TY  - JOUR
A1  - Wang, Jianbin
A1  - Friedman, Geoffrey
A1  - Doyon, Yannick
A1  - Wang, Nathaniel S.
A1  - Li, Carrie Jiaxin
A1  - Miller, Jeffrey C.
A1  - Hua, Kevin L.
A1  - Yan, Jenny Jiacheng
A1  - Babiarz, Joshua E.
A1  - Gregory, Philip D.
A1  - Holmes, Michael C.
T1  - Targeted gene addition to a predetermined site in the human genome using a ZFN-based nicking enzyme
Y1  - 2012/03/20 
JF  - Genome Research 
JO  - Genome Research 
DO  - 10.1101/gr.122879.111 
UR  - http://genome.cshlp.org/content/early/2012/04/10/gr.122879.111.abstract 
N2  - Zinc-finger nucleases (ZFNs) drive highly efficient genome editing by generating a site-specific DNA double-strand break (DSB) at a predetermined site in the genome. Subsequent repair of this break via the nonhomologous end-joining (NHEJ) or homology-directed repair (HDR) pathways results in targeted gene disruption or gene addition, respectively. Here, we report that ZFNs can be engineered to induce a site-specific DNA single-strand break (SSB) or nick. Using the CCR5-specific ZFNs as a model system, we show that introduction of a nick at this target site stimulates gene addition using a homologous donor template but fails to induce significant levels of the small insertions and deletions (indels) characteristic of repair via NHEJ. Gene addition by these CCR5-targeted zinc finger nickases (ZFNickases) occurs in both transformed and primary human cells at efficiencies of up to ∼1%–8%. Interestingly, ZFNickases targeting the AAVS1 “safe harbor” locus revealed similar in vitro nicking activity, a marked reduction of indels characteristic of NHEJ, but stimulated far lower levels of gene addition—suggesting that other, yet to be identified mediators of nick-induced gene targeting exist. Introduction of site-specific nicks at distinct endogenous loci provide an important tool for the study of DNA repair. Moreover, the potential for a SSB to direct repair pathway choice (i.e., HDR but not NHEJ) may prove advantageous for certain therapeutic applications such as the targeted correction of human disease-causing mutations. 
ER  -