A Synthetic CRISPR-Cas9 System for Homology-directed Repair
Poster May 23, 2016
John A. Schiel, Maren M. Gross, Emily M. Anderson*, Eldon T. Chou, Anja van Brabant Smith Dharmacon, part of GE Healthcare, 2650 Crescent Drive, Lafayette, CO 80026, USA
CRISPR-Cas9 is increasingly becoming the most widely used genome engineering tool due to its ease of use and ability to cause double strand breaks (DSBs) at almost any locus of interest. The cellular homology-directed repair (HDR) pathway can be used to introduce exogenous genetic content, although this application of CRISPR-Cas9 is not as straightforward as exploiting the endogenous non-homologous end joining (NHEJ) pathway to create gene knockouts. We use a synthetic dual-RNA approach based on the natural bacterial CRISPR-Cas9 system for genome editing in mammalian cells that allows for rapid analysis of multiple chemically synthesized guide RNAs. Here we demonstrate the utility of this system to HDR genomic engineering applications and provide guidelines for improving CRISPR Cas9-assisted HDR. We present examples and design recommendations for the use of short, single-stranded DNAs as donor templates for small insertions. We show that homology arm length of the single-stranded donor DNA affects the efficiency of HDR. We also demonstrate the use of plasmid DNA donor templates for large insertions, using fluorescent protein fusions as a model. Lastly, we outline techniques and methods for characterization of HDR-generated cell lines for precise genomic engineering.
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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
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The nuclear receptors pregnane X receptor (PXR) and constitutive androstane receptor (CAR) are closely related transcription factors that regulate the expression of phase I (cytochrome P450s), phase II metabolizing enzymes and transporter genes in response to xenobiotics, including prescription drugs.READ MORE
Psychiatric Risk Gene Cacna1c and Early Life Stress: Potential Gene-Environment interactions?Poster
Early life stress (ELS) is highly associated with development of psychopathology
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