Genetic Recording in Yeast Using CRISPR-Cas9
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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
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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.
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Genome editing methods such as ZFNs, TALENs, and CRISPR/Cas9 use engineered nucleases to create targeted double-stranded breaks in DNA which are then repaired through endogenous cellular processes. These nucleases can be used to modify genomes through techniques like gene knockout, targeted mutation insertion/deletion/correction, and studying gene function. CRISPR/Cas9 uses a guide RNA and Cas9 nuclease to target specific DNA sequences for editing. The four main steps for CRISPR are: 1) selecting target sequences near a PAM site, 2) designing and cloning gRNA, 3) delivering Cas9 and gRNA into cells, and 4) DNA repair after cleavage results in gene modification
Crispr
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.
Genome Editing Comes of Age; CRISPR, rAAV and the new landscape of molecular ...
Information is no longer a bottleneck, emphasis is shifting to the ‘what does it all mean’
In a translational context we hope that by answering that question we will be able to is to characterise the genetics that drive disease, and indeed develop drugs and diagnostics that are personalised to patients.
Genome editing provides the link between the information here, and this outcome here, by allowing scientists to recapitulate specific genetic alterations in any gene in any living tissue to probe function, develop disease models and identify therapeutic strategies. So, not only do we now have unparalleled access to genetic information, but we now have the tools to most accuartely understand what this genetic information – with genome editing allowing us to explore the genetic drivers of disease in physiological models.
AAV is a single-stranded, linear DNA virus with a a 4.7 kb genome which for the purpose of genome editing is replaced almost in entirety with the targeting vector sequence (except for the iTRs)
It is in effect a highly effective DNA delivery mechanism
After entry of the vector into the cell, target-specific homologous DNA is believed to activate and recruit HR-dependent repair factors can induce HR at rates approximately 1,000 times greater than plasmid based double stranded DNA vectors, but the mechanism by which it achieves this is still largely unknown
By including a selection cassette can select for cells that have integrated the targeting vector, and then screen for clones which have undergone targeted insetion rather than random integration, which will generally be around 1%.
Increase efficiency of genome editing with the Alt-R™ CRISPR-Cas9 System: Des...
Increase efficiency of genome editing with the Alt-R™ CRISPR-Cas9 System: Des...Integrated DNA Technologies
The CRISPR/Cas9 system has emerged as one of the leading tools for modifying genomes of organisms ranging from E. coli to humans. In a recent webinar, "New RNA Tools for Optimized CRISPR/Cas 9 Genome Editing", we presented how we developed the Alt-R™ CRISPR-Cas9 System for genome editing. Here, we take a look at designing your target sequences and ordering them as Alt-R CRISPR crRNA. We review the other components of the system and walk through the experimental process step by step, from design to evaluation of editing potency. We also discuss challenges and potential pitfalls and provide tips and guidance towards successful genome editing experiments. Learn more: http://www.idtdna.com/crisprCRISPR technology -A revolutionary discovery
CRISPR technology allows for genome editing using a prokaryotic immune system called CRISPR/Cas. The system works by adapting spacers from viral DNA, producing CRISPR RNA, and targeting matching sequences. It is being applied in industry to make bacterial cultures virus-resistant, in labs for genetic engineering, and in medicine for treating genetic diseases and developing more specific antibiotics.
Genome Editing CRISPR-Cas9
The document discusses CRISPR-Cas9 genome editing. It begins by explaining why genome editing is useful for applications like disease modeling, gene therapy, and agriculture. It then provides details on the CRISPR-Cas9 system, describing how it uses the Cas9 enzyme guided by a short RNA to introduce targeted double-stranded breaks in DNA. The document outlines several uses of CRISPR-Cas9 in research, including generating animal models of disease and correcting genetic defects in human cells and stem cells. It also discusses approaches for screening mammalian cells using libraries of guide RNAs to induce mutations.
Applications of crispr cas system
CRISPR-Cas is a genome editing technique derived from bacterial immune systems that allows for precise genomic modifications. The document discusses applications of CRISPR-Cas in plants, animals, and bacteria, including developing pest and disease resistant crops and livestock, modifying stem cells and embryos, targeting antibiotic resistant bacteria, and controlling gene expression.
