CRISPR-Cas9 genome editing utilizing chemically synthesized RNA
Poster Oct 06, 2016
Michael Delaney, Kaizhang He, Eldon Chou, Amanda Haas, Žaklina Strezoska, Melissa L. Kelley, and Anja van Brabant Smith Dharmacon, part of GE Healthcare, 2650 Crescent Drive, Lafayette, CO 80026, USA
The CRISPR-Cas9 system allows researchers to quickly edit genes for functional gene knockout in mammalian, fish, and plant genomes, among others. Consequently, this has dramatically transformed biological research. Cas9 nuclease and a guide RNA are required for CRISPR-Cas9 genome engineering; however, these components can be utilized in different reagent formats, depending upon the application. Vector-based guide RNA reagents utilize an expressed chimeric single guide RNA (sgRNA), while synthetic reagents can be either sgRNA or a two-RNA system of CRISPR RNA (crRNA) and tracrRNA as the guide RNA component. Chemical RNA synthesis has been applied for the rapid generation of either crRNA and tracrRNA or sgRNA. This allows for direct delivery into cells for unique gene editing applications such as high throughput arrayed screening or experiments that benefit from DNA-free components.
Genome-wide association studies (GWAS) have identified more than 100 genetic loci associated with type 2 diabetes. The majority of these are located in the intergenic or intragenic regions suggesting that the implicated variants may alter chromatin conformation. This, in turn, is likely to influence the expression of nearby or more remotely located genes to alter beta cell function. At present, however, detailed molecular and functional analyses are still lacking for most of these variants. We recently analysed one of these loci and mapped five causal variants in an islet-specific enhancer cluster within the STARD10 gene locus. Here, we aimed to understand how these causal variants influence b-cell function by alteration of the chromatin structure of enhancer clusterREAD MORE
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.
We utilized paired synthetic crRNAs coupled with our synthetic tracrRNA in cells transduced with lentiviral Cas9 to perform a functional knockout on hsa-miR-221. This three-part system (crRNA, tracrRNA and Cas9) has demonstrated efficient gene editing when used with only one guide RNA, but the goal was to use two crRNAs to remove the entire stem-loop.READ MORE