LAUSR

Here we describe an expansion of the DNA size limitations associated with CRISPR knock-in technology, more specifically, the physical extent to which mouse genomic DNA can be replaced with donor (in this case, human) DNA at an orthologous locus. Driving our efforts was the desire to create a whole animal model inserting a 25-kbp segment of a human tumor-suppressor gene in place of 17 kbp of its corresponding mouse orthologue. Included within the human fragment is a conditionally removable (by Cre-loxP) segment of the gene9s second intron, a cryptic exon immediately 39 of this, and a native exon some 20 kbp downstream. Using two methods, we first carried out the replacement by employing a combination of bacterial artificial chromosome recombineering, classic ES cell targeting, dual selection, and recombinase-driven cassette removal (traditional approach). Using a unique second method, we employed the same vector (devoid of its selectable marker cassettes), microinjecting it along with CRISPR RNA guides and Cas9 into mouse zygotes (CRISPR approach). In both instances, we were able to achieve humanization of the tumor suppressor to the extent designed, remove all selection cassettes, and demonstrate the functionality of the conditionally removable, loxP-flanked, intronic segment. Citation Format: Tiffany Leidy-Davis, Kai Cheng, Leslie Goodwin, Judith Morgan, Wen Chun Juan, Xavier Roca, Sin-Tiong Ong, David E. Bergstrom. Knock-in of a human tumor suppressor (25-kilobase pairs) by traditional and CRISPR/Cas9-stimulated homologous recombination [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5097.