Emerging Clinical Applications of CRISPR-Cas9 as Promising Strategies in Gene...Chi-Ping Day
CRISPR/Cas9 gene editing shows promise for correcting diseases through gene therapy and targeted changes. Current applications include editing T-cells to produce CAR T-cells for cancer therapy and editing retinal cells in rats to treat retinitis pigmentosa. Additional areas of focus include eliminating HIV from cells, correcting the Fah mutation in mice, and overcoming issues like delivery efficiency and off-target effects. Looking ahead, cell therapies and local treatments for eye/ear diseases appear promising applications for CRISPR's precise gene editing capabilities.
Genome Editing and CRISPR-Cas 9 by Maliha Rashid.pptxMaliha Rashid
An extensive presentation on the article: "Mechanism and Applications of CRISPR/
Cas-9-Mediated Genome Editing". DOI: https://doi.org/10.2147/BTT.S326422
CONTENTS:
Components of CRISPR
Mechanism of CRISPR/Cas 9 Genome editing
Applications of CRISPR-Cas-9
Role in gene therapy
Therapeutic Role
Role in agriculture
Role in gene silencing and activation
Base Editors
Prime Editors
Challenges for CRISPR/Cas -9 application
Recent advances
Conclusion
This document summarizes a presentation on using CRISPR-Cas9 for crop improvement. It begins with an introduction to CRISPR-Cas9 and how it is used to edit genomes by removing, adding, or altering DNA sequences. It then discusses the mechanism of the CRISPR-Cas9 complex and how it creates breaks in DNA that are repaired. The document reviews several case studies where CRISPR was used to modify crops, including creating low-gluten wheat and improving rice. It finds that CRISPR can efficiently edit multiple genes simultaneously with few off-target effects. The conclusion states that CRISPR is revolutionizing agriculture by enabling the creation of higher yielding, more resistant crop varieties without transgenes.
Gene therapy involves introducing genetic material into host cells to treat diseases caused by genetic mutations. It has been used to treat several conditions like ADA-SCID, cystic fibrosis, and inherited retinal diseases. Various gene delivery systems exist including viral vectors like adenovirus, AAV, and retroviruses. Strategies for gene therapy include gene augmentation, inhibition, targeting, assisted killing, and prodrug therapy. While promising, gene therapy still faces challenges like improving delivery methods and reducing immune responses.
The document provides an overview of the CRISPR/Cas9 gene editing technology. It discusses the history and components of the CRISPR system, how it works, applications in various fields like microbiology, biomedicine, agriculture, and therapeutics. Recent advances expand its use for transcriptional regulation, epigenetic editing, and live imaging. While powerful, it faces challenges like off-target effects that require further research to optimize its safe and ethical application.
This document discusses various topics related to gene therapy including its applications in cancer, HIV, and hemophilia. It discusses CAR receptor technology, the HACE lipoplex method, CRISPR/Cas9, mRNA vaccines, and the immune response to gene therapy. It also summarizes the case of Jesse Gelsinger who died in a gene therapy trial for a rare metabolic disorder and methods of gene knockout.
Application of crispr in cancer therapykamran javidi
Many bacterial clustered regularly interspaced short palindromic repeats (CRISPR)–CRISPR-associated (Cas) systems employ the dual RNA–guided DNA endonuclease Cas9 to defend against invading phages and conjugative plasmids by introducing site-specific double-stranded breaks in target DNA. Target recognition strictly requires the presence of a short protospacer adjacent motif (PAM) flanking the target site, and subsequent R-loop formation and strand scission are driven by complementary base pairing between the guide RNA and target DNA, Cas9–DNA interactions, and associated conformational changes. The use of CRISPR–Cas9 as an RNA-programmable
DNA targeting and editing platform is simplified by a synthetic single-guide RNA (sgRNA) mimicking the natural dual trans-activating CRISPR RNA (tracrRNA)–CRISPR RNA (crRNA) structure
Emerging Clinical Applications of CRISPR-Cas9 as Promising Strategies in Gene...Chi-Ping Day
CRISPR/Cas9 gene editing shows promise for correcting diseases through gene therapy and targeted changes. Current applications include editing T-cells to produce CAR T-cells for cancer therapy and editing retinal cells in rats to treat retinitis pigmentosa. Additional areas of focus include eliminating HIV from cells, correcting the Fah mutation in mice, and overcoming issues like delivery efficiency and off-target effects. Looking ahead, cell therapies and local treatments for eye/ear diseases appear promising applications for CRISPR's precise gene editing capabilities.
Genome Editing and CRISPR-Cas 9 by Maliha Rashid.pptxMaliha Rashid
An extensive presentation on the article: "Mechanism and Applications of CRISPR/
Cas-9-Mediated Genome Editing". DOI: https://doi.org/10.2147/BTT.S326422
CONTENTS:
Components of CRISPR
Mechanism of CRISPR/Cas 9 Genome editing
Applications of CRISPR-Cas-9
Role in gene therapy
Therapeutic Role
Role in agriculture
Role in gene silencing and activation
Base Editors
Prime Editors
Challenges for CRISPR/Cas -9 application
Recent advances
Conclusion
This document summarizes a presentation on using CRISPR-Cas9 for crop improvement. It begins with an introduction to CRISPR-Cas9 and how it is used to edit genomes by removing, adding, or altering DNA sequences. It then discusses the mechanism of the CRISPR-Cas9 complex and how it creates breaks in DNA that are repaired. The document reviews several case studies where CRISPR was used to modify crops, including creating low-gluten wheat and improving rice. It finds that CRISPR can efficiently edit multiple genes simultaneously with few off-target effects. The conclusion states that CRISPR is revolutionizing agriculture by enabling the creation of higher yielding, more resistant crop varieties without transgenes.
