Homology-directed repair with Dharmacon™ Edit-R™ CRISPR-Cas9 and single-stranded DNA oligos
Poster Aug 17, 2015
John A. Schiel, Eldon T. Chou, Maren Mayer, Emily M. Anderson , and Anja van Brabant Smith | Dharmacon, now part of GE Healthcare, 2650 Crescent Drive, Suite #100, Lafayette, CO 80026, US
The CRISPR-Cas9 system derived from Streptococcus pyogenes uses the Cas9 nuclease protein that complexes with a tracrRNA and a targeting crRNA containing a 20 nucleotide guide sequence complementary to the genomic target of interest, to create double-strand DNA breaks. Once the double-strand break occurs, the mammalian cell utilizes endogenous mechanisms to repair the broken genomic DNA. In the presence of a donor sequence, the double-strand break can be repaired precisely using homology-directed repair (HDR) resulting in the desired insertion or knockin. Here we demonstrate the use of synthetic single-stranded DNA oligo donors in a novel gene editing (Dharmacon™Edit-R™) platform comprised of synthetic tracrRNA and crRNAs which program Cas9 nuclease to perform HDR, resulting in precise insertion of short DNA sequences. By carefully optimizing lipid-based transfection conditions, we can utilize this platform to create knockins with efficiencies as high as 25%. We evaluate several parameters that affect the HDR efficiency including the length of homology arms needed in the single-stranded DNA oligo donor. Our data show that HDR is able to perform insertion of 10-12 nucleotide sequences with as little as 20 nucleotide homology arms. We additionally provide experimental workflows to perform simple and effective lipid-based HDR transfections in a 96-well plate format. The methods presented within can be applied to HDR-based insertion of epitope tags such as a FLAG tag, SNPs, precise stop codons, and amino acid changes in the active site of enzymes.
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