The document discusses CRISPR-Cas9 as a potential tool for genome editing. It describes how CRISPR was originally discovered in bacteria and archaea as a mechanism for adaptive immunity against viruses. The CRISPR-Cas9 system uses guide RNA to direct an endonuclease called Cas9 to introduce targeted double-strand breaks in DNA, which can then be repaired through non-homologous end joining or homology directed repair for genome editing. Applications discussed include using CRISPR-Cas9 for disease modeling in animals and cell lines more efficiently compared to previous methods, as well as for drug development by generating gene knockouts and mutations for target validation.
This document summarizes a presentation on CRISPR/Cas genome editing. It defines CRISPR/Cas as a type of genetic engineering that uses artificially engineered nucleases to make specific cuts in DNA. It describes the CRISPR/Cas system's origins and components, including Cas9, guide RNA, and PAM sequences. Applications discussed include genome editing in animals and plants, as well as concerns over off-target effects. Companies offering CRISPR services or kits are also mentioned.
This document provides an overview of CRISPR-Cas9 technology. It defines CRISPR as DNA sequences in bacteria that contain snippets of viral DNA used to detect and destroy invading viruses. The CRISPR system contains Cas9 proteins that cut DNA at specific locations guided by RNA, and RNA guide molecules. It works by integrating viral DNA into the bacteria's CRISPR loci, which are then transcribed into RNA to guide Cas9 to cleave invading viral DNA. Applications of CRISPR include disease modeling, cancer research, and correcting mutations in human embryos.
This document provides an overview of the CRISPR-Cas immune system in bacteria and its applications. It discusses:
1. The history and components of the CRISPR-Cas system, including Cas proteins, CRISPR RNA, and protospacer adjacent motifs.
2. The three main types of CRISPR-Cas systems and their mechanisms of targeting DNA or RNA.
3. How engineered versions of Cas9 can be used for targeted genome editing and modulation of gene expression in mammalian cells.
4. Applications of CRISPR-Cas in microbiology such as genetic engineering of bacteria, gene repression/activation using deactivated Cas9, and developing sequence-specific antimicrobials.
1. CRISPR is a bacterial immune system that provides immunity against viruses. It works by matching DNA sequences from viruses to DNA spacers and cleaving invading DNA.
2. The type II CRISPR system is the most well studied and requires only three components - Cas9 protein, CRISPR RNA (crRNA), and trans-activating CRISPR RNA (tracrRNA) - to function. Combining crRNA and tracrRNA into a single RNA molecule called sgRNA was crucial for developing the CRISPR technique.
3. CRISPR technology has become a powerful genome editing tool due to its simplicity, high efficiency, low cost, and ease of use. It allows targeted
Crispr-Cas9 system works on the concept of bacterial defence mechanism. The idea of which was replicated in eukaryotic cell in in- vitro condition by the researchers.
CRISPR-Cas9 is a revolutionary genome editing tool that allows targeted modifications to DNA. It utilizes the Cas9 endonuclease enzyme, which is guided to a specific location in the genome by a short RNA molecule. When the Cas9 enzyme cuts the DNA, it triggers the cell's repair mechanisms which can introduce changes to the genome at that location. CRISPR-Cas9 has significant advantages over previous genome editing techniques in terms of efficiency and ease of use. It holds promise for curing genetic diseases, advancing biomedical research, and improving crops and livestock. Future directions include optimizing delivery methods and enhancing the precision and control of genome alterations.
An Introduction to Crispr Genome Editing
Crispr cas: A new tool of genome editing
CRISPRs (Clustered Regularly Interspaced Short Palindromic Repeats) are part of an adaptive defense mechanism in bacteria and archaea. Use of the CRISPR/Cas9 system for genome editing has been a major technological breakthrough, making genome modification in cells or organisms fast, more efficient, and much more robust than previous genome editing methods. Single guide RNAs (sgRNAs) or guide RNAs (gRNAs) direct and activate the Cas9 endonuclease at a specific genomic sequence. Cas9 then cleaves the target DNA, making it available for repair by the non-homologous end joining (NHEJ) system or for creating an insertion site for exogenous donor DNA by homologous recombination.
This document summarizes a presentation on CRISPR/Cas genome editing. It defines CRISPR/Cas as a type of genetic engineering that uses artificially engineered nucleases to make specific cuts in DNA. It describes the CRISPR/Cas system's origins and components, including Cas9, guide RNA, and PAM sequences. Applications discussed include genome editing in animals and plants, as well as concerns over off-target effects. Companies offering CRISPR services or kits are also mentioned.
This document provides an overview of CRISPR-Cas9 technology. It defines CRISPR as DNA sequences in bacteria that contain snippets of viral DNA used to detect and destroy invading viruses. The CRISPR system contains Cas9 proteins that cut DNA at specific locations guided by RNA, and RNA guide molecules. It works by integrating viral DNA into the bacteria's CRISPR loci, which are then transcribed into RNA to guide Cas9 to cleave invading viral DNA. Applications of CRISPR include disease modeling, cancer research, and correcting mutations in human embryos.
This document provides an overview of the CRISPR-Cas immune system in bacteria and its applications. It discusses:
1. The history and components of the CRISPR-Cas system, including Cas proteins, CRISPR RNA, and protospacer adjacent motifs.
2. The three main types of CRISPR-Cas systems and their mechanisms of targeting DNA or RNA.
3. How engineered versions of Cas9 can be used for targeted genome editing and modulation of gene expression in mammalian cells.
4. Applications of CRISPR-Cas in microbiology such as genetic engineering of bacteria, gene repression/activation using deactivated Cas9, and developing sequence-specific antimicrobials.
1. CRISPR is a bacterial immune system that provides immunity against viruses. It works by matching DNA sequences from viruses to DNA spacers and cleaving invading DNA.
2. The type II CRISPR system is the most well studied and requires only three components - Cas9 protein, CRISPR RNA (crRNA), and trans-activating CRISPR RNA (tracrRNA) - to function. Combining crRNA and tracrRNA into a single RNA molecule called sgRNA was crucial for developing the CRISPR technique.
3. CRISPR technology has become a powerful genome editing tool due to its simplicity, high efficiency, low cost, and ease of use. It allows targeted
Crispr-Cas9 system works on the concept of bacterial defence mechanism. The idea of which was replicated in eukaryotic cell in in- vitro condition by the researchers.
CRISPR-Cas9 is a revolutionary genome editing tool that allows targeted modifications to DNA. It utilizes the Cas9 endonuclease enzyme, which is guided to a specific location in the genome by a short RNA molecule. When the Cas9 enzyme cuts the DNA, it triggers the cell's repair mechanisms which can introduce changes to the genome at that location. CRISPR-Cas9 has significant advantages over previous genome editing techniques in terms of efficiency and ease of use. It holds promise for curing genetic diseases, advancing biomedical research, and improving crops and livestock. Future directions include optimizing delivery methods and enhancing the precision and control of genome alterations.
An Introduction to Crispr Genome Editing
Crispr cas: A new tool of genome editing
CRISPRs (Clustered Regularly Interspaced Short Palindromic Repeats) are part of an adaptive defense mechanism in bacteria and archaea. Use of the CRISPR/Cas9 system for genome editing has been a major technological breakthrough, making genome modification in cells or organisms fast, more efficient, and much more robust than previous genome editing methods. Single guide RNAs (sgRNAs) or guide RNAs (gRNAs) direct and activate the Cas9 endonuclease at a specific genomic sequence. Cas9 then cleaves the target DNA, making it available for repair by the non-homologous end joining (NHEJ) system or for creating an insertion site for exogenous donor DNA by homologous recombination.