Gene therapy involves introducing genetic material into host cells to treat diseases caused by genetic mutations. It has been used to treat several conditions like ADA-SCID, cystic fibrosis, and inherited retinal diseases. Various gene delivery systems exist including viral vectors like adenovirus, AAV, and retroviruses. Strategies for gene therapy include gene augmentation, inhibition, targeting, assisted killing, and prodrug therapy. While promising, gene therapy still faces challenges like improving delivery methods and reducing immune responses.
The document provides an overview of the CRISPR/Cas9 gene editing technology. It discusses the history and components of the CRISPR system, how it works, applications in various fields like microbiology, biomedicine, agriculture, and therapeutics. Recent advances expand its use for transcriptional regulation, epigenetic editing, and live imaging. While powerful, it faces challenges like off-target effects that require further research to optimize its safe and ethical application.
This document discusses various topics related to gene therapy including its applications in cancer, HIV, and hemophilia. It discusses CAR receptor technology, the HACE lipoplex method, CRISPR/Cas9, mRNA vaccines, and the immune response to gene therapy. It also summarizes the case of Jesse Gelsinger who died in a gene therapy trial for a rare metabolic disorder and methods of gene knockout.
Application of crispr in cancer therapykamran javidi
Many bacterial clustered regularly interspaced short palindromic repeats (CRISPR)–CRISPR-associated (Cas) systems employ the dual RNA–guided DNA endonuclease Cas9 to defend against invading phages and conjugative plasmids by introducing site-specific double-stranded breaks in target DNA. Target recognition strictly requires the presence of a short protospacer adjacent motif (PAM) flanking the target site, and subsequent R-loop formation and strand scission are driven by complementary base pairing between the guide RNA and target DNA, Cas9–DNA interactions, and associated conformational changes. The use of CRISPR–Cas9 as an RNA-programmable
DNA targeting and editing platform is simplified by a synthetic single-guide RNA (sgRNA) mimicking the natural dual trans-activating CRISPR RNA (tracrRNA)–CRISPR RNA (crRNA) structure
This document summarizes information about the CRISPR Cas9 genome editing tool. It discusses how CRISPR Cas9 uses guide RNA and the Cas9 enzyme to create targeted double-strand breaks in DNA, allowing genes to be knocked out or altered. The document outlines the history and mechanism of CRISPR Cas9, compares it to other genome editing tools, discusses its applications in plant breeding including reducing off-target effects, and provides an example of using it to create parthenocarpic tomato plants.
Dr. Chris Lowe presented on Horizon Discovery's precision genome editing platform called GENESISTM. The presentation discussed optimizing GENESISTM by combining CRISPR and rAAV technologies to improve gene targeting efficiency. Custom cell line development services are offered to modify genes of interest in various cell lines for applications such as generating disease models and studying drug sensitivity. Key considerations for successful gene editing experiments include factors like gene/cell line selection, gRNA design/activity, donor design, screening/validation approaches. Case studies demonstrated applications of engineered cell lines.
Genome Editing & Gene Therapy by Eric KelsicImpact.Tech
Slides from the Genome editing & gene therapy Impact.tech seminar, hosted by Eric Kelsic on June 11th, 2019.
The seminar covers the experiments and inventions that led to the development of genome editing technologies. These inventions were derived from life itself: isolated from natural organisms and adapted for scientific and therapeutic goals. You will learn the history of how genome engineering tools, including CRISPR, and delivery technology, including AAV capsids, were created in their modern form. The seminar explores how genome editing and gene therapy technologies are giving individuals control over their own genomes, focusing on the treatment of genetic diseases. It will describe major companies and emerging trends in the gene therapy industry. Finally, the seminar will discuss how and where new discoveries, including accelerated algorithms for genetic engineering, will lead us in the near and distant future.
Eric Kelsic, PhD, is the founder and CEO of Dyno Therapeutics, a VC-backed biotech located in Cambridge, Massachusetts. Dyno is leading a machine learning revolution to develop enhanced capsid proteins that enable new gene and genome editing therapies. Eric co-developed the technology underlying Dyno’s machine-guided protein engineering platform as a Staff Scientist in George Church’s lab at the Wyss Institute of Harvard Medical School. He holds a PhD in Systems Biology from Harvard University and a BS in Physics from Caltech.
Intellia Therapeutics is a biotechnology company focused on developing gene editing treatments using CRISPR/Cas9 technology. They have over 100 employees and are located in Cambridge, Massachusetts. Their initial focus is on delivering CRISPR to the liver to treat diseases like transthyretin amyloidosis, alpha-1 antitrypsin deficiency, and hepatitis B. They are also exploring delivering CRISPR to other tissues and cells, both in vivo and ex vivo, to treat diseases like hemoglobinopathies and cancer. They face technical, regulatory, and public perception challenges in developing these novel genetic medicines.
CRISPR Cas9 is a genome editing technology that allows genetic material to be added, removed, or altered from a genome. It originated as a bacterial immune system but can now be directed to make precise edits to DNA. The technology has wide applications for gene therapy, agriculture, research, and more, but also raises ethical concerns if misused. CRISPR offers promising possibilities but also challenges that must be addressed regarding safety, accuracy, and societal effects.
This document provides an introduction and overview of a refresher course in molecular biology and bioinformatics. The course aims to provide a solid introduction to molecular biology techniques and bioinformatics tools currently used to investigate molecular mechanisms and their application to disease diagnosis and treatment. It will introduce concepts like genomics, transcriptomics, proteomics, epigenetics, genome editing with CRISPR, molecular cloning, and genome-wide association studies. The course also discusses various molecular biology techniques like DNA sequencing, PCR, immunohistochemistry and their role in furthering our understanding of human disease at the molecular level to improve patient care through more accurate diagnosis and targeted therapies.
