Targeted gene addition to a predetermined site in the human genome using a ZFN-based nicking enzyme

Jianbin Wang, Geoffrey Friedman, Yannick Doyon, Nathaniel S. Wang, Carrie Jiaxin Li, Jeffrey C. Miller, Kevin L. Hua, Jenny Jiacheng Yan, Joshua E. Babiarz, Philip D. Gregory, and Michael C. Holmes
Source: Genome Res
Publication Date: (2012)
Issue: 22 (7): 1316-26
Cells used in publication:
Species: human
Tissue Origin: blood
Nucleofector® I/II/2b
4D-Nucleofector® 96-well Systems
1–2 X 106 K562 cells were nucleofected with ZFN and donor plasmids using either the Nucleofector or Amaxa 96-well shuttle system. 1.25–2.5 ug of each ZFN expression plasmid and 12.5–25.0 ug of the donor constructs were used. For nucleofection of 1–2 X 105 cells with the Amaxa 96-well shuttle system, 0.4 mg of each ZFN expression plasmid and 0.8 mg of the donor constructs were used.
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.