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CRISPR/Cas9 Videos
Learn more about Crispr/Cas9 and his application
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CRISPR Cas9 - A Brief Introduction
CRISPR Cas9 system is a versatile and robust genome editing system with showing success in various model organisms. In this video, you will be introduced to this technology and its various applications and its impact on genome engineering. Learn more about:
- Technology Breakdown
- Mechanism of Gene Editing
- Variation to the CRISPR Systems
- Cas9 Nickase
- Cas9 Null Mutant
- CRISPR Research
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2) CRISPR Cas9 - Methods and Tools
CRISPR Cas9 system is a versatile and robust genome editing system with showing success in various model organisms. In this video, you will learn more about methods and tools, compromising:
- Expression
- Non-viral based Gene Delivery (Lipid-mediated transfection, Calcium phosphate transfection, Electroporation)
- Variation to the CRISPR Systems
- Viral based Gene Delivery (Lentivirus, Adenovirus, Adeno-associated Virus)
- Cas9
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3) CRISPR Cas9 - gRNA Design
The RNA guided CRISPR Cas9 system has revolutionized genetic engineering by allowing targeted genomic modification through the simple design of a 20 bp sequence. This technology utilizes a short guide RNA to direct the Cas9 nuclease to a specific genomic locus. The guide RNA is responsible for the specificity of the CRISPR Cas9 system. Many considerations should be taken through the design process. Learn more about:
- crRNA, tracerRNA and gRNA
- Basic considerations
- Variation to the CRISPR Systems
- Desired genomic modification
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4) CRISPR Cas9 - Gene Regulation with dCas9
The CRISPR Cas9 system has revolutionized the field of genetic engineering with its simplicity and versatility. Now, scientists have cleverly created a catalytically dead Cas9 (dCas9) that is able to bind its target with high specificity without cutting any DNA. This new CRISPR Cas9 system expands the field a step further as a novel new tool for reversible gene regulation. As you will learn in this video, dCas9 can act as a modular platform upon which activators or repressors can be easily fused and targeted to the gene of interest.
- Quick review of the CRISPR Cas9 system
- Introducing dCas9
- dCas9 systems for gene activation and repression
- dCas9 systems for epigenetic editing
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How to perform a CRISPR Knockin Experiment
Are you looking for a reliable and affordable way to knockin a gene? In this video, the CRISPR Cas9 gene editing system is used to knockin the RFP gene into the AAVS1 Safe Harbor site in HEK Cells. The Safe Harbor site is a preferred site for gene knockins as it enables robust transgene expression with minimal toxic effects. First, an All-in-One CRISPR Cas9 sgRNA vector construct with the sgRNA targeted to the human AAVS1 locus is designed. A donor plasmid containing the template for homology directed repair is also designed. After successful transfection, the success of the CRISPR Cas9 Knockin is verified using a PCR-based technique. This technique utilizes custom designed primers targeting a site within the RFP knockin. Knockin is confirmed when a PCR product is observed.
- Design of an All-in-One CRISPR Cas9 sgRNA vector construct with the sgRNA targeted to the human AAVS1 locus
- Design of a donor plasmid containing the template for homology directed repair
- Transfection, selection and expansion of successfully edited cells
- PCR Genotyping to confirm Knock-in
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How to perform a CRISPR Knockout Experiment
Due to CRISPR's unparalleled ease-of-use and affordability, gene knockout experiments are now more feasible than ever before! In this video, it is shown how the CRISPR Cas9 system is used to knockout the LIF gene in a colon carcinoma cell line. This is accomplished by the delivery of a Cas9 and sgRNA lentivector into the cell line followed by drug selection and monoclonal isolation. More importantly, you see a demonstration how to characterize and verify your CRISPR Knockout using the PCR-based Surveyor Assay, Sanger Sequencing, and Next Generation Sequencing. This complete process of validation ensures that your final CRISPR Knockout cell line is 100% successful. This case study teaches you the following steps:
- Delivery of Cas9 and sgRNA
- First round of selection
- Sequence analysis
- Monoclonal selection
- Confirmation of KO by NGS (Next Generation Sequencing)
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How to perform a Bacterial CRISPR Cas9 Knockout Experiment
Although the CRISPR system originated in bacteria, it is more commonly used to edit eukaryotic genomes rather than bacterial genomes. This is because most bacteria are unable to effectively repair double-stranded DNA breaks created by Cas9. Scientists have therefore developed a method that combines both recombineering technology and CRISPR Cas9 to allow us to be able to edit bacterial genomes! To demonstrate how this can be done, the CRISPR Cas9 system is used to knockout the chloramphenicol resistance casette (CAT) gene in E. coli (previously introduced into the genome at the yeeR locus). This process involved:
- Designing and delivering sgRNAs against the target site
- Designing and delivering a repair template
- Delivering phage-derived (lambda red) recombinases alongside Cas9 to enhance homologous recombination in bacteria
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How a mini CRISPR Cas9 system is revolutionizing gene therapy!
CRISPR saCas9: It's the greatness of spCas9 "miniaturized"! Have you heard of saCas9? It's a newly discovered nuclease that could change the game for CRISPR-based gene therapy. This "little" nuclease is small enough to fit into an adeno-associated virus and has already been busy editing the genomes of living organisms. Exciting, right? Watch our video to learn more about this CRISPR Cas9 system! Learn more about saCas9:
- saCas9 structure
- saCas9 in vivo performance
- saCas9 disease treatment
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CRISPR Cas9 vs. Cpf1: 5 ways the Cpf1 nuclease outsnips Cas9
What's the difference between CRISPR Cas9 and Cpf1? In this episode, you are introduced to Cpf1, a CRISPR nuclease discovered by the Zhang lab which is available at Hölzel Diagnostika. Cpf1 is smaller than Cas9 and targets complementary areas of the genome, making it an excellent addition to the CRISPR dream team. Watch our video to learn more about why Cpf1 deserves its own fan following! Learn more about:
- structure of Cpf1
- Cpf1 advantages
- differences between Cas9 and Cpf1
- opportunities using Cpf1