Understanding and controlling for sample and platform biases in NGS assaysCandy Smellie
What is the impact of assay failure in your laboratory and how do you monitor for it?
The advancement of next-generation sequencing has provided invaluable resources to researchers in multiple industries and disciplines, and will be a major driver during the personalized medicine revolution that is upon us. However, while the cost of generating sequencing data continues to decrease this does not take into account the significant costs associated with the infrastructure and expertise that are required to develop a robust, routine NGS pipeline.
Specifically, as predicted by Sboner, et al in 2011, the cost of the sequencing portion of the experiment continues to decrease and the costs associated with upfront experimental design and downstream analysis dominate the cost of each assay. This is true whether you are performing a pre-clinical R&D project, and perhaps even more so for clinical assays. In the paper, the authors note the unpredictable and considerable ‘human time’ spent on the upstream design and downstream analysis. Here at Horizon, we aim to develop tools that help researchers and clinicians optimize these workflows to make NGS more reliable and ultimately, more affordable by streamlining these resource intensive areas.
Genome editing with engineered nucleasesKrishan Kumar
Genome editing uses engineered nucleases to insert, replace or remove DNA from the genome. These nucleases create targeted double-strand breaks which are repaired through natural DNA repair processes, allowing for changes to the genome sequence. Three main engineered nuclease systems for genome editing are ZFNs, TALENs, and CRISPR-Cas9. CRISPR uses a guide RNA and Cas9 nuclease to make precise cuts at targeted DNA sequences for editing. It has advantages over ZFNs and TALENs in being cheaper, easier to design, and more efficient. Genome editing holds promise for applications in crops, medicine, and research.
Molecular diagnostic techniques involve manipulating and analyzing DNA, RNA, and proteins. These techniques are used in areas like neoplastic disorders, infectious diseases, and inherited conditions. Common techniques include amplification methods like PCR, blotting techniques such as Southern blot and Western blot, and hybridization methods like in situ hybridization and microarrays. Molecular diagnostics provides advantages like automation, speed, reliability, and sensitivity, though limitations include potential errors and sensitivity to inhibitors. These techniques have revolutionized fields like medicine, agriculture, forensics, and more by enabling abundant production of DNA and detection of genetic disorders.
CRISPR is genome editing technique mainly based on a natural immune process used by bacteria to defend themselves against invading viruses. Genome editing is the use of various technologies to make a permanent changes in the genomic DNA sequence of a cell or organism.
CRISPR in crop Improvement, CRISPR/Cas Genome editing toolParthasarathiG2
This document discusses the use of CRISPR-Cas9 genome editing in crop improvement. It begins with an introduction to CRISPR-Cas9 and its mechanism of action. It then discusses the discovery of CRISPR and key scientists involved. Several case studies on using CRISPR to edit rice genes for disease resistance and hybrid seed production are summarized. Achievements using CRISPR in rice, horticulture crops, and other field crops are briefly outlined. The document concludes that CRISPR provides a simple and efficient tool for genome editing in plants.
This document provides information on CRISPR Cas9 genome editing. It discusses the history and discovery of CRISPR dating back to 1987. It describes the key components of the CRISPR Cas9 system including Cas9 proteins, CRISPR RNA, protospacers, and PAM sequences. The mechanisms of how CRISPR Cas9 edits genomes through double strand breaks is explained. Finally, applications of CRISPR Cas9 are summarized, including using it to correct genetic mutations causing diseases in animals and potential applications in humans.
Gene therapy involves introducing genetic material into cells to treat or prevent disease. It has the potential to cure genetic disorders by correcting the underlying genetic defect. There are two main types - somatic gene therapy, which affects only targeted cells and is safer, and germline gene therapy which can permanently alter the genes and be passed to offspring. Recent advances include FDA-approved CAR-T immunotherapies for cancer and the first gene therapy approved for an inherited retinal disease. Challenges remain regarding delivery methods, safety, and ethical issues.
This document discusses newer drug delivery systems that provide targeted and sustained drug release. It begins by introducing drug delivery systems and their basic parameters of route of entry and dosage form. It then discusses the need for multidisciplinary approaches to drug delivery due to slow progress treating severe diseases. Newer systems aim for targeted delivery and sustained release formulations. These systems increase efficacy, provide site-specific delivery, decrease toxicity and side effects, and improve convenience and patient compliance. The document goes on to describe various drug delivery systems including carrier-based, transdermal, mucoadhesive, and osmotically-controlled systems. It provides examples of specific drug delivery carriers, formulations, and medical devices. In general, these newer systems provide improvements
CRISPR-Cas9 system a tool for gene editing presentation RashmiSharma304
CRISPR Cas9 System for Gene Editing
The document summarizes CRISPR Cas9 gene editing. It discusses the timeline of CRISPR discoveries from 1987-2019. It describes the classification, structure, and mechanism of the CRISPR Cas9 system. Applications discussed include using CRISPR in bacteria, wheat, and to alter muscle mass in humans. Ethical concerns regarding uses in human embryos and potential for non-medical enhancement are also covered.
Chronic granulomatous disease is a rare inherited disorder characterized by defects in the NADPH oxidase system, which leads to recurrent infections. It is caused by mutations affecting components of the NADPH oxidase enzyme complex, resulting in the inability of phagocytes to produce reactive oxygen species to kill certain bacteria and fungi. Patients present with recurrent infections of the lungs, skin, lymph nodes, liver or bones by catalase-positive organisms. Treatment involves lifelong antibiotic prophylaxis, with hematopoietic stem cell transplantation or gene therapy as curative options.