1) The document discusses the CRISPR-Cas9 system of genome editing and its applications.
2) CRISPR-Cas9 allows for accurate and multiplex gene modification guided by RNA and is an advanced technique compared to earlier tools like ZFNs and TALENs.
3) The document covers the mechanism of CRISPR-Cas9 immunity in bacteria, the general protocol for genome editing using CRISPR-Cas9, and new developments like modified Cas9 enzymes and the Cpf1 protein.
The document provides an introduction to the CRISPR/Cas9 genome editing technique. It discusses that CRISPR/Cas9 uses guide RNAs to direct the Cas9 nuclease to cut DNA at specific locations, and this double strand break can be repaired through nonhomologous end joining or homology directed repair to knock out or knock in genes. It also explains that CRISPR/Cas9 is more efficient, less expensive, and easier to use than previous genome editing techniques like ZFNs and TALENs. The document outlines several applications of CRISPR/Cas9 in biomedical research areas such as immunology, stem cell research, and generating transgenic animals.
The document discusses the CRISPR/Cas9 system. It describes how CRISPR/Cas9 uses a Cas9 protein guided by a single guide RNA to recognize and cut target DNA. The system has three stages: adaptation, expression and processing of CRISPR RNA, and interference where the Cas9 protein complex cuts the target DNA. CRISPR/Cas9 can be engineered to act as a nuclease, nickase, or inactive dead Cas9 for gene regulation applications like activation or repression. It provides a gift from nature for precise genome editing and regulation.
This document provides an overview of CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) and its role as an adaptive immune system in prokaryotes. It describes the components and function of the CRISPR-Cas system, including how it provides immunity against viruses and plasmids. Applications of CRISPR technology discussed include phage resistance in bacteria, gene regulation, and bacterial strain typing. Potential future uses involve harnessing CRISPR biology for applications like transcriptional control.
CRISPR is a new mechanism\tool to edit genes and in coming future it will provide us many new levels of success in curing of genetic disorders and modifying genes for human benifit
This document provides an overview of CRISPR/Cas9 genome editing. It discusses how CRISPR/Cas9 enables precise modification of DNA sequences, outlines the timeline of key discoveries in CRISPR research, and describes the molecular mechanism and potential applications of this technology, including in microbial research, crop improvement, and human gene therapy. It also notes some limitations of the CRISPR/Cas9 system and concludes by emphasizing the opportunities it provides to advance research and address challenges like food security.
CRISPR-Cas is a natural defense system in bacteria that uses CRISPR sequences and Cas proteins to target and degrade foreign DNA such as from viruses. It has been adapted for genome editing in other organisms using a Cas9 protein guided by a synthetic single guide RNA to introduce targeted double-strand breaks. This system allows for precise genome modifications and has applications in biomedical research, disease treatment, and engineering of plants and other organisms. However, off-target effects and delivery methods require further optimization.
The document discusses the CRISPR-Cas9 genome editing tool. CRISPR-Cas9 uses an enzyme called Cas9 and a guide RNA to cut DNA at a specific location, allowing DNA to be removed, added, or altered. It was developed based on the bacterial immune system and provides a simple, precise way to edit genomes. While promising for treating genetic diseases, its use in germline editing raises ethical concerns that require further discussion.
A simple version of the CRISPR/Cas system, CRISPR/Cas9, has been modified to edit genomes. By delivering the Cas9 nuclease complexed with a synthetic guide RNA (gRNA) into a cell, the cell's genome can be cut at a desired location, allowing existing genes to be removed and/or new ones added.
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 overview of CRISPR/Cas9 genome editing. It discusses the history and limitations of prior genome engineering techniques like recombinant DNA and zinc finger nucleases. It then explains how CRISPR/Cas9 works as a RNA-guided DNA endonuclease and how this allows it to efficiently and specifically edit genomes. The document outlines several applications of CRISPR/Cas9 like generating knockout animals and cell lines. It also notes some concerns about using the technique for human genome editing.
This document summarizes a seminar given on CRISPR technology. It discusses the history of CRISPR research from its discovery in bacteria in 1987 to its development as a genome editing tool in 2012. Key events and applications are outlined, including the founding of early biotech companies utilizing CRISPR. The core concepts of how CRISPR induces double strand breaks and DNA repair are explained. Recent advances in engineered Cas9 variants and discovery of Cpf1 are also summarized.
CRISPR-Cas9 is a genome editing tool that is creating a buzz in the science world. It is faster, cheaper and more accurate than previous techniques of editing DNA and has a wide range of potential applications.
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.
This document discusses the CRISPR-Cas9 genome editing technology. It begins by explaining that genome editing involves inserting, deleting, or replacing DNA in a genome using engineered molecular scissors. It then describes CRISPR-Cas9 specifically, noting that it is a bacterial immune system that utilizes CRISPR sequences and Cas9 proteins to cut viral DNA. The document outlines the components of the CRISPR-Cas9 system, including guide RNAs that locate DNA sections and Cas9 proteins that modify the DNA. It also discusses applications of CRISPR-Cas9 such as curing genetic diseases and editing crops, as well as noting that further investigation is still needed to ensure its safe therapeutic use.
CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)Akshay Deshmukh
clustered regularly interspaced short palindromic repeats is a family of DNA sequences found in the genomes of prokaryotic organisms such as bacteria. Now CRISPR use as genome editing tool in different Plant Breeder to manipulate the DNA of the crop
CRISPR/Cas9 is a genome editing technique that allows for highly specific modification of DNA. It involves a Cas9 protein guiding a customized RNA to a target location in the genome to cut DNA. Scientists adapted the natural CRISPR immune system found in bacteria for genome editing. CRISPR/Cas9 holds promise for treating genetic diseases and developing crops but also raises ethical concerns when applied to human germline cells due to heritable effects. Researchers continue improving targeting specificity and developing new Cas proteins for additional applications in genome editing and gene regulation.
This document describes the construction of a high capacity adenoviral vector (HCAdV) devoid of all viral genes that can be used to deliver the CRISPR/Cas9 system for genome editing. Researchers developed an intermediate shuttle plasmid containing the CRISPR/Cas9 genes and guide RNAs that could then be inserted into the HCAdV genome. The CRISPR/Cas9 system utilizes the natural bacterial immune system to precisely cut DNA at targeted locations guided by guide RNAs. This vector aims to improve the efficiency of delivering genome edited DNA to cells for applications such as gene therapy.
Crispr cas: A new tool of genome editing palaabhay
The document summarizes a presentation on CRISPR cas9, a new genome editing tool. It discusses the history of CRISPR, how CRISPR functions in bacteria, the classification and components of CRISPR systems, and the mechanism of CRISPR cas9. It then covers applications of CRISPR cas9 in genome editing, databases of CRISPR sequences, case studies using the technology, and future directions of CRISPR research.
1) The document discusses the CRISPR-Cas9 system of genome editing and its applications.
2) CRISPR-Cas9 allows for accurate and multiplex gene modification guided by RNA and is an advanced technique compared to earlier tools like ZFNs and TALENs.
3) The document covers the mechanism of CRISPR-Cas9 immunity in bacteria, the general protocol for genome editing using CRISPR-Cas9, and new developments like modified Cas9 enzymes and the Cpf1 protein.
The document provides an introduction to the CRISPR/Cas9 genome editing technique. It discusses that CRISPR/Cas9 uses guide RNAs to direct the Cas9 nuclease to cut DNA at specific locations, and this double strand break can be repaired through nonhomologous end joining or homology directed repair to knock out or knock in genes. It also explains that CRISPR/Cas9 is more efficient, less expensive, and easier to use than previous genome editing techniques like ZFNs and TALENs. The document outlines several applications of CRISPR/Cas9 in biomedical research areas such as immunology, stem cell research, and generating transgenic animals.
