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.
Psychiatric Risk Gene Cacna1c and Early Life Stress: Potential Gene-Environment interactions?Poster
Early life stress (ELS) is highly associated with development of psychopathology
and mood disorders in adulthood. Genetic studies have identified variation in the gene calcium voltage-gated channel subunit alpha1C (CACNA1C) to increase risk for several psychiatric disorders. This poster assessed the expression of Cacna1c following prepubertal stress.
Novel Role of the Innate Immune DNA Sensor IFI16 (Interferon Gamma Inducible Protein 16) as a Major Epigenetic Modulator During KSHV Infection and Lytic ReactivationPoster
Studies have shown that IFI16 acts as an antiviral restriction factor against a number of DNA viruses, by inhibiting viral replication or transcription through epigenetic modifications. However, till date, no specific epigenetic function of IFI16 has been identified. Here, we have discovered that IFI16 recruits two histone methyltransferases on the KSHV episome leading to altered Histone H3K9 methylation, thus regulating its lifecycle.READ MORE
A new method for generating arrayed RNAi screening tools for any organismPoster
RNA interference (RNAi) using small interfering RNAs (siRNAs) is an important technology for down-regulation of gene expression and a powerful tool to study cellular processes and pathways. Previously, large collections of siRNAs were available only for traditional experimental model systems, such as human and mouse, and predominantly provided as chemically synthesized libraries.READ MORE