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genome editing technique CRISPR-Cas9 - Copy.pptx
- 1. GENOME EDITING TECHNIQUE CRISPR-CAS9 PRESENTED ACADEMIC YEAR: 2022/2023 MODULE: CHROMOSOMAL AND GENETIC ANOMALY MINISTRY OF HIGHER EDUCATION AND SCIENTIFIC RESE ARCH FACULT Y OF NATURAL AND LIFE SCIENCES M1 BIOTECHNOLOGY AND MOLECUL AR PATHOLOGY
- 2. INTRODUCTION Gene editing refers to the use of advanced biotechnologies to make specific alterations to an organism's DNA. The primary goal of gene editing is to modify the genetic material of an organism in a precise and targeted manner, which can lead to changes in its physical and biochemical characteristics. This technology allows scientists to add, delete, or modify the DNA sequence of an organism's genome with great precision. There are several techniques used for gene editing, but the most widely used is the CRISPR-Cas9 system. This system involves using a bacterial protein called Cas9, which can be programmed to target and cut specific DNA sequences. Once the DNA is cut, the cell's natural repair mechanisms can be utilized to add, delete or replace genetic material. So how does this work mechanistically, and what are its applications?
- 3. WHAT IS ? CRISPR is a gene-editing technology that allows scientists to selectively modify specific genes within an organism's DNA. The CRISPR system works by using an RNA molecule to guide an enzyme called Cas9. CRISPR : Clustered Regularly Interspaced Short Palindromic Repeats cas9 is a protein that is part of the CRISPR gene editing system. Specifically, Cas9 is an enzyme that can cut DNA at specific locations, as guided by a small RNA molecule called a guide RNA. The Cas9 protein is derived from a type of bacteria that uses the CRISPR system as a defense mechanism against invading viruses. Cas9 : CRISPR Associated Proteins 2D AND 3D STRUCTURE OF CAS9 PROTEINE Cas 9 Target DNA sgRNA Target DNA Cas9 gRNA
- 4. what does each word mean ? Clustered mean that these repeating sequences and spacers are usually found together in a cluster (bound together) Regularly refers to the fact that the repeating sequences are usually of a consistent length and pattern In 1987, Yoshizumi Ishino and his team of researchers from the Osaka University in Japan first reported the presence of CRISPR, in the Escherichia coli genome. These refer to short, repeated sequences of DNA nucleotides found within the genome of prokaryotes. A BRIEF HISTORY OF CRISPR-CAS9 AS A GENOME- EDITING TOOLS REPORTE ABOUT THE PRESENCE OF CRISPR IN BACTERIA 1987 Short / Repeats same DNA sequence Interspaced different DNA sequence Palindromic it mean these sequences are the same when read from 5' to 3' on one strand of DNA and from 5' to 3’ on the other strand genome of prokaryotes spacers spacers spacers / / / / / / Clustered /Regularly / /
- 5. DISCOVERY OF CRISPR- ASSOCIATED (CAS) 2002 in 2002, Ruud Jansen and his team coined the term CRISPR and discovered CRISPR-associated (Cas) genes. Scientists eventually reported that the CRISPR spacers were derived from invading phage and extrachromosomal DNA, which led to the discovery of the CRISPR/Cas system's role in adaptive immunity in prokaryotes. (Cas) CRISPR Associated Proteins The CRISPR system was originally discovered as a bacterial immune system, and it works by using RNA molecules to guide Cas proteins to specific DNA sequences where they can make precise cuts or modifications. There are several different types of Cas proteins, each with their own unique properties and functions. For example, Cas9 is one of the most commonly used Cas proteins in gene editing applications Ruud Jansen 2002 even unicellular bacteria have a very basic immune system. As an daptive immunity BACTERIOPHAGE CRISPR CAS PROTEINS GENES The CAS proteins in general are going to be: HELICASES, those are proteins that unwind DNA. Or NUCLEASES, those that cut the DNA.
- 6. IDENTIFICATION OF THE CRISPER CAS9 SYSTEM AS A DNA-EDITING TOOL 2012 In 2012, Jennifer Doudna, Emmanuelle Charpentier, discovered that by designing guide RNA to target a specific region in the genome, “the CRISPR-Cas9 system can be used as a “cut-and-paste” tool to modify genomes. As a DNA-editing tool, CRISPR-Cas9 can remove or introduce new genes as well as silence or activate genes. CRISPR-Cas9 has been used to switch off genes that limit the production. George Church: he developed the first direct genomic sequencing method, which resulted in the first genome sequence (the human pathogen). Feng Zhang(2013):Feng Zhang and his colleagues at the Broad Institute of MIT and Harvard show that CRISPR-Cas9 can be used to edit genes in human cells. They demonstrate that the technique can be used to target multiple genes at once and to create knockout mutations After years of speculation over who would be recognized for the pioneering work on the gene editing tool CRISPR– Cas9, the Nobel Prize in Chemistry has finally been awarded to Emmanuelle Charpentier and Jennifer Doudna.in 2020 Jennifer Doudna Emmanuelle Charpentier Feng Zhang George Church Emmanuelle Charpentier Jennifer Doudna
- 7. CRISPR PROKARYOTIC ANTIVIRAL DEFENSE MECHANISM 1.Adaptation When a virus or other foreign DNA enters a bacterial cell, the CRISPR system recognizes and cuts a short fragment of the foreign DNA, called a spacer. This spacer is then integrated into the bacterial genome between the CRISPR repeats, providing a record of the invading virus. 2.Expression The CRISPR array is transcribed into a long precursor RNA molecule, which is then processed into small CRISPR RNA molecules (crRNAs) by cellular enzymes. 3.Interference The crRNA molecules combine with a set of Cas proteins to form a CRISPR-associated ribonucleoprotein (crRNP) complex. This complex scans incoming DNA and RNA for complementary sequences to the crRNA. If a match is found, the Cas proteins cut the target DNA or RNA, thereby preventing viral replication.
