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Homology-directed repair using synthetic crRNA and tracrRNA with single-stranded DNA oligos
Poster Title: Homology-directed repair using synthetic crRNA and tracrRNA with single-stranded DNA oligos
Submitted on 17 Dec 2015
Author(s): John A. Schiel, Eldon T. Chou, Maren Mayer, Emily M. Anderson, James Goldmeyer, and Anja van Brabant Smith | Dharmacon, part of GE Healthcare, 2650 Crescent Drive, Suite #100, Lafayette, CO 80026, US
Affiliations: Dharmacon (part of GE Healthcare)
This poster was presented at ASCB
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Poster Information
Abstract: Here we demonstrate the use of synthetic single-stranded DNA (ssDNA) oligo donors in a novel gene editing platform comprised of synthetic crRNA and tracrRNA that program Cas9 nuclease to perform HDR, resulting in precise insertion of short DNA sequences. By carefully optimizing lipid-based transfection conditions, including ssDNA oligo concentration, we can utilize this platform to create knockins. We evaluated several parameters that affect the HDR efficiency including the length of homology arms needed in the ssDNA oligo donor. We tested homology arm lengths of 10, 20, 30, 40, 50, 60, and 70 nucleotides and found that HDR is able to perform insertion of 10-12 nucleotide sequences with homology arms as short as 20 nucleotides. Interestingly, we also observed that longer ssDNA homology arms become less efficient.Summary: We demonstrate the use of ssDNA oligo donors with synthetic crRNA:tracrRNA and Cas9 nuclease to perform HDR and precisely insert short DNA sequences.References: Report abuse »
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