SEP 30, 2020 10:30 AM PDT

Straightforward CRISPR knockout: Leveraging predesigned synthetic sgRNAs for highly functional and specific gene knockout

Presented at: CRISPR 2020
Sponsored by: Horizon Discovery
C.E. Credits: P.A.C.E. CE Florida CE
Speaker

Abstract

CRISPR-based genome editing has accelerated biological research and holds great potential for studying and treating human diseases. The CRISPR-Cas9 system requires a Cas9 nuclease and a guide RNA, which may consist of either a CRISPR RNA (crRNA) coupled with a trans-activating crRNA (tracrRNA), or a single guide RNA (sgRNA) that combines the crRNA and tracrRNA in a single molecule. Both guide RNA formats can be chemically synthesized, offering advantages over expression systems. Synthetic guide RNAs are amenable to chemical modifications for increased stability, eliminate time-consuming steps of cloning and sequencing, and do not have the inherent immune response and cytotoxicity of in vitro transcribed guide RNAs. They can be readily delivered into cells for high-throughput arrayed screening applications and expand the types of phenotypic readouts that can be leveraged. In this webinar we will present data around the development and application of Horizon Edit-R synthetic sgRNA reagents for gene specific CRISPR knockout. We will discuss the importance of rational design, chemical modifications, delivery methods, and comparison of functionality with existing reagents. The functionality of individual guide RNA’s as well as a pool of all three guide RNAs, was tested in U2OS and primary human T cells. We performed viability assays for the knockout of essential genes and immuno-fluorescence analysis to demonstrate protein knockout. We also measured the phenotypic effects of gene knockout by flow cytometry showing depletion of cell surface proteins. Both sgRNA and crRNA:tracrRNA synthetic guide RNAs efficiently knockout the targeted proteins as measured by functional assays and sequence analysis. Pooling of the synthetic guide RNAs provides robust functional gene knockout and further simplifies high-throughput loss-of-function screening in arrayed format.

Learning Objectives:

1. Understand the usefulness of CRISPR Cas9 gene editing 

2. Understand the different tools available for gene silencing, knockout and editing

3. Understand the potential research and therapeutic applications for gene edited cell lines


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