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Experimental design considerations for homology-directed repair  utilizing a CRISPR-Cas9 system with synthetic crRNA and tracrRNA
EP24568
Experimental design considerations for homology-directed repair utilizing a CRISPR-Cas9 system with synthetic crRNA and tracrRNA
Submitted on 06 Oct 2016

Hidevaldo B. Machado, John A. Schiel, Maren Mayer-Gross, Eldon T. Chou, Megan Basila, Melissa L. Kelley, Anja van Brabant Smith Dharmacon, part of GE Healthcare, 2650 Crescent Drive, Lafayette, CO 80026, USA
GE Healthcare Dharmacon, Inc.
This poster was presented at Discovery On Target 2016
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Poster Abstract
Since its application in mammalian cells, CRISPR-Cas9 has rapidly become a common technology for gene editing experiments. The most common use of the CRISPR-Cas9 technology has been to cause gene knockouts that are generated as a result of imperfect repair of a targeted double-strand DNA break by the non-homologous end joining (NHEJ) pathway. Precise editing can also be obtained with the CRISPR-Cas9 system by providing a donor template to be incorporated into the cell’s genome by homology-directed repair (HDR). The donor template can be provided in the form of a single-stranded DNA oligonucleotide for short inserts or double-stranded DNA in the form of a plasmid or linear DNA fragment for larger inserts. Efficiency of gene knockin is much lower compared to gene knockout since NHEJ is active throughout the cell cycle, especially G1 phase, while the HDR pathway is only active during the S and G2 phases. Therefore, it is imperative to optimize the efficiency of gene editing experiment to increase the likelihood of successful knockin. We evaluated the efficiency of single-stranded oligonucleotide and double-stranded DNA plasmid donors for gene knockin. We will demonstrate the characterization of clonal cell lines and additional design and analysis considerations for precise genome engineering with HDR and the CRISPR-Cas9 systemReport abuse »
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