The document discusses the CRISPR/Cas9 system. It describes how CRISPR/Cas9 uses a Cas9 protein guided by a single guide RNA to recognize and cut target DNA. The system has three stages: adaptation, expression and processing of CRISPR RNA, and interference where the Cas9 protein complex cuts the target DNA. CRISPR/Cas9 can be engineered to act as a nuclease, nickase, or inactive dead Cas9 for gene regulation applications like activation or repression. It provides a gift from nature for precise genome editing and regulation.
This document provides an overview of CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) and its role as an adaptive immune system in prokaryotes. It describes the components and function of the CRISPR-Cas system, including how it provides immunity against viruses and plasmids. Applications of CRISPR technology discussed include phage resistance in bacteria, gene regulation, and bacterial strain typing. Potential future uses involve harnessing CRISPR biology for applications like transcriptional control.
CRISPR is a new mechanism\tool to edit genes and in coming future it will provide us many new levels of success in curing of genetic disorders and modifying genes for human benifit
This document provides an overview of CRISPR/Cas9 genome editing. It discusses how CRISPR/Cas9 enables precise modification of DNA sequences, outlines the timeline of key discoveries in CRISPR research, and describes the molecular mechanism and potential applications of this technology, including in microbial research, crop improvement, and human gene therapy. It also notes some limitations of the CRISPR/Cas9 system and concludes by emphasizing the opportunities it provides to advance research and address challenges like food security.
CRISPR-Cas is a natural defense system in bacteria that uses CRISPR sequences and Cas proteins to target and degrade foreign DNA such as from viruses. It has been adapted for genome editing in other organisms using a Cas9 protein guided by a synthetic single guide RNA to introduce targeted double-strand breaks. This system allows for precise genome modifications and has applications in biomedical research, disease treatment, and engineering of plants and other organisms. However, off-target effects and delivery methods require further optimization.
The document discusses the CRISPR-Cas9 genome editing tool. CRISPR-Cas9 uses an enzyme called Cas9 and a guide RNA to cut DNA at a specific location, allowing DNA to be removed, added, or altered. It was developed based on the bacterial immune system and provides a simple, precise way to edit genomes. While promising for treating genetic diseases, its use in germline editing raises ethical concerns that require further discussion.
A simple version of the CRISPR/Cas system, CRISPR/Cas9, has been modified to edit genomes. By delivering the Cas9 nuclease complexed with a synthetic guide RNA (gRNA) into a cell, the cell's genome can be cut at a desired location, allowing existing genes to be removed and/or new ones added.
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 overview of CRISPR/Cas9 genome editing. It discusses the history and limitations of prior genome engineering techniques like recombinant DNA and zinc finger nucleases. It then explains how CRISPR/Cas9 works as a RNA-guided DNA endonuclease and how this allows it to efficiently and specifically edit genomes. The document outlines several applications of CRISPR/Cas9 like generating knockout animals and cell lines. It also notes some concerns about using the technique for human genome editing.
This document summarizes a seminar given on CRISPR technology. It discusses the history of CRISPR research from its discovery in bacteria in 1987 to its development as a genome editing tool in 2012. Key events and applications are outlined, including the founding of early biotech companies utilizing CRISPR. The core concepts of how CRISPR induces double strand breaks and DNA repair are explained. Recent advances in engineered Cas9 variants and discovery of Cpf1 are also summarized.
CRISPR-Cas9 is a genome editing tool that is creating a buzz in the science world. It is faster, cheaper and more accurate than previous techniques of editing DNA and has a wide range of potential applications.
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.
This document discusses the CRISPR-Cas9 genome editing technology. It begins by explaining that genome editing involves inserting, deleting, or replacing DNA in a genome using engineered molecular scissors. It then describes CRISPR-Cas9 specifically, noting that it is a bacterial immune system that utilizes CRISPR sequences and Cas9 proteins to cut viral DNA. The document outlines the components of the CRISPR-Cas9 system, including guide RNAs that locate DNA sections and Cas9 proteins that modify the DNA. It also discusses applications of CRISPR-Cas9 such as curing genetic diseases and editing crops, as well as noting that further investigation is still needed to ensure its safe therapeutic use.
CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)Akshay Deshmukh
clustered regularly interspaced short palindromic repeats is a family of DNA sequences found in the genomes of prokaryotic organisms such as bacteria. Now CRISPR use as genome editing tool in different Plant Breeder to manipulate the DNA of the crop
CRISPR/Cas9 is a genome editing technique that allows for highly specific modification of DNA. It involves a Cas9 protein guiding a customized RNA to a target location in the genome to cut DNA. Scientists adapted the natural CRISPR immune system found in bacteria for genome editing. CRISPR/Cas9 holds promise for treating genetic diseases and developing crops but also raises ethical concerns when applied to human germline cells due to heritable effects. Researchers continue improving targeting specificity and developing new Cas proteins for additional applications in genome editing and gene regulation.
This document describes the construction of a high capacity adenoviral vector (HCAdV) devoid of all viral genes that can be used to deliver the CRISPR/Cas9 system for genome editing. Researchers developed an intermediate shuttle plasmid containing the CRISPR/Cas9 genes and guide RNAs that could then be inserted into the HCAdV genome. The CRISPR/Cas9 system utilizes the natural bacterial immune system to precisely cut DNA at targeted locations guided by guide RNAs. This vector aims to improve the efficiency of delivering genome edited DNA to cells for applications such as gene therapy.
Crispr cas: A new tool of genome editing palaabhay
The document summarizes a presentation on CRISPR cas9, a new genome editing tool. It discusses the history of CRISPR, how CRISPR functions in bacteria, the classification and components of CRISPR systems, and the mechanism of CRISPR cas9. It then covers applications of CRISPR cas9 in genome editing, databases of CRISPR sequences, case studies using the technology, and future directions of CRISPR research.
An Introduction to Crispr Genome EditingChris Thorne
In this short presentation, I make a case for doing genome editing vs some of the approaches that have gone before, describe some of the tools available, and the focus on CRISPR-Cas9, what it is, where it's come from and how it works.
The CRISPR/Cas9 system has emerged as one of the leading tools for modifying the genomes of organisms ranging from E. coli to humans. In this presentation, we discuss various methods for generating the crRNA and tracrRNA components that are required for guiding the Cas9 endonuclease to genomic targets. You will also learn how to optimize a new 2-part CRISPR RNA system from IDT that offers multiple benefits over other technologies.
Have you considered that protein over-expression or inefficient mRNA knockdown may be masking physiological effects in your assays? Increasingly scientists are moving to endogenous gene-editing to characterise the function of their genes of interest.
Dr Chris Thorne from Cambridge Biotech Horizon Discovery discusses the ground breaking gene-editing technology CRISPR. The simplicity of experimental design has led to rapid adoption of the technology across the scientific community. However, challenges remain.
This Slidedeck focuses specifically on implementing CRISPR experiments, and explore a number of key considerations crucial to maximising chances of targeting success, whether your goal is to generate a knock-out or a knock-in. Chris also takes a look at some of the alternative uses of CRISPR, including sgRNA genome wide synthetic lethality screens.
The slides aim to support those researchers either planning to or already using CRISPR gene-editing in their lab. Horizon Discovery have also recently launched a program aimed specifically at academic cell biologists to promote the adoption of CRISPR by offering FREE CRISPR Reagents for knock-out cell line generation - more information available here. http://www.horizondiscovery.com/what-we-do/discovery-toolbox/genassist-crispr--raav-genome-editing-tools
This document summarizes research analyzing the CRISPR-associated elements in two novel Propionibacterium acnes bacteriophages called Lauchelly and Attacne. Transmission electron micrographs show that the phages have a siphoviridae morphology. Genome analyses found conserved CRISPR-associated domains that may function as an anti-CRISPR mechanism. Host range tests found the phages could infect most P. acnes strains, though one strain appeared resistant to Attacne.