CRISPR/Cas9 is an advanced genome editing technology that can be used to develop plant disease resistance. It involves a Cas9 enzyme that acts like molecular scissors to cut DNA at specific locations guided by CRISPR RNA. This triggers DNA repair that can introduce changes to genes. Researchers have used CRISPR/Cas9 to develop resistance in plants against viruses, fungi, and bacteria by editing genes involved in host-pathogen interaction and disease susceptibility. It provides a precise and efficient way to edit plant genomes to improve crop resistance compared to previous tools. Scientists continue working to enhance the specificity and control of CRISPR/Cas9 for genome editing applications in agriculture.
This study aimed to optimize the CRISPR/Cas9 genome editing protocol for efficient homozygous gene knock-in in human induced pluripotent stem cells (iPSCs). The researchers targeted the CD90 locus for replacement with the mouse ortholog Cd90 and tested various experimental conditions. After optimization, CRISPR efficiency increased from 0.28% to 11.8% homozygous knock-in as determined by flow cytometry. Key conditions implicated in higher efficiency included plasmid concentrations and quality, Cas9 delivery method, nucleofection device, recovery conditions, and cell concentration during nucleofection.
This document provides an introduction to CRISPR-Cas9 technology. It discusses the history of CRISPR discovery from 1987 onwards. CRISPR-Cas9 allows for efficient and precise genome editing in bacteria and eukaryotic cells. It works by using CRISPR sequences and a Cas9 enzyme to cut DNA at specific sites. The document outlines the different types of CRISPR systems and Cas9 nucleases, and describes the biological mechanism of Cas9. It lists applications of CRISPR including gene silencing and editing. In conclusion, CRISPR is a powerful new tool for gene editing but also raises safety, ethical and regulatory questions.
Basavarajeeyam is a Sreshta Sangraha grantha (Compiled book ), written by Neelkanta kotturu Basavaraja Virachita. It contains 25 Prakaranas, First 24 Chapters related to Rogas& 25th to Rasadravyas.
These lecture slides, by Dr Sidra Arshad, offer a quick overview of the physiological basis of a normal electrocardiogram.
Learning objectives:
1. Define an electrocardiogram (ECG) and electrocardiography
2. Describe how dipoles generated by the heart produce the waveforms of the ECG
3. Describe the components of a normal electrocardiogram of a typical bipolar lead (limb II)
4. Differentiate between intervals and segments
5. Enlist some common indications for obtaining an ECG
6. Describe the flow of current around the heart during the cardiac cycle
7. Discuss the placement and polarity of the leads of electrocardiograph
8. Describe the normal electrocardiograms recorded from the limb leads and explain the physiological basis of the different records that are obtained
9. Define mean electrical vector (axis) of the heart and give the normal range
10. Define the mean QRS vector
11. Describe the axes of leads (hexagonal reference system)
12. Comprehend the vectorial analysis of the normal ECG
13. Determine the mean electrical axis of the ventricular QRS and appreciate the mean axis deviation
14. Explain the concepts of current of injury, J point, and their significance
Study Resources:
1. Chapter 11, Guyton and Hall Textbook of Medical Physiology, 14th edition
2. Chapter 9, Human Physiology - From Cells to Systems, Lauralee Sherwood, 9th edition
3. Chapter 29, Ganong’s Review of Medical Physiology, 26th edition
4. Electrocardiogram, StatPearls - https://www.ncbi.nlm.nih.gov/books/NBK549803/
5. ECG in Medical Practice by ABM Abdullah, 4th edition
6. Chapter 3, Cardiology Explained, https://www.ncbi.nlm.nih.gov/books/NBK2214/
7. ECG Basics, http://www.nataliescasebook.com/tag/e-c-g-basics
This document summarizes information about the CRISPR Cas9 genome editing tool. It discusses how CRISPR Cas9 uses guide RNA and the Cas9 enzyme to create targeted double-strand breaks in DNA, allowing genes to be knocked out or altered. The document outlines the history and mechanism of CRISPR Cas9, compares it to other genome editing tools, discusses its applications in plant breeding including reducing off-target effects, and provides an example of using it to create parthenocarpic tomato plants.
Dr. Chris Lowe presented on Horizon Discovery's precision genome editing platform called GENESISTM. The presentation discussed optimizing GENESISTM by combining CRISPR and rAAV technologies to improve gene targeting efficiency. Custom cell line development services are offered to modify genes of interest in various cell lines for applications such as generating disease models and studying drug sensitivity. Key considerations for successful gene editing experiments include factors like gene/cell line selection, gRNA design/activity, donor design, screening/validation approaches. Case studies demonstrated applications of engineered cell lines.
Genome Editing & Gene Therapy by Eric KelsicImpact.Tech
Slides from the Genome editing & gene therapy Impact.tech seminar, hosted by Eric Kelsic on June 11th, 2019.
The seminar covers the experiments and inventions that led to the development of genome editing technologies. These inventions were derived from life itself: isolated from natural organisms and adapted for scientific and therapeutic goals. You will learn the history of how genome engineering tools, including CRISPR, and delivery technology, including AAV capsids, were created in their modern form. The seminar explores how genome editing and gene therapy technologies are giving individuals control over their own genomes, focusing on the treatment of genetic diseases. It will describe major companies and emerging trends in the gene therapy industry. Finally, the seminar will discuss how and where new discoveries, including accelerated algorithms for genetic engineering, will lead us in the near and distant future.
