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Berkeley MCELLBI 140 - Targeted gene knockout in mammalian cells

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Targeted gene knockout in mammalian cells usingengineered zinc-finger nucleasesYolanda 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 LineDevelopment, 700 Chesterfield Parkway West, Chesterfield, MO 63017; and§Medical Research Council Laboratory of Molecular Biology, Hills Road,Cambridge CB2 2QH, United KingdomContributed by Aaron Klug, January 30, 2008 (sent for review November 14, 2007)Gene knockout is the most powerful tool for determining genefunction or permanently modifying the phenotypic characteristicsof a cell. Existing methods for gene disruption are limited by theirefficiency, time to completion, and/or the potential for confound-ing off-target effects. Here, we demonstrate a rapid single-stepapproach to targeted gene knockout in mammalian cells, usingengineered zinc-finger nucleases (ZFNs). ZFNs can be designed totarget a chosen locus with high specificity. Upon transient expres-sion of these nucleases the target gene is first cleaved by the ZFNsand then repaired by a natural— but imperfect—DNA repair pro-cess, nonhomologous end joining. This often results in the gener-ation of mutant (null) alleles. As proof of concept for this approachwe designed ZFNs to target the dihydrofolate reductase (DHFR)gene in a Chinese hamster ovary (CHO) cell line. We observedbiallelic gene disruption at frequencies >1%, thus obviating theneed for selection markers. Three new genetically distinct DHFRⴚ/ⴚcell lines were generated. Each new line exhibited growth andfunctional properties consistent with the specific knockout of theDHFR gene. Importantly, target gene disruption is complete within2–3 days of transient ZFN delivery, thus enabling the isolation ofthe resultant DHFRⴚ/ⴚcell lines within 1 month. These datademonstrate further the utility of ZFNs for rapid mammalian cellline engineering and establish a new method for gene knockoutwith application to reverse genetics, functional genomics, drugdiscovery, and therapeutic recombinant protein production.genetic engineering 兩 zinc-finger proteinsThe use of gene k nockouts in basic research, functionalgenomics, and industrial cell line engineering is severelylimited by an absence of methods for rapid targeting anddisr uption of an investigator-specified gene. Early approaches tosomatic cell gene disruption used genome-wide nontargetedmethods, including ionizing radiation and chemical-inducedmut agenesis (1, 2) whereas more recent methods used targetedhomologous rec ombination (HR) (3). However, the ⬎1,000-foldlower frequency of the targeted HR event relative to randomintegration in most mammalian cell lines (beyond mouse EScells) can necessitate screening thousands of clones and takeseveral months to identify a biallelic targeted gene k nockout.Strategies including positive and negative marker selection andpromoter-trap can boost efficiencies considerably, althoughthese approaches present their own technical challenges and arenot always successful in achieving high efficiency targeting (4, 5).A lthough advances with adeno-associated viral delivery strate-gies continue to improve the efficiency of knockouts (6, 7), thef requency is still very low and the time required to achievebiallelic gene knockout remains a barrier to its routine adoption.Here, we present a general solution for rapid gene knockout inmammalian cells.The repair of double strand DNA breaks (DSB) in mammaliancells occurs via the distinct mechanisms of homology directedrepair (HDR) or nonhomologous end joining (NHEJ) (8).A lthough HDR typically uses the sister chromatid of the dam-aged DNA as a template from which to perform perfect repairof the genetic lesion, NHEJ is an imperfect repair process thatof ten results in changes to the DNA sequence at the site of theDSB. During NHEJ, the cleaved DNA is further resected byexonuclease activity, and more bases may be added in anirregular fashion before the t wo ends of the severed DNA arerejoined (9). In mammalian systems, such as Chinese hamsterovary (CHO) cells, the ratio of HDR to NHEJ-based repair hasbeen found to be 9:13 (10). Studies in Drosophila (11) and laterin both plants and worms (12, 13) showed that DSBs generatedby site-specific zinc-finger nucleases (ZFNs) resulted in t argetedmut agenesis consistent with repair by NHEJ. In this article, wenow extend the ZFN approach to mammalian systems. We makeuse of the process of NHEJ to carry out targeted gene knockoutin CHO cells by using transiently expressed site-specific ZFNs togenerate the DSB in the gene that is being targeted (for reviews,see refs. 14 and 15).ZFNs employ a heterologous zinc-finger protein (ZFP) DNAbinding domain (which specifically binds to the designated targetsequence) fused to the cataly tic domain of the endonucleaseFokI (16). Dimerization of this FokI domain is required for itsDNA binding-dependent endonuclease activity. Thus, two indi-vidual ZFNs are designed as a pair to bind to the target DNAstretch with precise sequence specificity, spacing, and orienta-tion to facilitate dimerization and subsequent DNA cleavage (11,16). When expressed transiently in cells, the ZFNs generate asite-specific DSB in the endogenous target gene that subse-quently can be repaired via NHEJ. The precise nature of themut ations generated by NHEJ-based repair of the ZFN-inducedDSB cannot be predetermined and, indeed, need not be known.In this article, we show that the frequenc y of gene disr uptingmut ations generated by this stochastic process is more thansuf ficient for utility as a method for gene knockout.To demonstrate the ZFN approach to gene k nockout, weelected to disrupt the function of the dihydrofolate reductase(DHFR) gene in a Chinese hamster ovary cell line (CHO-S) thatis diploid for functional DHFR. CHO cells are the dominantsystem for the production of therapeutic recombinant proteins(17). DHFR is one of the most widely used and best character-Author 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. designedresearch; 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


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Berkeley MCELLBI 140 - Targeted gene knockout in mammalian cells

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