Increasing Gene Editing Efficiencies in Eukaryotic Cell Lines by Selection of Appropriate CRISPR-Cas9 Reagents
Poster Jun 12, 2015
Melissa L. Kelley, Žaklina Strezoska, Elena Maksimova, Hidevaldo Machado, Emily M. Anderson, Maren Mayer, Annaleen Vermeulen, Shawn McClelland, Anja van Brabant Smith
Genetic engineering of living cells is critical for understanding gene function in normal and diseased states. The CRISPR-Cas9 system is widely utilized because of its ease-of-use compared to other gene editing methods. This system requires a complex of Cas9 protein with tracrRNA and a gene-targeting crRNA to introduce double-strand DNA breaks at specific locations in the genome to disrupt protein translation and knockout gene function. To achieve high gene editing efficiencies, it is essential to choose the best CRISPR-Cas9 reagents for delivery and expression in the cells of interest. Depending on the transfectablity of specific cells, CRISPR components can be delivered using plasmid transfection or lentiviral transduction. Plasmid-expressed Cas9 can be co-transfected with synthetic crRNA and tracrRNA for efficient gene editing in cells amenable to lipid delivery. Cas9 that is packaged into lentiviral particles can be transduced into cells that are refractory to transfection. Lentiviral Cas9 can also be used to generate stable cell lines, which can then be transfected or transduced by synthetic or lentiviral-based CRISPR RNA components; this is particularly useful for screening applications. Importantly, expressing humanized Cas9 from different promoters (e.g., human and mouse CMV and EF1α, PGK, CAG) in different cell types results in varying levels of Cas9 expression and consequently, varying efficiencies of gene editing. In addition, cells transfected or transduced with gene editing reagents can be enriched by antibiotic selection or FACS using reagents in which the Cas9 gene is co-expressed with either an antibiotic resistance marker or a fluorescent protein reporter. This enrichment facilitates the isolation of clonal cells containing the desired mutation. Presented here are data demonstrating improvement of gene editing efficiencies in cells of interest by using the most effective delivery and selection approaches with the optimal CRISPR-Cas9 reagents.
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