Eric Kelsic, PhD, is the founder and CEO of Dyno Therapeutics, a VC-backed biotech located in Cambridge, Massachusetts. Dyno is leading a machine learning revolution to develop enhanced capsid proteins that enable new gene and genome editing therapies. Eric co-developed the technology underlying Dyno’s machine-guided protein engineering platform as a Staff Scientist in George Church’s lab at the Wyss Institute of Harvard Medical School. He holds a PhD in Systems Biology from Harvard University and a BS in Physics from Caltech.
Intellia Therapeutics is a biotechnology company focused on developing gene editing treatments using CRISPR/Cas9 technology. They have over 100 employees and are located in Cambridge, Massachusetts. Their initial focus is on delivering CRISPR to the liver to treat diseases like transthyretin amyloidosis, alpha-1 antitrypsin deficiency, and hepatitis B. They are also exploring delivering CRISPR to other tissues and cells, both in vivo and ex vivo, to treat diseases like hemoglobinopathies and cancer. They face technical, regulatory, and public perception challenges in developing these novel genetic medicines.
CRISPR Cas9 is a genome editing technology that allows genetic material to be added, removed, or altered from a genome. It originated as a bacterial immune system but can now be directed to make precise edits to DNA. The technology has wide applications for gene therapy, agriculture, research, and more, but also raises ethical concerns if misused. CRISPR offers promising possibilities but also challenges that must be addressed regarding safety, accuracy, and societal effects.
This document provides an introduction and overview of a refresher course in molecular biology and bioinformatics. The course aims to provide a solid introduction to molecular biology techniques and bioinformatics tools currently used to investigate molecular mechanisms and their application to disease diagnosis and treatment. It will introduce concepts like genomics, transcriptomics, proteomics, epigenetics, genome editing with CRISPR, molecular cloning, and genome-wide association studies. The course also discusses various molecular biology techniques like DNA sequencing, PCR, immunohistochemistry and their role in furthering our understanding of human disease at the molecular level to improve patient care through more accurate diagnosis and targeted therapies.
Understanding and controlling for sample and platform biases in NGS assaysCandy Smellie
What is the impact of assay failure in your laboratory and how do you monitor for it?
The advancement of next-generation sequencing has provided invaluable resources to researchers in multiple industries and disciplines, and will be a major driver during the personalized medicine revolution that is upon us. However, while the cost of generating sequencing data continues to decrease this does not take into account the significant costs associated with the infrastructure and expertise that are required to develop a robust, routine NGS pipeline.
Specifically, as predicted by Sboner, et al in 2011, the cost of the sequencing portion of the experiment continues to decrease and the costs associated with upfront experimental design and downstream analysis dominate the cost of each assay. This is true whether you are performing a pre-clinical R&D project, and perhaps even more so for clinical assays. In the paper, the authors note the unpredictable and considerable ‘human time’ spent on the upstream design and downstream analysis. Here at Horizon, we aim to develop tools that help researchers and clinicians optimize these workflows to make NGS more reliable and ultimately, more affordable by streamlining these resource intensive areas.
Genome editing with engineered nucleasesKrishan Kumar
Genome editing uses engineered nucleases to insert, replace or remove DNA from the genome. These nucleases create targeted double-strand breaks which are repaired through natural DNA repair processes, allowing for changes to the genome sequence. Three main engineered nuclease systems for genome editing are ZFNs, TALENs, and CRISPR-Cas9. CRISPR uses a guide RNA and Cas9 nuclease to make precise cuts at targeted DNA sequences for editing. It has advantages over ZFNs and TALENs in being cheaper, easier to design, and more efficient. Genome editing holds promise for applications in crops, medicine, and research.
Molecular diagnostic techniques involve manipulating and analyzing DNA, RNA, and proteins. These techniques are used in areas like neoplastic disorders, infectious diseases, and inherited conditions. Common techniques include amplification methods like PCR, blotting techniques such as Southern blot and Western blot, and hybridization methods like in situ hybridization and microarrays. Molecular diagnostics provides advantages like automation, speed, reliability, and sensitivity, though limitations include potential errors and sensitivity to inhibitors. These techniques have revolutionized fields like medicine, agriculture, forensics, and more by enabling abundant production of DNA and detection of genetic disorders.
CRISPR is genome editing technique mainly based on a natural immune process used by bacteria to defend themselves against invading viruses. Genome editing is the use of various technologies to make a permanent changes in the genomic DNA sequence of a cell or organism.
CRISPR in crop Improvement, CRISPR/Cas Genome editing toolParthasarathiG2
This document discusses the use of CRISPR-Cas9 genome editing in crop improvement. It begins with an introduction to CRISPR-Cas9 and its mechanism of action. It then discusses the discovery of CRISPR and key scientists involved. Several case studies on using CRISPR to edit rice genes for disease resistance and hybrid seed production are summarized. Achievements using CRISPR in rice, horticulture crops, and other field crops are briefly outlined. The document concludes that CRISPR provides a simple and efficient tool for genome editing in plants.
This document provides information on CRISPR Cas9 genome editing. It discusses the history and discovery of CRISPR dating back to 1987. It describes the key components of the CRISPR Cas9 system including Cas9 proteins, CRISPR RNA, protospacers, and PAM sequences. The mechanisms of how CRISPR Cas9 edits genomes through double strand breaks is explained. Finally, applications of CRISPR Cas9 are summarized, including using it to correct genetic mutations causing diseases in animals and potential applications in humans.
