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.
When there is a need to quickly analyze samples using a number of different PCR assays, it is likely that optimal conditions for each assay will not be the same. First, different assays often will require different annealing temperatures for their primers. In addition, amplicons may be designed to be of different lengths and therefore require varying durations of the extension step.READ MORE