New RNA tools for optimized CRISPR/Cas9 genome editing
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.
CRISPR-Cas9 Review: A potential tool for genome editing
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.
Gene Editing - Challenges and Future of CRISPR in Clinical Development
In this presentation, Medpace experts discuss the unique opportunity of evolving gene editing technology as it applies to human diseases
Crispr cas: A new tool of genome editing
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 Editing
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.
Crispr handbook 2015
This handbook describes CRISPR/Cas9 genome editing and other research applications for CRISPR technology.
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Lessons learned from high throughput CRISPR targeting in human cell lines
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Personalising CRISPR Genome Editing || Edward Perello || Disruptor Stories
Personalising CRISPR Genome Editing || Edward Perello || Disruptor Stories
Genome Editing Comes of Age; CRISPR, rAAV and the new landscape of molecular ...
Genome Editing Comes of Age; CRISPR, rAAV and the new landscape of molecular ...
Increase efficiency of genome editing with the Alt-R™ CRISPR-Cas9 System: Des...
Increase efficiency of genome editing with the Alt-R™ CRISPR-Cas9 System: Des...
New RNA tools for optimized CRISPR/Cas9 genome editing
New RNA tools for optimized CRISPR/Cas9 genome editing
CRISPR-Cas9 Review: A potential tool for genome editing
CRISPR-Cas9 Review: A potential tool for genome editing
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Gene Editing - Challenges and Future of CRISPR in Clinical Development
Similar to Genetic Recording in Yeast Using CRISPR-Cas9
Reducing off-target events in CRISPR genome editing applications with a novel...
Reducing off-target events in CRISPR genome editing applications with a novel...Integrated DNA Technologies
The CRISPR-Cas9 system demonstrates unparalleled genome editing efficiency in a broad range of species and cell types, but it suffers from concerns related to target specificity. Modified guide RNAs and mutant Cas9 proteins have been developed to reduce off-target editing but, in many cases, the alterations also significantly reduce on-target editing performance. In this presentation, Dr Chris Vakulskas discusses a novel, high-fidelity Cas9 protein that reduces off-target gene editing, while maintaining high on-target activity. Dr Vakulskas presents data from the development of the new Alt-R® S.p. HiFi Cas9 Nuclease 3NLS and describes its usefulness in mitigating unwanted off-target gene editing, without the issues associated with transfection of plasmid DNA.2.CRISPR .pptx
This document discusses CRISPR-Cas9 gene editing. It begins by defining gene/genome editing and describing what CRISPR is and how it functions in bacteria. CRISPR uses Cas9 proteins and guide RNA to create double-strand breaks in DNA at targeted locations. There are two main pathways for repairing double-strand breaks - nonhomologous end joining and homology-directed repair. The document then outlines the general workflow for CRISPR gene editing, including designing guide RNAs, selecting expression systems, transfecting cells, selecting clones, and characterizing gene editing through assays like sequencing and western blot.
CRISPR theory mechanism and applications || كرسبر النظريه وطريقه العمل والتطب...
CRISPR-Cas9 is a gene editing technique that allows DNA to be added, removed, or altered at specific locations in the genome. It involves using the Cas9 protein to cut DNA at a targeted site, which can be programmed by changing a short RNA sequence. CRISPR is simpler and easier than previous gene editing methods. It has enabled unprecedented efficiency and ease of use for gene therapy applications in correcting genetic defects and treating diseases. However, its rapid development has also led to an intensifying patent war and debate over its ethical use.
Crispr cas9
This document discusses the CRISPR-Cas9 genome editing technique. It begins with an overview of genome editing and provides a brief history. It then focuses on explaining CRISPR-Cas9, including its key components, how it was discovered as a natural bacterial immune system, and how it functions as a genomic tool. The document outlines the general CRISPR-Cas9 protocol and recent advances in the technique. It discusses applications in agriculture and for diseases. It also touches on advantages and limitations, as well as ethical issues. Two case studies are provided that demonstrate using CRISPR-Cas9 to modify genes in rice plants.
