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