Introduction and objectives
What is CRISPR/Cas9? What are the advantages of performing genome-wide knockouts using pooled libraries? What is the best way to identify the genes responsible for your observed phenotype?
Genome-wide sgRNA library screening made simple.
In this webinar, Thomas Quinn discusses the design and execution of genome-wide CRISPR/Cas9-based knockout screens using pooled lentiviral sgRNA libraries. He also describes the features and benefits of the Guide-it CRISPR Genome-Wide sgRNA Library System and NGS analysis kit, a complete solution developed with the aim of making lentiviral CRISPR/Cas9 screening accessible to all.
The webinar is displayed in the order it was given, but divided into bite-size sections, so you can jump right to the topic that interests you most.
About the presenter
Thomas Quinn, M.S.
Group Leader (Virology), Takara Bio USA, Inc.
Thomas received his B.S. degree in Zoology with a concentration in Genetics from Michigan State University in 1992. In 1997, he completed his M.S. in Molecular Medicine and Genetics from Wayne State University. His thesis work focused on the development of viral vector systems for application in CAR-T therapies directed at melanoma and lung malignancies. Since 1998, he has been working at Takara Bio USA, Inc. as a Group Leader focusing on the development of high-efficiency viral delivery products including adenoviral, retroviral, lentiviral and AAV systems, as well as CRISPR/Cas9 based genome editing products.
Introduction and objectives
What is CRISPR/Cas9? What are the advantages of performing genome-wide knockouts using pooled libraries? What is the best way to identify the genes responsible for your observed phenotype?
Overview of a genome-wide sgRNA library screen
What are some possible methods for performing a genome-wide screen and points to consider? Using a drug resistance phenotype screen as an example, learn how knocked-out genes that convey resistance or sensitivity to the drug can be identified by downstream NGS analysis.
Advantages and challenges of pooled libraries
What are the advantages and challenges of guide RNA libraries vs. RNAi and arrayed libraries vs. pooled libraries? Challenges include using effective guide RNA designs, making sure that the complexity of your library allows you to screen the entire human genome for targets, producing sufficient amounts of high-quality lentivirus, and maintaining library representation throughout all the handling steps.
Why we choose the Brunello algorithm, and how we tested its effectiveness
Learn how the lentiviral vector and guide RNAs in the library are designed for maximizing performance, and for predicting, selecting, and testing for active guide RNAs. Also learn about the advantages of the Brunello library algorithm, optimization of the number of guides per gene, and their representation relative to each other in the library. How does the representation of guide RNAs in the plasmid library correlate to their representation in the virus produced from it?
Workflow and timeline for a genome-wide library screen
Learn more about the workflow timeline, the steps for obtaining transduced cells that contain Cas9 and a single copy of guide RNA per cell, as well as the approximate time needed for each of these steps.
Creation of a screenable Cas9 stable cell line
Take a deeper look at the process for creating the perfect Cas9+ stable cell line prior to the screening. Why test different MOI levels? Is Cas9 expression consistent over time?
Library preparation, titration, and transduction
With this simple-to-use, all-in-one format of the guide RNA library all you need to do is add water! See math demonstrating how much virus you need and how many target cells you need to transduce. Learn how our library design reduces your workload.
After completion of the library screening
Learn about downstream processes such as large-scale DNA isolation, NGS library preparation, data analysis, and where to get sequence information for the library.
Demonstration of an actual screening experiment
Learn about a library screening we performed from start to finish to identify the genes involved in 6-thioguanine (6-TG) resistance. Also, see sequencing data and the description of useful basic analysis tools.
Webinar summary
Thomas Quinn summarizes the take-home messages from the webinar. Our optimized design overcomes many of the difficulties associated with library screening, such as maintaining the representation of guide RNAs and maximizing on-target editing.
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