- 8. CRISPR-CAS9 MECHANISM OF ACTION The CRISPR-Cas9 system consists of two key molecules that introduce a change (mutation?) into the DNA. These are: An enzyme called Cas9. This acts as a pair of ‘molecular scissors’ that can cut the two strands of DNA at a specific location in the genome so that bits of DNA can then be added or removed. A piece of RNA called guide RNA (gRNA). This consists of a small piece of pre-designed RNA sequence (about 20 bases long) located within a longer RNA scaffold. The scaffold part binds to DNA and the pre-designed sequence ‘guides’ Cas9 to the right part of the genome. This makes sure that the Cas9 enzyme cuts at the right point in the genome. The guide RNA is designed to find and bind to a specific sequence in the DNA. The guide RNA has RNA bases that are complementary to those of the target DNA sequence in the genome. This means that, at least in theory, the guide RNA will only bind to the target sequence and no other regions of the genome. The Cas9 follows the guide RNA to the same location in the DNA sequence and makes a cut across both strands of the DNA. At this stage the cell recognises that the DNA is damaged and tries to repair it.
- 9. APPLICATIONS OF CRISPR CAS9 To date, CRISPR-Cas9 has been commonly used to create gene editing in plants, animal, and human samples. This technique is widely used in various scientific fields, including medical science and therapeutics, as well as plant and animal sciences.
- 10. 1.CREATE ANTI-MALARIA MOSQUITOES Fighting malaria by eliminating the mosquitoes that carry out the disease genetically are modified to not infect humans using crispr technology. Mosquitoes performing disinfection. Genetically modified mosquitoes that do not infect humans are inherited by nearly 100% of the offspring.
- 11. 2.CONCEIVING “IMPROVED” BABIES 3.CULTIVATING HUMAN ORGANS IN PIGS 4.BRING EXTINCT SPECIES BACK TO LIFE 5.CURE DISEASES 6.BOOST PLANTS AND IMPROVE THEIR QUALITIES the first baby genetically modified to immunize him against the AIDS virus by deactivating a gene called CCR5 Human organs grown in genetically modified pigs. Bringing the mammoth, the Tasmanian tiger or the back to life thanks to CRISPR-Cas9. injecting genetically modified T cells into a patient with lung cancer so that they recognize and attack tumor modifying plants without integrating foreign genes, CRISPR could revolutionize varietal improvement while avoiding the
- 12. DANGERS OF CRISPR CAS9 UNINTENDED CONSEQUENCES INCOMPLETE EDITS OFF-TARGET EFFECTS IMMUNE RESPONSE
- 13. E TH ICS O F C R ISPR CAS9 Safety: One of the most pressing ethical concerns related to CRISPR-Cas9 is safety. Ethical limits: Finally, there is a broader ethical question about the appropriate use of CRISPR- Cas9. Some argue that there should be limits on the use of the technology Access to treatment: There is also concern about unequal access to CRISPR-Cas9 treatment. Germline editing: The use of CRISPR-Cas9 to edit the genes of embryos or sperm and eggs raises concerns about the long- term effects of such changes. Informed consent: Another important ethical consideration is informed consent. Patients who receive CRISPR-Cas9 treatment must be fully informed about the risks and benefits of the treatment
- 14. CONCLUSION The CRISPR-Cas9 system offers several advantages over other genome editing techniques, including its ease of use and high specificity, meaning it can target specific genes without affecting other parts of the genome. As a result, it has become a popular tool for biomedical research and is being explored as a potential treatment for genetic diseases. With so many invigorating possibilities for this exciting new technology, it will be fascinating to see which of these major diseases and issues will be solved first, signaling the dawn of a new era in molecular biology
- 15. THANK YOU.
Editor's Notes
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- In gene editing, the Cas9 enzyme is programmed to recognize and cut specific DNA sequences, allowing researchers to selectively modify or delete genes in living organisms. The Cas9 enzyme is one of the key components of the CRISPR system and has revolutionized the field of genetic engineering.
