« Back
A Synthetic CRISPR-Cas9 System for Homology-directed Repair
Poster Title: A Synthetic CRISPR-Cas9 System for Homology-directed Repair
Submitted on 23 May 2016
Author(s): John A. Schiel, Maren M. Gross, Emily M. Anderson*, Eldon T. Chou, Anja van Brabant Smith Dharmacon, part of GE Healthcare, 2650 Crescent Drive, Lafayette, CO 80026, USA
Affiliations: Dharmacon (part of GE Healthcare)
This poster was presented at CRISPR 2016 - Israel
Poster Views: 2,673
View poster »

Poster Information
Abstract: CRISPR-Cas9 is increasingly becoming the most widely used genome engineering tool due to its ease of use and ability to cause double strand breaks (DSBs) at almost any locus of interest. The cellular homology-directed repair (HDR) pathway can be used to introduce exogenous genetic content, although this application of CRISPR-Cas9 is not as straightforward as exploiting the endogenous non-homologous end joining (NHEJ) pathway to create gene knockouts. We use a synthetic dual-RNA approach based on the natural bacterial CRISPR-Cas9 system for genome editing in mammalian cells that allows for rapid analysis of multiple chemically synthesized guide RNAs. Here we demonstrate the utility of this system to HDR genomic engineering applications and provide guidelines for improving CRISPR Cas9-assisted HDR. We present examples and design recommendations for the use of short, single-stranded DNAs as donor templates for small insertions. We show that homology arm length of the single-stranded donor DNA affects the efficiency of HDR. We also demonstrate the use of plasmid DNA donor templates for large insertions, using fluorescent protein fusions as a model. Lastly, we outline techniques and methods for characterization of HDR-generated cell lines for precise genomic engineering.Summary: Synthetic, dual-RNA-encoded Cas9 is used for precise homology-directed repair (HDR) gene engineering. Both short and long (GFP) inserts are covered.Report abuse »
Ask the author a question about this poster.
Ask a Question »

Creative Commons

Related Posters

Single-cell studies: Focus on reverse transcription
Zucha Daniel, Androvic Peter, Kubista Mikael, Valihrach Lukas

Osteolytic jaw lesions
Dr. Amal Hassan Zakarya, Sarah Ahmed Mohamed, Noura Mohamed Kamal, Heba Ahmed Saleh, Ali Abd Elwahed

Oral Soft Tissue Lumps and Bumps
Dr. Amal Hassan Zakarya, Sarah Ahmed Mohamed, Noura Mohamed Kamal, Heba Ahmed Saleh

Potential therapeutic effects of induced pluripotent stem cells on induced salivary gland cancer in experimental rats
Yasmine Alaa El-Din1, Dina Sabry2, Amal Hassan Abdelrahman3, and Safa Fathy3

Correlation between tissue expression of miRNA137 and CD8 in oral lichen planus
Sanaa aghbari, olfat shaker, shaimaa Omar, and el Rahman, atria attia