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.
Inhibition of The Auto-inflammation Suppressor Protein ISG15 Triggers Preeclampsia by Blocking Trophoblast Migration and InvasionPoster
In summary, ISG15 expression levels are crucial for trophoblast morphology and function (migration/invasion). By blocking trophoblast invasion, reduced ISG15 levels could contribute to impaired spiral artery transformation that reduces utero-placental blood flow in preeclampsia. Thus, agents inducing ISG15 expression are likely to be therapeutic in preeclampsia.
An Emerging Phenotype of Partial RAG 1/2 Deficiency Among Young Children with Autoimmunity and Viral InfectionsPoster
We describe the natural history of a cohort of 12 patients with confirmed partial RAG1/2 mutations and autoimmunity at a young age. We were seeking the link between viral infections and autoimmunity and tested candidate biomarkers that may reflect the underlying RAG1/2 protein deficiency.READ MORE
Complete alignment identification of CRISPR-Cas9 genomic off-targets using Edit-R CRISPR specificity tool and a comprehensive analysis of positional mismatch tolerancePoster
A web tool that performs complete crRNA specificity checking is introduced. In addition, we evaluated positional off-targeting of the CRISPR system.READ MORE
Comments | 0 ADD COMMENT
World Advanced Therapies & Regenerative Medicine Congress
May 16 - May 18, 2018