Adding to the Genome: Accuate Targeting and Effecient Insertion of Long ssDNA

The development of CRISPR-mediated genome targeting is one of the most disruptive technologies of this decade, however the usefulness of this system alone is limited to the inactivation of genetic information. Homology mediated repair allows the creation and repair of genetic information at the CRISPR break, and is most efficient when single stranded template is used.

Using novel technologies we demonstrate the creation of ssDNA kilobases in size, and the insertion of such DNA into targeted break sites with a variety of mammalian cell lines and animal models, often with as little as 50 bases of homology. The efficiency of this method is augmented by the use of a chemically modified crRNA/tracrRNA/Cas9. Data summarizing the methods of design, transfection, and screening using this system will be presented.