Knockout of microRNAs Using the CRISPR-Cas9 System with Paired Synthetic crRNAs
Poster Apr 24, 2018
Eldon T. Chou, John A. Schiel, Elena Maksimova, Travis Hardcastle, Emily A. Anderson, Annaleen Vermeulen, Anja van Brabant Smith
The CRISPR (clustered regularly interspaced short palindromic repeats)-Cas9 (CRISPR-associated 9) system derived from Streptococcus pyogenes uses a Cas9 nuclease directed by a guide RNA (gRNA) to create a DNA double-strand break (DSB) at the target site. The gRNAs can be dual synthetic molecules, like the native bacterial system containing a CRISPR RNA (crRNA) and a trans-activating CRISPR RNA (tracrRNA) (Figure 1), or a single synthetic guide RNA (sgRNA). The DSB is most often repaired by either nonhomologous end joining (NHEJ) or homology directed repair (HDR) through endogenous mechanisms within mammalian cells. NHEJ can result in insertions or deletions (indels) that produce functional gene knockouts through nonsense mutations or introduction of a stop codon. When using CRISPR-Cas9 components targeting coding genes, there are typically multiple protospacer adjacent motif (PAM) sequences (NGG for S. pyogenes) to choose from along the gene to design a gRNA. For most CRISPR Cas9 genome engineering experiments, one targeting gRNA is sufficient to generate the desired functional gene knockout. However, for some applications, it may be advantageous to use two gRNAs to generate a larger deletion and ensure gene knockout or to remove an exon, long non-coding RNA (lncRNA), or transcriptional regulatory element.
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
Identification of genes involved in cell cycle regulation using arrayed synthetic CRISPR RNA libraries in a multiparameter high-content assayPoster
Gene knockout using CRISPR-Cas9 has dramatically transformed biological research and has been rapidly applied to loss-of-function screening primarily using pooled lentiviral sgRNA libraries. A synthetic CRISPR RNA (crRNA) approach is amenable to screening in arrayed, well-by-well fashion and expands the types of phenotypic readouts that can be used, including high-content and morphology-based assays.READ MORE
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2nd Annual Artificial Intelligence in Drug Development Congress
Sep 20 - Sep 21, 2018