Increasing genome editing efficiency with optimized CRISPR-Cas enzymes
Genome editing by CRISPR systems has proven to be groundbreaking for basic biomedical research with significant implications for the treatment of human diseases. While the CRISPR-Cas9 and CRISPR-Cas12a (Cpf1) systems enable genome editing in a broad range of host species and cell types, both can exhibit poor editing efficiencies at specific target sites or in systems where delivery of CRISPR reagents is difficult. There are concerns about target specificity of the CRISPR-Cas9 system and, in many cases, typical remedies such as modified guide RNAs or mutant Cas9 proteins cause loss of genome editing efficiency. Many of these solutions for improving specificity were developed for delivery of the Cas9-gRNA complex via plasmid DNA vectors rather than delivery as ribonucleoproteins (RNPs). However, RNP delivery of CRISPR reagents is being increasingly used because of the risk of unwanted stimulation of the immune system by plasmid delivery.
In this webinar, Dr Vakulskas discusses improved Cas9 and Cas12a (Cpf1) nucleases that have been optimized to significantly increase editing efficiency in living cells. He also presents data showing that IDT’s latest high-fidelity Cas9, Alt-R HiFi S.p. Cas9 V3, increases on-target editing efficiency and dramatically reduces off-target editing.
Crispr cas9
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.
SIP Presenation
This document summarizes the generation of AFAP1L1 knockout cell lines using CRISPR/Cas9 technology. Sarcoma and U2OS cell lines were used. The CRISPR plasmid was able to successfully target AFAP1L1 and an mCherry repair plasmid confirmed integration. After co-transfection, 99% of cells expressed red fluorescence, indicating successful knockout. Future work will include sequencing clones to verify editing and observing cell morphology without AFAP1L1, including potential in vivo mouse models to study metastatic effects.
Alt-R™ CRISPR-Cas9 System: Ribonucleoprotein delivery optimization for improv...
Alt-R™ CRISPR-Cas9 System: Ribonucleoprotein delivery optimization for improv...Integrated DNA Technologies
Struggling with low editing efficiency or delivery problems? IDT has developed a simple and affordable CRISPR-Cas9 solution that outperforms other methods. In this presentation we present the advantages of using a Cas9:tracrRNA:crRNA ribonucleoprotein (RNP) complex in genome editing experiments, and explain why it is the most efficient driver for genome editing compared to alternative methods, such as expression plasmids or the use of sgRNAs. We also review RNP delivery using cationic lipids and electroporation, and provide tips for optimized transfection in your system. Full-length cDNA Sequencing.pdf
This document discusses methods for sequencing full-length cDNA using the PacBio RS system. It compares two common cDNA library preparation kits and shows they produce libraries with expected size distributions but differ in input requirements and stringency. It also demonstrates that normalization during cDNA preparation increases coverage breadth by detecting more genes. Size selection or targeted enrichment methods like SureSelect can be used to focus on specific transcript sizes or gene subsets. Analysis options include aligning reads to transcript or genome references to characterize isoforms or detect novel splice variants.
CRISPR CAS9.pptx
This document provides an overview of designing CRISPR/Cas9 based genome editing in crop plants. It discusses selecting a target gene, constructing the CRISPR/Cas9 system using Cas9 protein and guide RNA, adding gene regulatory elements like promoters and terminators, designing constructs, transforming plants using various methods, and validating genome edits using techniques like sequencing, phenotyping, and molecular assays. The goal is to use this gene editing tool to introduce traits like disease resistance, drought tolerance, and improved nutrition in crop plants.
Crispr cas9
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.
Genome editing
This presentation highlights the basics and application of genome editing strategies in plants, strategies to reduce off-target mutation, identification of mutant analysis etc.
CRISPR - gene-editing for everyone
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
CRISPR.pdf
CRISPR/Cas9 is a powerful genome editing tool that allows genetic material to be added, altered or removed at specific locations in the genome. It involves a bacterial adaptive immune system where CRISPR sequences and Cas genes work together. The Cas9 protein uses a guide RNA to introduce double stranded breaks at targeted DNA sequences. This enables precise genome editing through non-homologous end joining or homology directed repair. CRISPR/Cas9 provides a simple and accurate way to modify genes for applications in research, medicine, agriculture and more. While it holds great promise, there are also limitations and concerns regarding off-target effects that researchers continue working to address.
