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

Abstract

Gene knockout is the most powerful tool for determining gene function or permanently modifying the phenotypic characteristics of a cell. Existing methods for gene disruption are limited by their efficiency, time to completion, and/or the potential for confounding off-target effects. Here, we demonstrate a rapid single-step approach to targeted gene knockout in mammalian cells, using engineered zinc-finger nucleases (ZFNs). ZFNs can be designed to target a chosen locus with high specificity. Upon transient expression of these nucleases the target gene is first cleaved by the ZFNs and then repaired by a natural-but imperfect-DNA repair process, nonhomologous end joining. This often results in the generation of mutant (null) alleles. As proof of concept for this approach we designed ZFNs to target the dihydrofolate reductase (DHFR) gene in a Chinese hamster ovary (CHO) cell line. We observed biallelic gene disruption at frequencies >1%, thus obviating the need for selection markers. Three new genetically distinct DHFR ؊/؊ cell lines were generated. Each new line exhibited growth and functional properties consistent with the specific knockout of the DHFR gene. Importantly, target gene disruption is complete within 2-3 days of transient ZFN delivery, thus enabling the isolation of the resultant DHFR ؊/؊ cell lines within 1 month. These data demonstrate further the utility of ZFNs for rapid mammalian cell line engineering and establish a new method for gene knockout with application to reverse genetics, functional genomics, drug discovery, and therapeutic recombinant protein production.genetic engineering ͉ zinc-finger proteins T he use of gene knockouts in basic research, functional genomics, and industrial cell line engineering is severely limited by an absence of methods for rapid targeting and disruption of an investigator-specified gene. Early approaches to somatic cell gene disruption used genome-wide nontargeted methods, including ionizing radiation and chemical-induced mutagenesis (1, 2) whereas more recent methods used targeted homologous recombination (HR) (3). However, the Ͼ1,000-fold lower frequency of the targeted HR event relative to random integration in most mammalian cell lines (beyond mouse ES cells) can necessitate screening thousands of clones and take several months to identify a biallelic targeted gene knockout. Strategies including positive and negative marker selection and promoter-trap can boost efficiencies considerably, although these approaches present their own technical challenges and are not always successful in achieving high efficiency targeting (4, 5). Although advances with adeno-associated viral delivery strategies continue to improve the efficiency of knockouts (6, 7), the frequency is still very low and the time required to achieve biallelic gene knockout remains a barrier to its routine adoption. Here, we present a general solution for rapid gene knockout in mammalian cells.The repair of double strand DNA breaks (DSB) in mammalian cells occurs via the distinct mechanisms of homol...

Synthesis of programmable integrases

“…In particular, aberrant cleavage of unknown off-target chromosomal sequences can result in significant toxicity in treated cells (33). Additionally, repeated cleavage at both the target site and off-target sites stimulates error-prone DNA repair mechanisms that lead to random mutations at these sites (23,24,29). These events are unpredictable and extremely difficult to comprehensively characterize experimentally and 1 recent study found that off-target modifications occurred with a frequency of Ͼ13% (23).…”

“…Synthetic zinc finger DNA-binding proteins have been generated for many sequences, using several approaches (15)(16)(17). Capitalizing on this work, researchers have incorporated synthetic zinc finger proteins into a wide variety of molecular tools (7,(18)(19)(20)(21)(22)(23)(24)(25)(26)(27)(28)(29).…”

Subfunctionalization of Paralogous Aryl Hydrocarbon Receptors from the FrogXenopus Laevis: Distinct Target Genes and Differential Responses to Specific Agonists in a Single Cell Type

“…The complex mixture of indels and wild-type alleles we found in many strains likely resulted from a combination of both incomplete cutting and TALEN activity in daughter cells, following division of the single cell progenitor. Similarly, zinc finger nucleases can elicit both heterozygous and homozygous mutations in cultured mammalian cells (Liu et al, 2010;Santiago et al, 2008), and TALEN gene editing in mouse embryos produces heterozygous and mosaic mice (eg, Davies et al, 2013;Sung et al, 2013). To verify clonality, mutant strains were subjected to 2 rounds of clonal dilution and genotyping.…”

Genome-Editing Technologies: Principles and Applications

“…However, with the advent of cost-effective and userfriendly gene-editing technologies, custom cell lines carrying nearly any genomic modification can now be generated in simply a matter of weeks. Examples of some of the outcomes that have become routine because of the emergence of targeted nucleases include gene knockout (Santiago et al 2008;Mali et al 2013b), gene deletion (Lee et al 2010), gene inversion (Xiao et al 2013), gene correction (Urnov et al 2005;Ran et al 2013), gene addition (Moehle et al 2007;Hockemeyer et al 2011;Hou et al 2013), and even chromosomal translocation (Fig. 2) (Torres et al 2014).…”