PT - JOURNAL ARTICLE AU - Jonathan M. Geisinger AU - Sören Turan AU - Sophia Hernandez AU - Laura P. Spector AU - Michele P. Calos TI - <em>In Vivo</em> Blunt-End Cloning Through CRISPR/Cas9-Facilitated Non-Homologous End-Joining AID - 10.1101/019570 DP - 2015 Jan 01 TA - bioRxiv PG - 019570 4099 - http://biorxiv.org/content/early/2015/05/21/019570.short 4100 - http://biorxiv.org/content/early/2015/05/21/019570.full AB - The ability to precisely modify the genome in a site-specific manner is extremely useful. The CRISPR/Cas9 system facilitates precise modifications by generating RNA-guided double-strand breaks. We demonstrate that guide RNA pairs generate deletions that are repaired with a high level of precision by non-homologous end-joining in mammalian cells. We present a method called knock-in blunt ligation for exploiting this excision and repair to insert exogenous sequences in a homology-independent manner without loss of additional nucleotides. We successfully utilize this method in a human immortalized cell line and induced pluripotent stem cells to insert fluorescent protein cassettes into various loci, with efficiencies up to 35.8% in HEK293 cells. We also present a version of Cas9 fused to the FKBP12-L106P destabilization domain for investigating repair dynamics of Cas9-induced double-strand breaks. Our in vivo blunt-end cloning method and destabilization-domain-fused Cas9 variant increase the repertoire of precision genome engineering approaches.