@article {Geisinger019570, author = {Jonathan M. Geisinger and S{\"o}ren Turan and Sophia Hernandez and Laura P. Spector and Michele P. Calos}, title = {In Vivo Blunt-End Cloning Through CRISPR/Cas9-Facilitated Non-Homologous End-Joining}, elocation-id = {019570}, year = {2015}, doi = {10.1101/019570}, publisher = {Cold Spring Harbor Laboratory}, abstract = {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.}, URL = {https://www.biorxiv.org/content/early/2015/05/27/019570}, eprint = {https://www.biorxiv.org/content/early/2015/05/27/019570.full.pdf}, journal = {bioRxiv} }