Gene therapy involves introducing genetic material into cells to treat or prevent disease. It has the potential to cure genetic disorders by correcting the underlying genetic defect. There are two main types - somatic gene therapy, which affects only targeted cells and is safer, and germline gene therapy which can permanently alter the genes and be passed to offspring. Recent advances include FDA-approved CAR-T immunotherapies for cancer and the first gene therapy approved for an inherited retinal disease. Challenges remain regarding delivery methods, safety, and ethical issues.
This document discusses newer drug delivery systems that provide targeted and sustained drug release. It begins by introducing drug delivery systems and their basic parameters of route of entry and dosage form. It then discusses the need for multidisciplinary approaches to drug delivery due to slow progress treating severe diseases. Newer systems aim for targeted delivery and sustained release formulations. These systems increase efficacy, provide site-specific delivery, decrease toxicity and side effects, and improve convenience and patient compliance. The document goes on to describe various drug delivery systems including carrier-based, transdermal, mucoadhesive, and osmotically-controlled systems. It provides examples of specific drug delivery carriers, formulations, and medical devices. In general, these newer systems provide improvements
CRISPR-Cas9 system a tool for gene editing presentation RashmiSharma304
CRISPR Cas9 System for Gene Editing
The document summarizes CRISPR Cas9 gene editing. It discusses the timeline of CRISPR discoveries from 1987-2019. It describes the classification, structure, and mechanism of the CRISPR Cas9 system. Applications discussed include using CRISPR in bacteria, wheat, and to alter muscle mass in humans. Ethical concerns regarding uses in human embryos and potential for non-medical enhancement are also covered.
Chronic granulomatous disease is a rare inherited disorder characterized by defects in the NADPH oxidase system, which leads to recurrent infections. It is caused by mutations affecting components of the NADPH oxidase enzyme complex, resulting in the inability of phagocytes to produce reactive oxygen species to kill certain bacteria and fungi. Patients present with recurrent infections of the lungs, skin, lymph nodes, liver or bones by catalase-positive organisms. Treatment involves lifelong antibiotic prophylaxis, with hematopoietic stem cell transplantation or gene therapy as curative options.
CRISPR/Cas9 is an advanced genome editing technology that can be used to develop plant disease resistance. It involves a Cas9 enzyme that acts like molecular scissors to cut DNA at specific locations guided by CRISPR RNA. This triggers DNA repair that can introduce changes to genes. Researchers have used CRISPR/Cas9 to develop resistance in plants against viruses, fungi, and bacteria by editing genes involved in host-pathogen interaction and disease susceptibility. It provides a precise and efficient way to edit plant genomes to improve crop resistance compared to previous tools. Scientists continue working to enhance the specificity and control of CRISPR/Cas9 for genome editing applications in agriculture.
This study aimed to optimize the CRISPR/Cas9 genome editing protocol for efficient homozygous gene knock-in in human induced pluripotent stem cells (iPSCs). The researchers targeted the CD90 locus for replacement with the mouse ortholog Cd90 and tested various experimental conditions. After optimization, CRISPR efficiency increased from 0.28% to 11.8% homozygous knock-in as determined by flow cytometry. Key conditions implicated in higher efficiency included plasmid concentrations and quality, Cas9 delivery method, nucleofection device, recovery conditions, and cell concentration during nucleofection.
This document provides an introduction to CRISPR-Cas9 technology. It discusses the history of CRISPR discovery from 1987 onwards. CRISPR-Cas9 allows for efficient and precise genome editing in bacteria and eukaryotic cells. It works by using CRISPR sequences and a Cas9 enzyme to cut DNA at specific sites. The document outlines the different types of CRISPR systems and Cas9 nucleases, and describes the biological mechanism of Cas9. It lists applications of CRISPR including gene silencing and editing. In conclusion, CRISPR is a powerful new tool for gene editing but also raises safety, ethical and regulatory questions.
Basavarajeeyam is a Sreshta Sangraha grantha (Compiled book ), written by Neelkanta kotturu Basavaraja Virachita. It contains 25 Prakaranas, First 24 Chapters related to Rogas& 25th to Rasadravyas.
These lecture slides, by Dr Sidra Arshad, offer a quick overview of the physiological basis of a normal electrocardiogram.
Learning objectives:
1. Define an electrocardiogram (ECG) and electrocardiography
2. Describe how dipoles generated by the heart produce the waveforms of the ECG
3. Describe the components of a normal electrocardiogram of a typical bipolar lead (limb II)
4. Differentiate between intervals and segments
5. Enlist some common indications for obtaining an ECG
6. Describe the flow of current around the heart during the cardiac cycle
7. Discuss the placement and polarity of the leads of electrocardiograph
8. Describe the normal electrocardiograms recorded from the limb leads and explain the physiological basis of the different records that are obtained
9. Define mean electrical vector (axis) of the heart and give the normal range
10. Define the mean QRS vector
11. Describe the axes of leads (hexagonal reference system)
12. Comprehend the vectorial analysis of the normal ECG
13. Determine the mean electrical axis of the ventricular QRS and appreciate the mean axis deviation
14. Explain the concepts of current of injury, J point, and their significance
Study Resources:
1. Chapter 11, Guyton and Hall Textbook of Medical Physiology, 14th edition
2. Chapter 9, Human Physiology - From Cells to Systems, Lauralee Sherwood, 9th edition
3. Chapter 29, Ganong’s Review of Medical Physiology, 26th edition
4. Electrocardiogram, StatPearls - https://www.ncbi.nlm.nih.gov/books/NBK549803/
5. ECG in Medical Practice by ABM Abdullah, 4th edition
6. Chapter 3, Cardiology Explained, https://www.ncbi.nlm.nih.gov/books/NBK2214/
7. ECG Basics, http://www.nataliescasebook.com/tag/e-c-g-basics
Integrating Ayurveda into Parkinson’s Management: A Holistic ApproachAyurveda ForAll
Explore the benefits of combining Ayurveda with conventional Parkinson's treatments. Learn how a holistic approach can manage symptoms, enhance well-being, and balance body energies. Discover the steps to safely integrate Ayurvedic practices into your Parkinson’s care plan, including expert guidance on diet, herbal remedies, and lifestyle modifications.