Bacterial Adaptive immunity, Gene drives and the genetic control of MalariaOlabode Onile-ere
This document discusses using CRISPR-Cas9 gene drive technology to genetically control malaria. It provides background on malaria, adaptive bacterial immunity, and how CRISPR works. CRISPR-Cas9 could be used to insert genes into mosquitoes that block malaria parasite development, achieving population replacement. It could also reduce mosquito reproductive fitness for population suppression. While this approach could eliminate malaria, issues around ethics, ecological effects, and the possibility of unintended outcomes require further exploration before real-world application. Gene drives hold promise but perfection of the technology and alignment with public values is needed.
This document summarizes an experiment on the effect of methyl viologen (MV) on the photoluminescence and scattering of gold nanorods. MV was added to gold nanorod solutions in concentrations of 5 mM and 1 M. The addition of MV significantly changed the scattering spectra through a red shift, widening, and slight decrease in intensity, but did not affect photoluminescence. This proves photoluminescence is more stable in electron withdrawing environments. The changes to scattering demonstrate the interaction between MV and the gold nanorods' surface plasmon resonance.
Precision medicine for oncology requires accurate and sensitive molecular characterization. However, sample degradation, polymerase errors, and sequencing errors reduce accuracy for sequencing genetic variants. By incorporating molecular tagged adapters in target enrichment, and using DNA probes that deliver extremely even and deep coverage, we are able to demonstrate a 300-fold reduction in false positives at or above 0.25% variant frequency. In this presentation, Dr Mirna Jarosz discusses these methods and how they can significantly reduce error rates in your sequencing data.
Recent breakthroughs in genome editing technology have led to a rapid adoption that parallels that seen with RNAi. And like RNAi, these methods are taking the scientific world by storm, with high profile publications in fields as diverse as HIV treatment, stem cell therapy, food crop modification and drug development to name but a few.
Critically, the endogenous modification of genes enables the study of their function in a physiological context. It also overcomes some of the artefacts that can result from established techniques such as transgenesis and RNAi, which have mislead researchers with false positives or negatives. Until recently however genome editing required considerable technical expertise, and consequently was a relatively niche pursuit.
In this talk we will look at how the latest developments in genome editing tools have changed this, with improvements in both ease-of-use and targeting efficiency, as well as a concomitant reduction in costs opening up these approaches to the wider scientific community.
Rapid adoption of the CRISPR/Cas9 system has for example led to a long list of organisms and tissues in which genetic changes have been made with high efficiency. Other technologies such as recombinant adeno-associated virus (rAAV) offer further precision, stimulating the cell’s high-fidelity DNA repair pathways to insert exogenous sequence with unrivalled specificity. Targeting efficiency can be improved still further by using the technologies in combination – genome cutting induced by CRISPR can significantly enhance homologous recombination mediated by rAAV.
Despite these rapid advances, some pitfalls remain, and so we’ll discuss some of the key considerations for avoiding these, ranging from simply picking the right tool for the job to designing an experiment that maximises chances of success.
Finally we’ll look at how genome editing is being applied to both basic and translational research, and in both a gene-specific and genome wide manner. For the study of disease associated genes and mutations scientists can now complement wide panels of tumour cells with genetically defined isogenic cell pairs identical in all but precise modifications in their gene of interest. The ease-of-design and efficiency of the CRISPR system is also being exploited for genome wide synthetic lethality screens, facilitating rapid drug target identification with significantly reduced risk of false negatives and off-target false positives. And again, further synergies are achieved when these approaches are combined to look for potential synthetic lethal targets in specific genomic contexts.
Genetic Recording in Yeast Using CRISPR-Cas9Robert Beem
This document describes a study on using CRISPR/Cas9 for genome editing in yeast (Saccharomyces cerevisiae). The goal was to analyze the activity of self-targeting guide RNAs (stgRNAs) in yeast. Methods included constructing plasmids containing Cas9 and an stgRNA in E. coli, then integrating them into the yeast genome using homologous recombination. Activity was assayed using a T7 endonuclease assay to detect mutations from Cas9 cutting the target sequence. Results showed the stgRNA was effective in yeast and that Cas9 was active even at low levels due to leaky expression from the inducible promoter. Future applications could include protein engineering and gene therapy research
(1) CRISPR-Cas9 is a new genetic editing technique that allows easier correction of faulty genes. It has potential for treating genetic diseases but also raises ethical concerns.
(2) The document discusses using CRISPR-Cas9 to edit somatic/adult cells (acceptable) vs germline/embryonic cells (controversial). Editing germline cells could affect future generations and paves the way for "designer babies".
(3) The proposed position is to continue CRISPR-Cas9 research on animals and adult cells but support a moratorium on human germline/embryonic editing and a permanent ban due to safety issues and concerns about human
The key considerations of crispr genome editingChris Thorne
While CRISPR is simple to use, widely applicable and often highly efficient, there are a number of things to keep in mind to maximise experimental success. Here's what we recommend...
Genome editing tools form the basis for personalized medicine, especially for therapies requiring change in genome. Currently there are four contenders to this – Meganucleases, ZNF Nucleases, TALENs and CRISPRs. Although, the technologies are many, there are very few commercial providers of this technology. This is attributed to the fact that select few possess the intellectual property rights of turning these technologies to valid form of therapy; for example, ZFN patent with Sangamo BioSciences and TALENs with Cellectis, Transposagen and Life Technologies.
Lessons learned from high throughput CRISPR targeting in human cell linesChris Thorne
In just a short period of time CRISPR-Cas9 technology has revolutionized the field of genome editing, and taken the scientific community by storm. Already our understanding of how best to apply this technology has advanced significantly and almost every week new publications appear showcasing its application in basic and translational research.
While CRISPR-Cas9 is applicable across many different cell types, we have found it particularly suited for genome editing in near-haploid human cell lines. This has allowed us to establish a robust pipeline for the inactivation of non-essential genes at unprecedented scale and efficiency.
We have now knocked out over 1500 human genes and have generated a resource that is, to the best of our knowledge, the largest collection of human knockout cell lines available, covering comprehensive subsets of genes clustered by biological pathway (e.g. the autophagy pathway, the JAK/STAT pathway) or by phylogenetic relationship (e.g. kinases, bromodomain-containing proteins).
In this talk we will discuss how, through more than 1500 genome editing experiments, we have started to unravel some of the general principles governing the use of CRISPR-Cas9 in mammalian cells. For example, we have analyzed the impact of variation in the guide RNA sequence on Cas9 cleavage efficiency and characterized the mutational signature arising from CRISPR-Cas9 cleavage.
We will also highlight (with examples) how these learnings are now being applied to introduce other genomic modifications in a high throughput manner, including chromosomal deletions, translocations, point mutations and endogenous gene tags.
Edward Perello, CBO of Desktop Genetics, joins us at the Science: Disrupt London Session on Disruptor Stories to talk machine learning, CRSIPR, pivoting and his startup story.
i explained about basics of genome engineering and crispr system.
CRISPR will change the world and it is just the beginning, are you ready to meet the future? you think its great and beautiful or.....?
please give your feedback to my email
pooyanaghshbandi@yahoo.com
i am starting to write a critical and fantastic review article about CRISPR, if you are interested to join please contact me.
