Targeted gene knockout in mammalian cells using engineered zinc finger nucleases Yolanda Santiago Edmond Chan Pei Qi Liu Salvatore Orlando Lin Zhang Fyodor D Urnov Michael C Holmes Dmitry Guschin Adam Waite Jeffrey C Miller Edward J Rebar Philip D Gregory Aaron Klug and Trevor N Collingwood Sangamo BioSciences Inc 501 Canal Boulevard Suite A100 Richmond CA 94804 Pfizer Inc Bioprocess Research and Development Cell Line Development 700 Chesterfield Parkway West Chesterfield MO 63017 and Medical Research Council Laboratory of Molecular Biology Hills Road Cambridge CB2 2QH United Kingdom 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 1 000 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 homology directed repair HDR or nonhomologous end joining NHEJ 8 www pnas org cgi doi 10 1073 pnas 0800940105 Although HDR typically uses the sister chromatid of the damaged DNA as a template from which to perform perfect repair of the genetic lesion NHEJ is an imperfect repair process that often results in changes to the DNA sequence at the site of the DSB During NHEJ the cleaved DNA is further resected by exonuclease activity and more bases may be added in an irregular fashion before the two ends of the severed DNA are rejoined 9 In mammalian systems such as Chinese hamster ovary CHO cells the ratio of HDR to NHEJ based repair has been found to be 9 13 10 Studies in Drosophila 11 and later in both plants and worms 12 13 showed that DSBs generated by site specific zinc finger nucleases ZFNs resulted in targeted mutagenesis consistent with repair by NHEJ In this article we now extend the ZFN approach to mammalian systems We make use of the process of NHEJ to carry out targeted gene knockout in CHO cells by using transiently expressed site specific ZFNs to generate the DSB in the gene that is being targeted for reviews see refs 14 and 15 ZFNs employ a heterologous zinc finger protein ZFP DNA binding domain which specifically binds to the designated target sequence fused to the catalytic domain of the endonuclease FokI 16 Dimerization of this FokI domain is required for its DNA binding dependent endonuclease activity Thus two individual ZFNs are designed as a pair to bind to the target DNA stretch with precise sequence specificity spacing and orientation to facilitate dimerization and subsequent DNA cleavage 11 16 When expressed transiently in cells the ZFNs generate a site specific DSB in the endogenous target gene that subsequently can be repaired via NHEJ The precise nature of the mutations generated by NHEJ based repair of the ZFN induced DSB cannot be predetermined and indeed need not be known In this article we show that the frequency of gene disrupting mutations generated by this stochastic process is more than sufficient for utility as a method for gene knockout To demonstrate the ZFN approach to gene knockout we elected to disrupt the function of the dihydrofolate reductase DHFR gene in a Chinese hamster ovary cell line CHO S that is diploid for functional DHFR CHO cells are the dominant system for the production of therapeutic recombinant proteins 17 DHFR is one of the most widely used and best characterAuthor contributions P Q L F D U M C H D G J C M E J R P D G and T N C designed research Y S E C P Q L S O A W and J C M performed research P Q L L Z E J R P D G and T N C analyzed data and P D G A K and T N C wrote the paper Conflict of interest statement Y S E C P Q L S O F D U M C H D G J C M E J R P D G and T N C are full time employees of Sangamo BioSciences Inc A K is a member of the scientific advisory board for Sangamo BioSciences Inc L Z is a full
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