TEST BANK For Basic and Clinical Pharmacology, 14th Edition by Bertram G. Kat...rightmanforbloodline
TEST BANK For Basic and Clinical Pharmacology, 14th Edition by Bertram G. Katzung, Verified Chapters 1 - 66, Complete Newest Version.
TEST BANK For Basic and Clinical Pharmacology, 14th Edition by Bertram G. Katzung, Verified Chapters 1 - 66, Complete Newest Version.
TEST BANK For Basic and Clinical Pharmacology, 14th Edition by Bertram G. Katzung, Verified Chapters 1 - 66, Complete Newest Version.
TEST BANK For Basic and Clinical Pharmacology, 14th Edition by Bertram G. Katzung, Verified Chapters 1 - 66, Complete Newest Version.
Osteoporosis - Definition , Evaluation and Management .pdfJim Jacob Roy
Osteoporosis is an increasing cause of morbidity among the elderly.
In this document , a brief outline of osteoporosis is given , including the risk factors of osteoporosis fractures , the indications for testing bone mineral density and the management of osteoporosis
Does Over-Masturbation Contribute to Chronic Prostatitis.pptxwalterHu5
In some case, your chronic prostatitis may be related to over-masturbation. Generally, natural medicine Diuretic and Anti-inflammatory Pill can help mee get a cure.
Rasamanikya is a excellent preparation in the field of Rasashastra, it is used in various Kushtha Roga, Shwasa, Vicharchika, Bhagandara, Vatarakta, and Phiranga Roga. In this article Preparation& Comparative analytical profile for both Formulationon i.e Rasamanikya prepared by Kushmanda swarasa & Churnodhaka Shodita Haratala. The study aims to provide insights into the comparative efficacy and analytical aspects of these formulations for enhanced therapeutic outcomes.
Cell Therapy Expansion and Challenges in Autoimmune DiseaseHealth Advances
There is increasing confidence that cell therapies will soon play a role in the treatment of autoimmune disorders, but the extent of this impact remains to be seen. Early readouts on autologous CAR-Ts in lupus are encouraging, but manufacturing and cost limitations are likely to restrict access to highly refractory patients. Allogeneic CAR-Ts have the potential to broaden access to earlier lines of treatment due to their inherent cost benefits, however they will need to demonstrate comparable or improved efficacy to established modalities.
In addition to infrastructure and capacity constraints, CAR-Ts face a very different risk-benefit dynamic in autoimmune compared to oncology, highlighting the need for tolerable therapies with low adverse event risk. CAR-NK and Treg-based therapies are also being developed in certain autoimmune disorders and may demonstrate favorable safety profiles. Several novel non-cell therapies such as bispecific antibodies, nanobodies, and RNAi drugs, may also offer future alternative competitive solutions with variable value propositions.
Widespread adoption of cell therapies will not only require strong efficacy and safety data, but also adapted pricing and access strategies. At oncology-based price points, CAR-Ts are unlikely to achieve broad market access in autoimmune disorders, with eligible patient populations that are potentially orders of magnitude greater than the number of currently addressable cancer patients. Developers have made strides towards reducing cell therapy COGS while improving manufacturing efficiency, but payors will inevitably restrict access until more sustainable pricing is achieved.
Despite these headwinds, industry leaders and investors remain confident that cell therapies are poised to address significant unmet need in patients suffering from autoimmune disorders. However, the extent of this impact on the treatment landscape remains to be seen, as the industry rapidly approaches an inflection point.
Muktapishti is a traditional Ayurvedic preparation made from Shoditha Mukta (Purified Pearl), is believed to help regulate thyroid function and reduce symptoms of hyperthyroidism due to its cooling and balancing properties. Clinical evidence on its efficacy remains limited, necessitating further research to validate its therapeutic benefits.