The document discusses CRISPR-Cas systems, which provide bacteria and archaea with adaptive immunity against viruses. It describes the key components and functions of various CRISPR-Cas systems, including Cas9 from type II systems. CRISPR-Cas9 has been engineered into a powerful tool for genome editing in mammalian cells by creating a single-guide RNA to direct Cas9 to cleave specific DNA sequences. The document also compares CRISPR-Cas9 to previous genome editing tools like zinc finger nucleases and TALENs, noting that CRISPR-Cas9 requires only changing the guide RNA sequence rather than re-engineering proteins.
This document summarizes a study on two genome editing tools: CRISPR/Cas9 and RNAi. CRISPR/Cas9 originated from the adaptive immune system of bacteria and allows for easy editing of DNA at specific locations using an RNA guide and Cas9 enzyme. RNAi utilizes small interfering RNAs to degrade mRNA and silence genes. The document discusses the mechanisms, applications in agriculture, disease modeling and gene therapy, and compares the advantages of CRISPR/Cas9 over RNAi.
This document summarizes the development of genome editing technologies leading up to the advent of CRISPR-Cas9. Early approaches relied on site-specific DNA recognition by oligonucleotides or proteins, but were limited. The discovery of CRISPR immune systems in bacteria revealed Cas9, an RNA-guided endonuclease that introduces targeted DNA double-strand breaks. Engineering the dual-RNA structure into a single guide RNA created a simple two-component system for programming Cas9 to cleave any DNA sequence. CRISPR-Cas9's simplicity, efficiency, and versatility has enabled widespread applications in biology research across many cell and organism types.
This document summarizes the history and development of CRISPR-Cas9 genome editing technology. It discusses how early approaches to targeted DNA cleavage relied on site-specific DNA recognition by oligonucleotides or proteins. Zinc finger nucleases and TAL effector nucleases were developed that allowed for genome editing but were difficult to design and validate. The discovery of CRISPR immune systems in bacteria provided the basis for a new approach using Cas9 guided by CRISPR RNA. Experiments showed Cas9 could be programmed to cleave DNA sequences using a simple RNA guide. This established the CRISPR-Cas9 system as an easy and efficient tool for genome editing that has enabled many new applications across biology.
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 document summarizes an experiment aiming to knockout the CTNNB1 gene in mouse embryonic stem cells using CRISPR-Cas9 genome editing. It describes designing a guide RNA targeting exon 10 of CTNNB1, cloning it into a vector, transforming E. coli, and testing primers for detecting knockout via PCR and qPCR. Prior studies showed CTNNB1 knockout embryos had defects in ectoderm and mesoderm development. The authors hypothesized knocking out CTNNB1 in stem cells would produce inviable embryos, similar to prior findings.
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.
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The document summarizes the CRISPR-Cas system, beginning with what CRISPR refers to as patterns of DNA sequences found in bacterial genomes. It describes the three stages of adaptive immunity in CRISPR-Cas systems: insertion of invading DNA as a spacer, transcription of precursor CRISPR RNA which is processed into individual CRISPR RNAs targeting the invader, and Cas protein-directed cleavage of foreign nucleic acid guided by the CRISPR RNA. Applications of CRISPR-Cas systems discussed include genome editing, gene regulation through catalytically inactive Cas9 fusion proteins, cargo delivery by fusing Cas9 to other proteins, and RNA cleavage by Type III CRISPR-Cas systems.
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This document summarizes research on the CRISPR/Cas9 system for genome editing in human embryos. It discusses efforts to understand DNA repair mechanisms after inducing double-strand breaks, reduce off-target mutations, and improve the specificity and efficiency of editing. While the technology shows promise, significant issues around off-target effects, mosaicism, and ethical concerns must still be addressed before any clinical applications. The document concludes that further basic research is needed to advance the field while also having open discussions on societal implications.
This document provides an overview of CRISPR/Cas9 gene editing technology. It discusses the history of CRISPR discoveries from 1993 onwards and how the technology has been adapted for genome editing. Previous gene editing methods like ZFNs, TALENs and rAAVs are also summarized. The document then explains in detail how the CRISPR/Cas9 system works to edit genomes using a Cas9 enzyme guided by CRISPR RNA. Applications of CRISPR like genomic editing through knockouts and gene silencing are highlighted.
CRISPR technologies have progressed by leaps and bounds over the past decade, not only having a transformative effect on
biomedical research but also yielding new therapies that are poised to enter the clinic. In this review, I give an overview of (i)
the various CRISPR DNA-editing technologies, including standard nuclease gene editing, base editing, prime editing, and epigenome editing, (ii) their impact on cardiovascular basic science research, including animal models, human pluripotent stem
cell models, and functional screens, and (iii) emerging therapeutic applications for patients with cardiovascular diseases, focusing on the examples of Hypercholesterolemia, transthyretin amyloidosis, and Duchenne muscular dystrophy.
CRISPR is a bacterial adaptive immune system that provides immunity against viruses. It has been adapted for genome editing using Cas9 nuclease guided by a synthetic single guide RNA. The Cas9-guide RNA complex binds target DNA and makes a double-strand break, which can be repaired by non-homologous end joining to disrupt a gene or by homology-directed repair with a donor template to edit the genome. This powerful new technology allows various genome editing applications such as gene knockouts, insertions, corrections, and regulation of gene expression.
Genome engineering uses programmable nucleases like CRISPR-Cas9 to make targeted modifications to DNA. CRISPR-Cas9 is an adaptive immune system in bacteria that uses Cas9, an RNA-guided DNA endonuclease, to cleave DNA when guided by CRISPR RNA (crRNA). The Cas9 protein uses crRNA and trans-activating CRISPR RNA (tracrRNA) to induce double-strand breaks in DNA matching the crRNA sequence. CRISPR-Cas9 allows for efficient, precise genome editing and has applications in gene therapy, agriculture, and research.
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.
1. Researchers used CRISPR/Cas9 to efficiently generate biallelic RAG1 knockout in mouse embryonic stem cells. They designed single-guide RNAs targeting RAG1 and transfected stem cells with Cas9, achieving indels in 92% of clones, including 59% with homozygous out-of-frame mutations.
2. The RAG1 knockout stem cell lines maintained pluripotent gene expression and normal morphology. CRISPR/Cas9 allowed faster generation of RAG1 knockout mice than previous methods by creating chimeric embryos.
3. Precisely designed single-guide RNAs and targeting multiple sites simultaneously enhanced CRISPR/Cas9's ability to introduce double-
CRISPR (clustered regularly interspaced short palindromic repeats) is a family of DNA sequences found within the genomes of prokaryotic organisms such as bacteria and archaea. These sequences are derived from DNA fragments of bacteriophages that have previously infected the prokaryote and are used to detect and destroy DNA from similar phages during subsequent infections. Hence these sequences play a key role in the antiviral defense system of prokaryotes.
Cas9 (CRISPR-associated protein 9) is an enzyme that uses CRISPR sequences as a guide to recognize and cleave specific strands of DNA that are complementary to the CRISPR sequence. Cas9 enzymes together with CRISPR sequences form the basis of a technology known as CRISPR-Cas9 that can be used to edit genes within organisms.This editing process has a wide variety of applications including basic biological research, development of biotechnology products, and treatment of diseases.
The CRISPR-Cas system is a prokaryotic immune system that confers resistance to foreign genetic elements such as those present within plasmids and phages that provides a form of acquired immunity. RNA harboring the spacer sequence helps Cas (CRISPR-associated) proteins recognize and cut foreign pathogenic DNA. Other RNA-guided Cas proteins cut foreign RNA. CRISPR are found in approximately 50% of sequenced bacterial genomes and nearly 90% of sequenced archaea.
