Details

Autor(en) / Beteiligte

• Wang, Jianbin
• Friedman, Geoffrey
• Doyon, Yannick
• Wang, Nathaniel S
• Li, Carrie Jiaxin
• Miller, Jeffrey C
• Hua, Kevin L
• Yan, Jenny Jiacheng
• Babiarz, Joshua E
• Gregory, Philip D
• Holmes, Michael C

Titel

Targeted Gene Addition to a Predetermined Site in the Human Genome Using a ZFN-Based Nicking Enzyme

Ist Teil von

• Molecular therapy, 2012-09, Vol.20 (9), p.2-2

Erscheinungsjahr

2012

Quelle

Alma/SFX Local Collection

Beschreibungen/Notizen

• 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 non-homologous 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 reveal 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 a 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.