4. To Discuss
• Gene therapy
• History of CRISPR
• Mechanism of CRISPR Cas 9 Technology
• Advantages of CRISPR
• Limitations of CRISPR
• Overcoming the Limitations
• Delivery of CRISPR gene therapy
• Application
• Summary 4
6. Why shift from traditional gene therapy to CRISPR
1. Immunotoxicity
2. Oncogenesis
• Examples :
1. Clinical trial ( in US ) non-mutated OTC ( Ornithine trans Carbamoylase ) gene was
delivered to the liver hepatic artery injection of the recombinant adenoviral vector
housing the therapeutic gene Jessie a 18 yr old with a mild form of OTC deficiency
participated in the trial 4 days after the trial he died ( Immunotoxicity from the
adenovirus vector )
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7. 2. Gene therapy trial
Use of retroviral vectors
Ex vivo delivery of therapeutic transgenes to autologous s CD34+ hematopoietic stem cells
5 Patients developed therapy related leukemia
( Integration of therapeutic gene into LMO2 protooncogene )
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16. Off target effects
• Off target effects : Unintended mutations at the site other than target
• Factors causing the off target effects :
1. Excess affinity between Cas 9 and the target DNA
2. Features in the sgRNA :
• Seed sequence : 10-12 bp region proximal to PAM ( Protospacer adjacent motif )
• GC content
3. Repair pathway involved ( NHEJ or HDR )
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17. Overcoming the off target effects
• SpCas9HF-1 variant ( High fidelity )
Introduces mutation to 4 residues involved in direct hydrogen bonding between Cas 9 and the
Phosphate backbone of target DNA
Reduces the excess affinity between Cas 9 and target DNA
• Evo Cas 9 and HiFi Cas 9
Altered amino acid residues in the Rec 3 domain ( Nucleotide recognition )
Increases the specificity to induce double stranded Breaks ( DSB ) 17
18. Overcoming the off target effects cont.…
• Cas9_R63A/Q768A Variant
R 63 A mutation destabilizes the R Loop formation In the presence of Mismatches
Q 768 Mutation increases the sensitivity to PAM Distal mismatches
• Optimize Guide design platforms such as :
1. E-Crisp
2. CRISPR-design
3. CasOFFinder
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19. Overcoming the DNA damage toxicity
• Crispr induced double stranded breaks :
1. Induce apoptosis rather than the desired gene edit ( p 53 Activation )
2. Large deletions and complex rearrangements
• Method of overcoming the DNA damage toxicity and off target effects is Precise genome
editing
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21. Overcoming Immunotoxicity
1. Using of Non Viral vectors ( Nanoparticles, Exosomes, Liposomes )
2. New Cas 9 Variants with reduced Immunogenic risk like Cj Cas 9 ( Cas 9 from
Campylobacter Jejuni )
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24. Advantages and Disadvantages of Ex Vivo Delivery
• Advantages :
1. Greater safety ( Patient not exposed to gene altering tool )
2. Technical feasibility
3. Tighter Quality control of the edited cells
• Disadvantages :
1. Survival and retention of in Vivo function of the cells outside the patient
2. Limitation of the method to certain cell type that can survive and be expanded in culture
(hematopoietic stem and progenitor cells (HSPCs) and T cells )
• This therapy provides benefits in Hematological disorder and Cancer immunotherapy24
25. Advantages and Disadvantages of In Vivo delivery
• Advantage :
• Expression of the gene editing toolkit can be controlled to target specific organs
• Disadvantage :
1. Degradation by circulating proteases or nucleases, opsonization by opsonin
2. Cargo must reach the target tissue and bypass the vascular endothelium (preventing
accessibility to larger delivery vehicles (>1 nm diameter).
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27. Disease Gene target CRISPR-Cas 9 mediated
intervention
Metastatic Non small cell lung
cancer
PDCD-1 CRISPR-Cas9 mediated PD-1
knockout-T cells
from autologous origin
Metastatic Renal Cell
Carcinoma
PDCD-1 CRISPR-Cas9 mediated PD-1
knockout-T cells from
autologous origin
Esophageal Cancer PDCD-1 CRISPR-Cas9 mediated PD-1
knockout-T cells from
autologous origin
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28. Covid-19
• PAC-MAN (prophylactic antiviral CRISPR in human cells) is a CRISPR-Cas13-based strategy
as a Therapeutic application against COVID 19
• RNA guided RNA endonuclease activity of Cas13d in human cells to eliminate the SARS-CoV-
2 virus
• CRISPR based detection tools such as SHERLOCK, DETECTR, and FELUDA for COVID
19
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29. Sickle cell anaemia
• Exagamglogene autotemcel ( Exa- cel ) by vertex pharmaceuticals for Sickle cell anaemia (
Stage III clinical trial )
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30. Summary
• CRISPR is Clustered regular interspaced short palindromic repeats
• It is a bacterial adaptive immune response
• CRISPR locus consist of trRNA ( trans RNA ), Cas ( CRISPR associated endonuclease ), PAM
(Protospacer adjacent Motif ), Spacer ( incorporated viral fragment ) which further transcribe
into crRNA ( CRISPR RNA )
• trRNA and crRNA can be combined to form sgRNA ( single guide RNA ) guides Cas to
cause Cleavage at the desired site ( recognition by PAM )
• It is easy, fast and Highly accurate technology
• Limitations include Off target effects, Immunotoxicity, and DNA damage toxicity
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31. • Off target effect can be reduced by certain Cas Variants such as SpCas9HF-1 variant, Evo Cas 9 and
HiFi Cas 9, Cas9_R63A/Q768A Variant
• Off target effects and DNA induced toxicity can also be reduced by Precise Gene editing
• Precise gene editing involves three methods : CRISPR/Cas 9 HDR ( Using ssODN ), Base editors (
Adenosine base editors and cytosine base editor ) and Prime editor ( Cas 9n and prime editor guide
RNA ( peg RNA ) )
• Delivery of CRISPR can be by Ex Vivo or In Vivo method
• Delivery agents include Physical method ( Electroporation, Microinjection ), Viral Vectors
(Adenovirus ) and Non viral vectors ( Nanoparticles )
• Applications Include in Cancer, COVID 19 ( Detection ( DETECTR ) and therapeutic ( PAC-MAN ) )
• Exagamglogene autotemcel ( Exa- cel ) for Sickle cell anaemia
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32. References
• Uddin F, Rudin M, Sen T. CRISPR Gene Therapy: Applications, Limitations, and Implications
for the Future. Front. Oncol.2020;10:1387.
• Zhang XH, Tee LY, Wang XG, Huang QS, Yang SH. Off-target Effects in CRISPR/Cas9-
mediated Genome Engineering. Mol Ther Nucleic Acids. 2015 Nov 17;4(11):e264.
• Zhu Y. Advances in CRISPR/Cas9. Biomed Res Int. 2022;2022:9978571.
• Liu W, Li L, Jiang J, Wu M, Lin P. Applications and challenges of CRISPR-Cas gene-editing to
disease treatment in clinics. Precis Clin Med. 2021;4(3):179-191.
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