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CRISPR-Cas9 Review: A potential tool for genome editing
1. CRISPR-Cas9: A Potential Tool for Genome Editing
1. Introduction
1.1. Origins
In the year 1987, a team of Japanese scientists were the first to describe an unusual locus
found in the E. coli genome, adjacent to the iap gene, having short palindromic repeats interspersed
by similarly sized non-repetitive DNA spacers (Fig. 1) [1]. This clustered, regularly interspersed,
short palindromic repeat locus is therefore termed “CRISPR”. Nearly a decade later, up to forty
percent of sequenced bacteria and ninety percent of archaea were found to harbor this CRISPR locus
[2] [3].
In the year 2002, bacterial strains that survived bacteriophage infection were observed to
express the CRISPR loci, suggesting that the particular region may have a role in prokaryotic adaptive
immunity [4]. This hypothesis was subsequently confirmed when phage-resistant bacterial strains had
specific CRISPR loci spacers disrupted, they acquired susceptibility to phage infection, while
insertion of novel spacers into wild type stains demonstrated acquired resistance [5].
The proteins involved in the prokaryotic immune system were found to be conserved and
encoded in close proximity to the CRISPR locus (Fig. 1) [6]. As such, these proteins are known as
CRISPR-associated, or in short, “Cas”.
1.2. Mechanism of Action
The bacterial CRISPR-based acquired immunity (Fig. 1) encompasses three processes,
namely, spacer addition, CRISPR-RNA (crRNA) maturation and target elimination [7][8][9].
Upon initial exposure to foreign phage DNA, bacterial Cas1 locates the DNA’s unique
protospacer adjacent motif (PAM) and cuts a short DNA fragment (protospacer) that is directly beside
[7]. Integration of this protospacer into the CIRSPR locus is directed by the Cas1-Cas2 complex [7].
Successful integration of protospacers into the host genome are thereafter referred to as spacers.
A pre-crRNA is a long mRNA transcript of the CRISPR locus containing an array of spacer
and repeat sequences. It hybridizes with multiple trans-activating crRNAs (tracrRNA) to form RNA
duplex structures that are targeted for cleavage by RNase III [10]. The cleaved, mature crRNA,
encodes for a particular spacer and repeat sequence which remains hybridized to a tracrRNA, and this
short duplex is called the guide RNA (gRNA) [7][8].
Each gRNA has a unique spacer sequence that recognizes its complementary protospacer of
the phage DNA [7][8]. When an immunized bacterial cell reencounters the same phage DNA, the
appropriate gRNA guides Cas9, an endonuclease, to specifically target and eliminate the invading
DNA by inducing a site-specific double strand break (DSB) [7][8]. The PAM ensures that Cas9
2. complexes locate the correct protospacer sequence [7]. As the PAM is only found on invading phage
DNA, the DSB mechanism is able to discriminate self from non-self [7].
Figure 1: Bacterial CRISPR-based acquired immunity. Phase 1 (immunization): Initial injection of phage double strand DNA into a
bacterial cell followed by Cas1-Cas2 complex (large teal oval with associated grey ovals) excision of the protospacer (yellow rectangle)
from the phage DNA. The Cas1-Cas2 complex inserts the protospacer into the Type II CRISPR locus region containing short palindromic
repeats (black) and novel spacers derived from other foreign phage DNA (purple, green and red rectangles). Phase 2 (immunity): Pre-crRNA
(multi-colored linear mRNA) and tracrRNA (purple stem-loop mRNA) are transcribed and processed into guide RNA (mature crRNA-
tracrRNA duplex) which subsequently guides Cas9 (small teal oval) to cleave the invading DNA. (Figure adapted from [9])
1.3. Tool for Genome Editing
There are four major tools capable of inducing site-specific double strand breaks (DSB); zinc
finger nuclease (ZFN), transcription activator-like effector nuclease (TALEN), meganuclease and
CRISPR-Cas complex [11]. Any of these platforms may be programmed for DSB-induced genome
editing in prokaryotes and eukaryotes by exploiting their endogenous DNA repair mechanisms [11].
Breaks in the genome are remediated via one of two main repair mechanisms; non-
homologous end-joining (NHEJ) or homology-directed repair (HDR) [11]. The NHEJ pathway does
not utilize a template to flank the break region, hence, there is random insertion and/or deletions
(indels) of nucleotides at the site of damage [11]. This error-prone repair mechanism results in
frameshifts at the break site (Fig. 2.), and if the region encodes for a gene, its function is knocked out
[11]. On the other hand, HDR employs a template sequence that is homologous to the break site [11].
This high fidelity repair mechanism is mainly active during the S and G2 phases of mammalian cell
cycle, and the sister chromatid is used as template for the break sites [11]. However, an exogenous
template may be introduced to flank these break sites to incorporate synthetic sequences (Fig. 2) [11].
The CRISPR-Cas system has been praised for its efficiency, precision, low cost and ease of
use [8]. Furthermore, for multiplexing purposes, this system has the ability to simultaneously edit
multiple target sites without the need of cumbersome protein engineering as required in ZFNs or
TALENs [7][8]. Three types of CRISPR systems have been identified, however, only the type II
CRISPR-Cas system is the least complex as it only requires three components to function; Cas
3. endonuclease, mature crRNA and auxiliary tracrRNA [2][7]. This system has been further simplified
by fusing the crRNA with the tracrRNA to form a single guide RNA (sgRNA) [7][11].
Figure 2: Genome double strand break (DSB) repair mechanism. Single endonuclease-induced DSB (left): Non-homologous end-joining
(NHEJ) knocks out gene function by altering the gene’s original stop codon location. Homology-directed repair (HDR) occurs via strand
invasion into complementary templates. HDR may knock-in point mutations when provided with a mutated exogenous flanking template
(light blue dsDNA with star). HDR may knock-in new gene function when the exogenous flanking template contains its coding sequence
(light blue dsDNA with orange regions). Dual endonuclease-induced DSB (right): NHEJ large sequence deletions. (Figure adapted from
[11])
The type II CRISPR-Cas9 system is derived from Streptococcus pyogenes, and this powerful
genome editing tool has already been employed to alter the genes of different organisms and cell
types ranging from microorganisms, insects, plants, fishes, to mammalian cell lines [12]. Here, I will
review a few CRISPR-Cas9 genome editing applications, limitations and optimizations.
2. Applications
2.1. Disease Modeling
Mice are routinely used to study mammalian genetics and associated diseases by knocking
out or knocking in specific genes through embryonic stem cell (ES) homologous DNA recombination
methods [13]. As these methods were first established in mouse ES cells, genome editing has been
limited to mice. This posed difficulties in editing other mammalian species that may be better suited
to model unique human diseases [13][14]. Furthermore, it may take up to a year or more to
successfully produce a genetically altered mouse [8].
In human cell lines, homologous recombination methods have had little rate of success such
that alternative approaches with short interfering RNA have become common [8]. These alternatives
also bring about their own set of limitations such as transient gene expression knock-downs and off
target effects contributing to experimental inaccuracies [8].
4. Recent developments in genome editing technologies have enabled researchers to circumvent
the limitations around animal and cell line based disease modeling. Advantages include the ability to
genetically tweak animals for which ES cell lines are unavailable, the potential to acquire
homozygous knock-outs within the first generation, and the opportunity to explore other types of
animal models [8].
