Cas9 Driven by an Optimal Promoter Improves Gene Editing in Eukaryotic Cell Lines when Paired with Synthetic crRNA and tracrRNA
Poster Mar 10, 2015
Amanda Haupt, Emily Anderson, Žaklina Strezoska, Hidevaldo Machado, Shawn McClelland, Maren Mayer, Adam Rocker, Annaleen Vermeulen, Amanda Birmingham, Melissa Kelley, Anja Smith
Interest in genome engineering of mammalian cells has been increasing in the past few years with the development of new tools to create DNA breaks at specific locations on the cell genome. Among these tools, the CRISPR (clustered regularly interspaced short palindromic repeats)-Cas9 (CRISPR associated protein 9) system has gained significant interest due to its relative simplicity and ease of use compared to other genome engineering technologies. The CRISPR-Cas9 system requires a complex of the Cas9 protein with a trans-activating RNA (tracrRNA) and a gene-targeting CRISPR RNA (crRNA) (Figure 1), or a single guide RNA (sgRNA, a chimeric form of tracrRNA with a crRNA).
Presented here are results on the efficiency of using synthetic crRNA and tracrRNA to introduce gene editing events when co-transfected with a plasmid expressing Cas9. We explored the use of antibiotic and fluorescence-activated cell sorting (FACS) methods for enrichment of cells that have undergone gene editing, and the use of multiple promoters to increase efficiency of gene editing with Cas9 and synthetic tracrRNA and crRNAs.
When there is a need to quickly analyze samples using a number of different PCR assays, it is likely that optimal conditions for each assay will not be the same. First, different assays often will require different annealing temperatures for their primers. In addition, amplicons may be designed to be of different lengths and therefore require varying durations of the extension step.READ MORE