- Atsuo Nakata is a researcher who has contributed to the development of the CRISPR-Cas gene editing technology. CRISPR-Cas is a revolutionary tool that allows scientists to modify DNA with unprecedented precision, and it has the potential to revolutionize fields such as medicine, agriculture, and biotechnology.
- Helicases are enzymes that play a crucial role in unwinding and separating the two strands of DNA or RNA molecules during various cellular processes, such as DNA replication, repair, recombination, and transcription. They belong to the class of enzymes known as nucleic acid motor proteins, which consume energy from ATP hydrolysis to perform mechanical work on nucleic acids. The helicases use the energy derived from ATP hydrolysis to destabilize the base pairs holding the two strands of nucleic acid together, resulting in the unwinding of the double-stranded structure. These enzymes typically have a characteristic motor domain that binds and hydrolyzes ATP, and a helicase domain that physically interacts with the nucleic acid and catalyzes the unwinding process. There are several types of helicases, each with a specific role in different cellular processes, such as DNA replication, DNA repair, RNA transcription, RNA splicing, and RNA decay. Helicases are essential for maintaining the integrity and stability of the genome, and their dysfunction has been linked to various diseases, including cancer and genetic disorders. Nucleases are enzymes that catalyze the breakdown of nucleic acids, which are the building blocks of DNA and RNA. These enzymes hydrolyze the phosphodiester bonds that connect the nucleotides in the nucleic acid chain, resulting in the cleavage of the chain into smaller fragments. There are two main types of nucleases: endonucleases and exonucleases. Endonucleases cleave the nucleic acid chain at specific internal sites, whereas exonucleases cleave the nucleic acid chain at the ends. Endonucleases are often used for genetic engineering and molecular biology applications, such as restriction enzymes used for DNA cloning, and CRISPR-Cas nucleases used for gene editing. Exonucleases, on the other hand, play a critical role in DNA replication, DNA repair, and RNA processing by removing nucleotides from the ends of the DNA or RNA molecule. For example, DNA polymerases, which replicate DNA, contain a 5' to 3' exonuclease activity that enables proofreading and correction of replication errors. Nucleases are also used in various diagnostic and therapeutic applications, such as in the detection of viral infections or in the treatment of cancer. In summary, nucleases play a crucial role in many biological processes and have a wide range of applications in research, biotechnology, and medicine.
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- The applications of CRISPR-Cas9 are vast and varied, ranging from basic research to potential clinical therapies. Some of the most promising applications include: Genome editing: CRISPR-Cas9 can be used to edit the genome of various organisms, including humans, to correct disease-causing mutations or introduce beneficial traits. Gene therapy: CRISPR-Cas9 can be used to directly edit genes in living cells, potentially leading to new therapies for genetic diseases. Disease modeling: CRISPR-Cas9 can be used to create animal models of human diseases, allowing researchers to study the underlying mechanisms of diseases and test potential therapies. Agricultural biotechnology: CRISPR-Cas9 can be used to modify crops to be more resistant to pests and environmental stresses or to improve their nutritional content. Biotechnology: CRISPR-Cas9 can be used to create new enzymes and proteins, potentially leading to new biotech products and industries. the potential applications of CRISPR-Cas9 are vast and varied, and its impact on fields such as medicine, agriculture, and biotechnology could be enormous
- Advantages: CRISPR-Cas9 has numerous advantages over previous genome editing technologies. Some of the key advantages include: Precision: CRISPR-Cas9 is incredibly precise and allows scientists to target specific DNA sequences with unprecedented accuracy. Simplicity: Compared to other genome editing techniques, CRISPR-Cas9 is relatively simple and easy to use, making it accessible to a wider range of researchers. Versatility: CRISPR-Cas9 can be used to edit the genomes of a wide range of organisms, including humans, animals, and plants. Efficiency: CRISPR-Cas9 is highly efficient, allowing researchers to achieve high rates of editing in a relatively short amount of time. Cost-effectiveness: Compared to other genome editing technologies, CRISPR-Cas9 is relatively inexpensive, making it more accessible to researchers with limited funding.
- . Limitations: Despite its many advantages, CRISPR-Cas9 also has some limitations that must be considered. Some of the key limitations include: Off-target effects: CRISPR-Cas9 can sometimes cut DNA at unintended locations, leading to unintended changes in the genome. Mosaicism: When editing embryos or other multicellular organisms, not all cells may be edited equally, leading to mosaicism and potential health concerns. Ethical concerns: The use of CRISPR-Cas9 in germline editing, or the modification of DNA that can be passed down to future generations, raises ethical concerns around the potential risks and implications of such modifications. Delivery challenges: Delivering CRISPR-Cas9 to specific cells or tissues can be challenging and may require the use of viral vectors, which can pose their own safety concerns. Patent disputes: There are ongoing patent disputes surrounding the use of CRISPR-Cas9, which could potentially limit access to the technology for certain researchers or companies