Targeted gene addition into a specified location in the human genome using designed zinc finger nucleases

Abstract

Efficient incorporation of novel DNA sequences into a specific site in the genome of living human cells remains a challenge despite its potential utility to genetic medicine, biotechnology, and basic research. We find that a precisely placed double-strand break induced by engineered zinc finger nucleases (ZFNs) can stimulate integration of long DNA stretches into a predetermined genomic location, resulting in high-efficiency site-specific gene addition. Using an extrachromosomal DNA donor carrying a 12-bp tag, a 900-bp ORF, or a 1.5-kb promoter-transcription unit flanked by locus-specific homology arms, we find targeted integration frequencies of 15%, 6%, and 5%, respectively, within 72 h of treatment, and with no selection for the desired event. Importantly, we find that the integration event occurs in a homology-directed manner and leads to the accurate reconstruction of the donor-specified genotype at the endogenous chromosomal locus, and hence presumably results from synthesis-dependent strand annealing repair of the break using the donor DNA as a template. This site-specific gene addition occurs with no measurable increase in the rate of random integration. Remarkably, we also find that ZFNs can drive the addition of an 8-kb sequence carrying three distinct promoter-transcription units into an endogenous locus at a frequency of 6%, also in the absence of any selection. These data reveal the surprising versatility of the specialized polymerase machinery involved in double-strand break repair, illuminate a powerful approach to mammalian cell engineering, and open the possibility of ZFN-driven gene addition therapy for human genetic disease.

Efficient genome engineering by targeted homologous recombination in mouse embryos using transcription activator-like effector nucleases

“…Factors that could influence integration efficiencies after injecting a targeting plasmid together with TALEN mRNA directly into oocytes may include the targeting strategy itself (for example, the choice of the targeted exon), the size of the targeted gene, the size and complexity of the targeting plasmid, the length of the homology arms, the efficiency of TALENs to induce DNA double-strand breaks and the position of the TALEN-induced DNA double-strand break relative to the homology arms 35 . Future work should be directed towards comparative studies using different genome engineering technologies to understand whether certain loci might be more accessible for one but not another genome-editing technology.…”

Targeted gene knockout in mammalian cells by using engineered zinc-finger nucleases

“…In previous reports, we have demonstrated that ZFNs are powerful tools for gene modification by harnessing homologous recombination from an extrachromosomal donor (16,25). Here, we have omitted the donor DNA, relying instead simply on NHEJderived modifications, which are effectively random, leading to a mutated gene with knocked out function.…”

Nonhomologous End Joining-Mediated Gene Replacement in Plant Cells

“…ZFNs are artificial hybrid restriction enzymes that are composed of a fusion between a designed zinc-finger protein DNA-binding domain and the cleavage domain of the FokI endonuclease (for review, see Porteus, 2009;Urnov et al, 2010;Weinthal et al, 2010;Tzfira et al, 2012). ZFNs have been designed to target a wide variety of native and artificial sequences in human, animal, and plant cells (Kumar et al, 2006;Porteus, 2006;Lombardo et al, 2007;Moehle et al, 2007;Doyon et al, 2008;Cai et al, 2009;Shukla et al, 2009;Liu et al, 2010;Zhang et al, 2010;de Pater et al, 2013). In many cases, ZFN-mediated DSBs have been used to stimulate the HR DNA-repair machinery for HR-mediated gene replacement and gene addition.…”