The CRISPR-Cas9 system is being widely used for genome engineering in many different biological applications. It was originally adapted from the bacterial Type II CRISPR system and uses a Cas9 endonuclease guided by RNA to introduce double-strand DNA breaks at specific locations in the genome. The Dharmacon Edit-R CRISPR-Cas9 Gene Engineering platform includes the three components required for gene editing in mammalian cells: (1) a plasmid expressing a Cas9 nuclease, (2) a chemically synthesized tracrRNA, and (3) a synthetic crRNA designed to the target site of interest. In this presentation, the complete workflow of the Edit-R platform is demonstrated to knock out the PPIB gene at the protein level in HEK293T cells. Data from the analysis of 42 edited clones are presented.
Attendees will learn:
- CRISPR-Cas9 background and crRNA design considerations
- Transfection optimization for maximal editing efficiency
- Enrichment of cell populations with gene editing events by FACS
- Clonal selection
- Detailed analysis of editing events
Emily Anderson, Ph.D.
Senior Scientist in Dharmacon Research and Development
Dr. Anderson received a PhD in biochemistry from the University of Colorado at Boulder, where she elucidated the structure and function of protein: single-strand DNA interactions at telomeres. Since joining Dharmacon in 2003, Anderson has worked extensively on the mechanism, function, specificity, and delivery of RNA interference (RNAi) and its applications. She manages projects involving the development and application of gene engineering and other gene-modulation technologies. Anderson is an inventor on several issued patents, played a role in the introduction of several product lines, and has authored peer-reviewed publications in the RNAi field.