With CRISPR-Cas9 multiplexed genome editing, multiple generations of interbreeding to
derive an animal model with many genetic modifications can be avoided. This multiplexing potential
has been demonstrated to knock-out two genes of a monkey in one step by co-injecting one-cell-stage
embryos with Cas9 mRNA and sgRNAs [15]. In mice, CRISPR-Cas9 multiplexed genome editing of
two genes produced biallelic mutations with eighty percent efficiency [16]. Also, the CRISPR-Cas9
system empowers researchers to study single nucleotide polymorphism-associated human diseases
efficiently in mice by introducing a donor template that encodes the mutated sequence (Fig. 2) [14].
These remarkable achievements suggest that there may be no technical barrier in using the CRISPR-
Cas9 system to model other animals or cell lines for the development of new pharmaceuticals.
2.2. Drug Development
Genome-wide association studies, a field of study in functional genomics, have provided us a
wealth of knowledge on polygenic diseases such as Alzheimer’s, schizophrenia, autism and diabetes
[7]. Drug development for these genetic diseases relies heavily on disease model studies in zebrafish,
drosophila, mice and even mammalian cell lines using RNA interference (RNAi) [17][18]. As
mentioned previously in the disease modeling section, RNAi techniques have the tendency for partial
knock-downs and unmeasurable phenotypes [17]. Despite these limitations, genome-wide screening
for novel therapeutic targets have been using subsets of the RNAi technology, specifically, short
hairpin RNA (shRNA) and small interfering RNA [17].
The advancement of programmable genome editing platforms has enabled the generation of
gene knock-outs and mutation knock-ins in clinically relevant animal and cell based models for target
validation and drug discovery [17]. Unlike RNAi, which silences a particular gene expression by
targeting its mRNA transcripts, the CRISPR-Cas system targets its DNA coding sequence directly,
leading to permanent and complete gene knock-out [18]. As amendments made to the genome via the
CRISPR-Cas9 system are irreversible, alternative methods have been explored to mimic the RNAi
gene silencing approach, but at the DNA level as it confers greater efficiency [18]. A team of
scientists managed to create a dead Cas9 (dCas9) by inactivating its DSB catalytic mechanism [19].
Therefore, when guided by an engineered sgRNA, the dCas9-sgRNA complex interferes with the
targeted gene’s transcription, strongly silencing its gene expression [19]. This CRISPR interference
(CRISPRi) method appears to be most analogous to the RNAi principle.
In summary, the CRISPR-Cas9 system has enabled the study of other clinically relevant
animal models. Also, the ability to perform multiplexed genome editing and interference has opened
5. up greater opportunities for novel therapeutic target screening. Overall, functional genomics has
uncharted territories for us to explore in terms of polygenic disease knowledge and novel drug
development.
2.3. Regenerative Medicine
Many genetic disorders require individuals to be placed under prolonged or lifelong
medication. Based on current developments in genetic engineering, gene therapy may soon become a
primary route of treatment for genetic diseases [7]. Several proof of concept studies have
demonstrated the potential of gene therapy to correct monogenic recessive disorders such as
hemophilia, cystic fibrosis and Duchenne muscular dystrophy (DMD) [7][11]. On the other hand,
non-genetic diseases may also be prevented by modifying one’s genome.
Knock-out gene therapy relies on the error-prone NHEJ repair mechanism to induce indels at
the targeted gene, causing a frameshift mutation (Fig. 2) [11]. Familial hypercholesterolemia is an
autosomal dominant genetic disease caused by a mutation in the PCSK9 gene [20]. This gene codes
for a proteinase that degrades low density lipoprotein receptors (LDLR) and therefore contributes to
decreased metabolism of LDL cholesterol by the liver, increasing the risk for cardiovascular diseases
[20]. Patients with this condition are continuously prescribed PCSK9 proteinase-antagonists, however,
a single co-injection of Cas9 together with PCSK9-targeted sgRNA into mice liver in vivo has shown
to knock-out the targeted gene and effectively lower cholesterol levels [21]. Another promising
application of knock-out gene therapy is by disrupting the CCR5 gene encoding a major co-receptor
essential for HIV-1 to infect CD4+
T-cells [7][11]. Successful clinical trials have been observed with
ZFN edited stem cells being reintroduced into patients [7]. Similarly, engrafting Cas9 edited human
hematopoietic stem and progenitor cells appears to be a powerful alternative to combat AIDS [22].
Knock-in gene therapy employs the high fidelity HDR mechanism of cells to rectify
mutations by providing an exogenous template encoding the correct gene sequence to flank the DSB
site (Fig. 2) [11]. In addition, an alternative error-free insertion of exogenous genetic elements up to
fifteen kilobases have been developed using NHEJ-mediated ligation [23]. This is achieved by using
nuclease-induced DSBs to generate compatible overhangs on both the exogenous template and the
endogenous target site [23]. Both methods are advantageous compared to conventional viral vector-
mediated gene therapy associated with random insertion mutagenesis [11].
In certain cases, where knock-out or knock-in gene therapies may be inappropriate to rectify a
particular genetic disease, deletion-based gene therapy may be explored. To delete large genetic
elements several megabases in size from the genome, two targeted DSBs flanking the region of
interest must be simultaneously administered (Fig. 2) [11]. Using the CRISPR-Cas9 platform, this
method has been proven to be helpful in treating hemoglobinopathis by removing an entire BCL11A
erythroid-specific enhancer region [24]. Additionally, in DMD, where internal gene deletions lead to
6. frameshifts and as a result causes protein dysfunction, deliberate targeted excision of its exons can
rectify the frameshifts to generate a truncated, partially functional protein [11].
Targeted gene therapy with the CRISPR-Cas9 system hold much promise in the upcoming
decade as numerous proof of concept studies have already been established [7][11], and it is only a
matter of time before a flood of new clinical trials using this system will be approved.
2.3. Medical Microbiology
The abuse of antibiotics in medicine and animal agriculture has led to great selection pressure
on bacteria such that multidrug-resistant strains, especially the human pathogens, are of growing
concern as our pre-existing arsenal of useful antibiotics is shrinking. Another issue is that these
antibiotics have broad-spectrum targets and does not discriminate pathogens from beneficial normal
flora in our systems. Alternative novel antimicrobials have been explored using the CRISPR-Cas9
system to circumvent these limitations [25].
The sgRNA in the CRISPR-Cas9 system can be engineered to target virtually any essential
genomic regions of the bacteria for killing. As for the mode of delivery, preliminary studies employed
bacteriophages with phagemids encoding the Streptococcus pyogenes Cas9, the engineered crRNA
and the auxiliary tracrRNA [26][27]. These studies demonstrated effective killing of pathogenic
Staphylococcus aureus, carbapenem-resistant Enterobacteriaceae and enterohemorrhagic Escherichia
coli by targeting their virulence genes [26][27]. Specificity was also observed in one of the study as
the sequence-specific Cas9 discriminatively targeted virulent from avirulent Staphylococcus aureus
[26].
The CRISPR-Cas9 system can also be re-programmed to target antibiotic resistance genes
residing within the bacterial genome or in plasmids [25]. This could possibly be a method to restrict
plasmid-borne antibiotic resistance between clinically relevant pathogenic strains. Also, re-sensitizing
pathogenic strains to previously obsolete antibiotics can re-expand our antimicrobial arsenal.
In summary, sequence-specific antimicrobials may be used to treat multidrug-resistant
infections without killing beneficial normal flora by either directly via genome disruption or indirectly
through re-sensitization to antibiotics. However, as with any antimicrobials, further studies have to be
conducted to evaluate the types of evolution this method could bring about due to selection pressure.
2.5. Industrial Microbiology
A number of industrially relevant fungi and yeast strains utilized for the production of biofuel
are tough to engineer because of their intricate genomes [28]. Saccharomyces cerevisiae, one of the
most commonly exploited microbial cell factories, have diploid or polyploidy stains that make
genome editing a tricky task. Conventional methods employ selection of markers that are co-
integrated into its genome for gene mutation, deletion and integration [28]. Metabolic engineering has
7. found the CRISPR-Cas9 system to be a versatile genome editing tool to overcome these types of
challenges [28].
