DNA-free CRISPR-Cas9 Genome Engineering in Zebrafish
Poster Mar 10, 2016
Amanda Haas, Alex J. Blasky*, Rytis Prekeris*, John A. Schiel, Melissa L. Kelley, and Anja van Brabant Smith | Dharmacon, now part of GE Healthcare, 2650 Crescent Drive, Suite 100, Lafayette, CO 80026, USA *University of Colorado - Anschutz Medical Campus, Department of Cell & Developmental Biology, Denver, CO, USA
The CRISPR-Cas9 system permits researchers to quickly edit genes for functional protein knockout in mammalian, fish and plant genomes, among others, and consequently has dramatically transformed biological research. The CRISPR-Cas9 system requires exogenous Cas9 nuclease to be delivered into the cell, which can be accomplished through transfection of an expression plasmid, mRNA or protein, or through transduction with lentiviral particles. DNA-based Cas9 constructs, while appropriate for many applications, may result in unwanted integration events. Lentiviral delivery results in integration of the Cas9 expression cassette into the cell’s genome, and transfection of a Cas9 plasmid may result in the insertion of vector sequence at the site of the double-stranded break when the genomic DNA is repaired through the NHEJ pathway. The use of Cas9 mRNA or protein avoids any unwanted integration, and in combination with using synthetic crRNA and tracrRNA, results in a completely DNA-free gene editing system. Here we demonstrate successful gene editing using DNA-free CRISPR-Cas9 reagents for gene knockout in zebrafish. Zebrafish embryos from a stable transgenic line were injected with Cas9 nuclease mRNA, synthetic tracrRNA, and crRNA designed to target GFP. A mismatch detection assay confirmed efficient gene editing, and successful functional protein knockout was confirmed by loss of GFP fluorescence.
Despite the developments in conventional PCR, the complexity of multiplex Real Time PCR is still limited due to the lack of sufficient detection channels. To achieve high-end multiplexing capacity on standard Real Time PCR machines, Anapa Biotech has developed the MeltPlex® technology (see box on right).READ MORE
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.