gloms onto RNA, not DNA, and an enzyme called Cas13a cuts the genetic material. Once Cas13a snips the target, it starts indiscriminately cutting any RNA it encounters. • A team lead by bioengineer James Collins and CRISPR genome-editing pioneer Feng Zhang, both from the Broad Institute in Cambridge, Massachusetts, has now shown that these “collateral” cuts can form the basis for the SHERLOCK
- 22. •The researchers demonstratethat SHERLOCK can detect viral and bacterial infections, cancer mutations found at low frequencies, and subtle DNA sequence variations known as single nucleotide polymorphisms that are linked to other diseases. •SHERLOCK, a somewhat strained acronym coined by the team, stands for specific high sensitivity enzymatic reporter unlocking. •To exploit SHERLOCK for detecting small amounts of virus, the researchers spiked samples containing Zika or dengue virus with so-called fluorescent reporter RNA. When this RNA is cut, it effectively shoots off fluorescent flares. •The team then unleashed Cas13a connected to a bit of RNA that targeted genetic sequences from either Zika or dengue. Once Cas13a found and sliced even a few viral sequences, it subsequently snipped the fluorescent reporters and created a detectable signal indicating the presence of the virus.
- 23. The detectio n sensitivit y of the new CRISPR- Cas13a system for specific genetic material is1 million times better than the most common ly used diagnost
- 24. CRISPR: TARGETS CANCER INFIRST HUMAN TRIAL •Normally, T cells survey the body to seek out and destroy abnormal cells that may be turning cancerous. These cells often have strange proteins on their surface that alert T cells that they’re up to no good. However, in an evolutionary approach, cancer cells often gain the ability to “switch off” any T cell that gets in their way, effectively blocking the attack. •Many of the most successful cancer therapies try to circumvent this response by boosting the immune system. A 2015 study led by Dr. Carl June of UPenn, who is advising the new trial, used an older, less efficient gene engineering technique called zinc finger nucleases to give T cells better ability to fight off HIV. The therapy
- 25. The Plan; •In all,scientists will recruit 18 patients with three types of cancer (myeloma, sarcoma or melanoma) who have stopped responding to existing treatment. The two-year trial will take place at three centers that are members of the Parker Institute For Cancer Immunotherapy, including UPenn, UC San Francisco and the University of Texas. •The researchers will remove T cells from the patients and, using a harmless virus to deliver the CRISPR machinery into the cells, perform three gene edits on them. The first edit will insert a gene for a protein called the NY-ESO-1 receptor. This protein gives T cells the power to better recognize and home in on cancerous cells. •Unfortunately, T cells have two native proteins that interfere with this process, so the second edit will remove these
- 26. •The third editgives T cells staying power: it will remove a gene that allows cancer cells to recognize the immune cell and prevent the cancer from shutting off the attack. •Because CRISPR doesn’t work every time, not all the cells will get every modification. In the end, the engineered cells will be a mixture with various combinations of the proposed changes. Only 3-4% may contain all three .After the edits, the researchers will infuse the cells back into the patients and closely monitor for any issues. •One of the biggest worries is that CRISPR might inadvertently snip other genes, potentially creating new cancer genes or triggering existing ones. Using various tests, the team plans to carefully measure the growth rate of the engineered T cells and test for genomic abnormalities.But the outlook is bright. •In a test run using T cells from healthy
- 27. •Another worry isthat the technique itself could activate the body’s immune response. CRISPR uses an enzyme called Cas9, which originates from bacteria, to do the snipping. Although there are ways to protect the edited cells from the immune system, they could still be attacked. • The last concern isn’t scientific, but pertains to UPenn’s potential conflict of interest. June, who will serve as an advisor to the trial, has several patents on T cell engineering for cancer and is involved in several companies
- 30. CONCLUSION: •The CRISPR/Cas 9technique is one of a number of gene-editing tools. Many favor the CRISPR/Cas9 technique because of its high degree of flexibility and accuracy in cutting and pasting DNA. •One of the reasons for its popularity is that it makes it possible to carry out genetic engineering on an unprecedented scale at a very low cost. •How it differs from previous genetic engineering techniques is that it allows for the introduction or removal of more than one gene at a time. •This makes it possible to manipulate many different genes in a cell line, plant or animal very quickly, reducing the process from taking a number of years to a matter of weeks. • It is also different in that it is not species-specific, so can be used on organisms previously resistant to genetic engineering.
- 31. •The technique isalready being explored for a wide number of applications in fields ranging from agriculture through to human health. • In agriculture it could help in the design of new grains, roots and fruits. Within the context of health it could pave the way to the development of new treatments for rare metabolic disorders and genetic diseases ranging from hemophilia through to Huntingdon's disease. • It is also being utilized in the creation of transgenic animals to produce organs for transplants into human patients. • The technology is also being investigated for gene therapy. Such therapy aims to insert normal genes into the cells of people who suffer from genetic disorders such as cystic fibrosis, hemophilia or Tay Sachs. •Several start-up companies have been founded to exploit the technology commercially and large pharmaceutical companies are also exploring its use for drug discovery and development purposes.
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