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.
Inhibition of The Auto-inflammation Suppressor Protein ISG15 Triggers Preeclampsia by Blocking Trophoblast Migration and InvasionPoster
In summary, ISG15 expression levels are crucial for trophoblast morphology and function (migration/invasion). By blocking trophoblast invasion, reduced ISG15 levels could contribute to impaired spiral artery transformation that reduces utero-placental blood flow in preeclampsia. Thus, agents inducing ISG15 expression are likely to be therapeutic in preeclampsia.
An Emerging Phenotype of Partial RAG 1/2 Deficiency Among Young Children with Autoimmunity and Viral InfectionsPoster
We describe the natural history of a cohort of 12 patients with confirmed partial RAG1/2 mutations and autoimmunity at a young age. We were seeking the link between viral infections and autoimmunity and tested candidate biomarkers that may reflect the underlying RAG1/2 protein deficiency.READ MORE
Complete alignment identification of CRISPR-Cas9 genomic off-targets using Edit-R CRISPR specificity tool and a comprehensive analysis of positional mismatch tolerancePoster
A web tool that performs complete crRNA specificity checking is introduced. In addition, we evaluated positional off-targeting of the CRISPR system.READ MORE