Animal bt group presentation
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: Gene editing for everyone
CRISPR/Cas9 gene editing is based on a microbial restriction system, that has been harnessed for genome targeting using only a short sequence of RNA as a guide.
The beauty of the system is that unlike protein binding based technologies such as Zinc Fingers and TALENs which require complex protein engineering, the design rules are very simple, and it is this fact that is allowing CRISPR to take genome engineering from a relatively niche persuit to the mainstream scientific community.
The principle of the system is that a short guide RNA, homologous to the target site recruits a nuclease – Cas9
This then cuts the dsDNA, triggering repair by either the low fidelity NHEJ pathway, or by HDR in the presence of an exogenous donor sequence.
High Efficiencies for both knockouts and knock-ins have been reported and whilst there are understandable concerns about specificity, new methodologies to address these are now being developed
The system itself is comprised of three key components
the Cas9 protein, which cuts/cleaves the DNA and
Two RNAs - a crispr RNA contains the sequence homologous to the target site and a trans-activating crisprRNA (or TracrRNA) which recruits the nuclease/crispr complex
For genome editing, the crisperRNA and TraceRNA are generally now constructed together into a single guideRNA or sgRNA
Genome editing is elicited through hybridization of the sgRNA with its matching genomic sequence, and the recruitment of the Cas9, which cleaves at the target site.
Gene Editing for everyone
CRISPR/Cas9 gene editing is based on a microbial restriction system, that has been harnessed for genome targeting using only a short sequence of RNA as a guide.
CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)
This Lecture is presented by our volunteer Talha Saleem, he is from Karachi Pakistan, and he is covering CRISPR topic.
For video: https://www.youtube.com/watch?v=lSp1j0dRfjQ&t=88s
Pcr primer design
Primer design is a key step in PCR that requires considering various factors to optimize the reaction. These include primer length, melting temperature, GC content, specificity, and potential for secondary structures. Well-designed primers are unique in targeting a single region, have compatible melting temperatures, and do not form hairpins or primer dimers that could inhibit the reaction. Choosing appropriate primers is essential for successful PCR amplification.
CRISPR in crop Improvement, CRISPR/Cas Genome editing tool
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.
Similar to Genetic Recording in Yeast Using CRISPR-Cas9 (20)
Reducing off-target events in CRISPR genome editing applications with a novel...
Reducing off-target events in CRISPR genome editing applications with a novel...
CRISPR theory mechanism and applications || كرسبر النظريه وطريقه العمل والتطب...
CRISPR theory mechanism and applications || كرسبر النظريه وطريقه العمل والتطب...
Increasing genome editing efficiency with optimized CRISPR-Cas enzymes
Increasing genome editing efficiency with optimized CRISPR-Cas enzymes
Alt-R™ CRISPR-Cas9 System: Ribonucleoprotein delivery optimization for improv...
Alt-R™ CRISPR-Cas9 System: Ribonucleoprotein delivery optimization for improv...
CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)
CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)
CRISPR in crop Improvement, CRISPR/Cas Genome editing tool
CRISPR in crop Improvement, CRISPR/Cas Genome editing tool
Genetic Recording in Yeast Using CRISPR-Cas9
- 1. Yeast Genome Editing Using CRISPR/Cas9 Robert Beem
- 2. Project Goal Analyze activity of self targeting guide RNA (stgRNA) in Saccharomyces cerevisiae
- 4. Motivations • Yeast grows faster amongst eukaryotes • Faster genomic evolution • Protein Evolution and Production • Study limitations of Cas9
- 5. Methods • Plasmid Construction in E. coli (DH5α) Antibiotic selection Cas9 (S. pyogenes) stgRNA
- 6. Methods • Yeast Genome Integration Homologous recombination - Tryptophan, Leucine markers W303-1A + Cas9 & stgRNA
- 7. Construction of Cas9 Plasmid RBM1* RBM3 RBM4 pTPGI dCas9 VP64 D10A H840A H840AD10A pTPGI dCas9 TAA pTPGI Cas9 TAA *starting plasmid NLS NLS NLS
- 8. pTPGI Inducible Promoter Ellis et al., Nature Biotechnology 2009 Gal + aTc TetR pTPGI Cas9 Gal aTc Cas9 Low Low Low Low High Low High Low Low High High High NLS
- 9. Construction of stgRNA Plasmid RBM2* RBM7 RBM8 pRPR1 gRNA scaffold RPR t2micron origin RBM2 pRPR1 gRNA scaffold RPR t pRS405 pRPR1 stgRNA scaffold RPR t pRS405 SDS TTGG AACC *starting plasmid
- 10. Cas9 trp1 trp1-1 Cas9 trp1-1trp1 Homologous Recombination in Yeast TRP: Bsu36I LEU: BfuAI
- 11. Cas9 Genome Integration trp1-1: nonsense mutation, glu83STOP RBY1 pTPGI Cas9 TAA W303-1A Chr IV
- 12. stgRNA Genome Integration leu2-3, 112: frameshift mutation, gly83 RBY2 W303-1A Chr III pRPR1 gRNA scaffold RPR t pRS405 SDS TTGG AACC
- 14. CRISPR/Cas Overview 3 steps: 1. Synthesis 2. Transcription 3. Targeting
- 15. CRISPR/Cas Synthesis Natural: Genome CRISPR/Cas gene Artificial: Integrated Plasmid stgRNA/Cas gene
- 17. CRISPR/Cas Targeting Natural: Cutting foreign DNA Artificial: SDS CCNNGG PAM stgRNA scaffold
- 18. SDS CCNNGG PAM SDS CCNNGG PAM NHEJ Original DNA Modified DNA
- 19. How do we assay Cas9 activity? • Sequence mutations • T7 endonuclease assay
- 20. New England Biolabs, Genome Editing 2015
- 21. T7 Endonuclease Assay • Shows SDS editing has occurred • Original Amplification: 1.4 kb • Edited Amplifications: – 800bp – 600bp
- 22. Experimental Steps Grow • Gal +/- aTc • 22 hours Amplify • Forward primer 600bp upstream • Reverse primer 800bp downstream Assay • T7 endonuclease • Gel Electrophoresis
- 24. stgRNA T7 Endonuclease Assay (20bp SDS) + aTc - no aTc
- 25. Summary • stgRNA is effective in S. cerevisiae • pTPGI lacking aTc “leaks” Cas9 • Cas9 is significant in low quantities
- 26. Future Research • Longer SDS • Expanding toolkit for genome engineering • Multiple promoters • Protein evolution using stgRNA • Applications: – Protein engineering – Gene therapy
Editor's Notes
- Motivations: Mention first that stgRNA has been tested in human cells Want to know if stgRNA is active in yeast Want to know if stgRNA can possibly be more efficient in yeast Yeast does not have as complex RNA processing machinery as human cells Speculate that stgRNA can be more effective in S. cerevisiae Other motivation is that yeast grow faster Hence you can perform genomic evolution on a faster timescale If it works in yeast we could leverage the system towards protein evolution
- Site cut: TRP: Bsu36I LEU: BfuAI
- Restate: RPR is unlike other promoters. RPR1 does not make a protein. RPR is an RNA polymerase III (RNAPIII) promoter. Gal1 and others are RNAPII promoters that produce mRNA with poly-A tails.
- Yeast machinery: reiterate: Cas9: RNA p 2 -> translatable stgRNA: RNA p 3 -> stays in nucleus
- Before explaining NHEJ say this: in phage if you cleave the DNA it will degrade but in yeast there would be a repair mechanism
- Mention: I got the desired colonies, and then grew in selective media + Glu first, and then did -/+ for experiment.
- Negative was brighter because the positive cells grew slower due to metabolic burden of expressing cas9 at high levels so what we notice is not just a drop in cas9 targeting efficiency but a systemic effect