Genome engineering of several different industrially relevant Saccharomyces cerevisiae
strains using the CRISPR-Cas9 platform has demonstrated successful biallelic disruption of a gene
with efficiency up to seventy-eight percent [29]. In addition, by harnessing the multiplex nature of the
CRISPR-Cas9 platform, genome engineering of up to five different genomic loci in Saccharomyces
cerevisiae has demonstrated to increase mevalonate titers up to forty-fold compared to wild-type
strains, although no overexpression of genes was done in the mevalonate pathway [30]. Therefore,
CRISPER-Cas9 can be used in strain optimization to yield greater amounts of important secondary
metabolites.
2.6. Agricultural Biotechnology
Genetically modified crops incorporated with transgene via the HDR mechanism undergoes
strict regulations, decreasing their commercial viability [7]. NHEJ knock-outs however, are said to be
non-transgenic and is beyond the United States Department of Agriculture (USDA) regulatory
authority [31]. Highlighted here are a few prominent CRISPR-Cas9 based crop and livestock genome
editing that have taken place over the past few years.
Engineering plants and crops to be resistant to diseases is one of the major goals for
agricultural biologists. Leveraging upon the superior gene silencing and multiplexing capabilities of
the CRISPR-Cas9 system over RNAi, a number of model plant and crop species have been further
studied [7]. Examples include Arabidopsis, tobacco, tomato, maize, rice and wheat [7]. The powdery
mildew disease is a deadly wheat infection caused by the fungus Blumeria graminis [7]. In bread
wheat, Triticum aestivum, a hexaploid crop, three mildew resistance locus (MLO) homoeoalleles were
mutated via CRISPR-Cas9 [32]. Subsequent self-fertilization yielded knock-outs for all six alleles,
conferring the bead wheat resistance to powdery mildew disease [32].
Biofortification is the process by which plants, crops and livestock are genetically engineered
to enhance their nutritional value. Improvements can be made directly by modulating the amount of
nutrient the foodstuff produces, or indirectly by eliminating anti-nutrients that decreases nutrient
bioavailability [7]. Goat, sheep, pig and cattle have already been successfully engineered using the
CRISPR-Cas9 system [7]. Other biofortification strategies using CRISPR-Cas9 are still under
development, and although ZNFs and TALENs have already been extensively used, the potential of
CRISPR technology to improve the nature of agricultural produce and livestock remains massive.
3. Limitations
3.1. Target Specificity
Off-targeting, whereby DSBs are made at sites other than the intended target region, has been
one of the greatest limitation of not only the CRISPR-Cas9 system, but also other genome editing
tools within the family of programmable nucleases [2][8]. Should off-target mutations occur
8. frequently using the CRISPR-Cas9 system, there may be a loss of genomic stability and functionality
of otherwise normal genes, reducing the reliability of the tool for biomedical and clinical applications.
The targeting specificity of Streptococcus pyogenes Cas9 is tightly controlled by the twenty-
nucleotide guide sequence in the sgRNA and the PAM (typically NGG) sequence located beside the
target region (Fig. 3) [2][8]. However, potential off-target DSBs could be induced if there is three to
five base pair mismatch in the PAM-distal region of the guide sequence (seed region) [2].
A genome-wide binding analysis in mouse embryonic stem cells using dCas9 and chromatin
immunoprecipitation followed by sequencing (ChIP-seq) revealed a distinct seed region for on-target
binding, but also multiple off-target binding sites [2]. However, majority of these off-target regions
were not cleaved by catalytically active Cas9 [2].
Based on the catalytic activity, exceptionally high or low GC content within the sgRNA could
cause the guided Cas9 to be less active [33]. Also, methylation of DNA at CpG sites have been
reported to decrease Cas9 binding efficiency [2].
Figure 3: Cas9 endonuclease guided by sgRNA to target sequence. Cas9 endonuclease (large green structure) containing the sgRNA
(small green loop structure) is shown interrogating the target DNA (blue double strand structure). Scissors indicate Cas9 cleavage site three
base pair upstream the PAM sequence. The single letter DNA code depicted encodes for (N=A, T, C, G; R=G or A). (Figure adapted from
[2])
3.2. Delivery Techniques
9. Lentiviral and Adeno-associated virus (AAV) vectors have been commonly used to transfer
genetic information into cells. However, they do not have sufficient capacity to shuttle an entire Cas9
genome editing infrastructure [7]. Although other viral vectors such as adenovirus possess greater
carrying-capacity, they are highly immunogenic and have limited cell-type infectivity [7].
4. Optimizations
4.1. System Modifications
Three different forms of modification have been made to the Cas9 system to increase
targeting fidelity. Firstly, inactivation of the RuvC nuclease domain generates a Cas9 with nickase
(nCas9) functionality, causing only targeted single strand breaks in DNA instead of DSBs [7].
Appropriately spaced nicks formed with two nCas9 can mimic the effects of DSB, reducing the off-
target frequency as compared to using only one endonuclease [7].
Secondly, as off-target DSBs are mainly due to the strong binding affinity between the
sgRNA and non-specific target site, a truncated guide sequence could reduce the binding affinity such
that mismatched base pairs are no longer well tolerated, decreasing off-target editing [7][8].
Lastly, dCas9 can be engineered with Fok I nuclease domain to generate fCas9 [7]. A paired
fCas9 functions analogously to the paired nCas9, and has up to 140-fold increase in specificity
compared to wild type Cas9 [7].
Earlier this year, researchers have reported the generation of a high-fidelity CRISPR-Cas9
system capable of precise genome edition with undetectable off-target genome disruption [36].
4.2. Alternative Delivery Techniques
To overcome the capacity limitations of viral vectors, the essential Cas9 genome editing
modules have been engineered into separate plasmids and co-transfected into cells [7]. For in vivo
disease modeling, mice have been engineered to express Cas9 in a Cre-driven manner, such that only
the sgRNA is required for efficient genome editing [7]. Another novel method to introduce Cas9
proteins into cells involve fusion with anionic supercharged GFP that are delivered to human cell
lines and mice using cationic liposomes [35].
Direct introduction of purified Cas9 proteins together with the engineered sgRNA into cells
have shown to minimize off-target DSBs compared to plasmid-based Cas9-sgRNA expression [34].
The increased fidelity was reported to be derived from the rapid degradation of these Cas9-sgRNA
complexes in cells [34].
5. Conclusion
In recent years, research and application of the CRISPR-Cas9 system have picked up pace.
The advantages associated with this system compared to other programmable nucleases are
efficiency, specificity, time and cost saving. Furthermore, its ability to conduct reliable multiplexed
10. applications have opened up the opportunity for us to explore the vast knowledge of functional
genomics unattainable previously with RNAi technology.
This genome editing tool is providing advancement in many scientific fields. Enhancing
agriculture, defending against pathogens and remediating human genetics are just few of the benefits
listed from the large pool of published studies. With the recent development of high-fidelity Cas9 that
generates no detectable genome-wide off targets, many human-based clinical trials harnessing this
powerful genome editing platform are expected to be approved sooner than later.
Many questions still remain about how the innate immune system of organisms targeted with
CRISPR-Cas9 will respond. As of now, little is known on the selection pressure or evolution these
types of genome editing can bring about. Nevertheless, as many genome editing preclinical studies
and clinical trials have been successful, there is much hope and optimism for the future of genome
editing with CRISPR-Cas9 technology.
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