Structure-based redesign of the dimerization interface reduces the toxicity of zinc-finger nucleases

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228

We report here an efficient method for targeted mutagenesis of Arabidopsis genes through regulated expression of zinc finger nucleases (ZFNs)-enzymes engineered to create DNA doublestrand breaks at specific target loci. ZFNs recognizing the Arabidopsis ADH1 and TT4 genes were made by Oligomerized Pool ENgineering (OPEN)-a publicly available, selection-based platform that yields high quality zinc finger arrays. The ADH1 and TT4 ZFNs were placed under control of an estrogen-inducible promoter and introduced into Arabidopsis plants by floral-dip transformation. Primary transgenic Arabidopsis seedlings induced to express the ADH1 or TT4 ZFNs exhibited somatic mutation frequencies of 7% or 16%, respectively. The induced mutations were typically insertions or deletions (1-142 bp) that were localized at the ZFN cleavage site and likely derived from imprecise repair of chromosome breaks by nonhomologous end-joining. Mutations were transmitted to the next generation for 69% of primary transgenics expressing the ADH1 ZFNs and 33% of transgenics expressing the TT4 ZFNs. Furthermore, ≈20% of the mutant-producing plants were homozygous for mutations at ADH1 or TT4, indicating that both alleles were disrupted. ADH1 and TT4 were chosen as targets for this study because of their selectable or screenable phenotypes (adh1, allyl alcohol resistance; tt4, lack of anthocyanins in the seed coat). However, the high frequency of observed ZFNinduced mutagenesis suggests that targeted mutations can readily be recovered by simply screening progeny of primary transgenic plants by PCR and DNA sequencing. Taken together, our results suggest that it should now be possible to obtain mutations in any Arabidopsis target gene regardless of its mutant phenotype.nonhomologous end-joining | gene knockout | alcohol dehydrogenase | chalcone synthase

Section: Discussionmentioning

confidence: 99%

High frequency targeted mutagenesis in Arabidopsis thaliana using zinc finger nucleases

Zhang

Maeder

Unger-Wallace

et al. 2010

341

228

“…20. These FokI dimerization variants provide an additional level of specificity to ZFN cleavage by effectively eliminating unwanted homodimerization events elsewhere in the genome (20,21). The ZFN9461/ZFN9684 pair used the ELKK FokI variants in all cases.…”

Section: Design Of Zinc Finger Nucleasesmentioning

confidence: 99%

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

Santiago

Chan²,

Liu³

et al. 2008

329

230

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...

“…The induction of homologous gene targeting by sequence specific endonuclease is seen today as an emerging technology with many applications (36). However, and increasing emphasis is being set on the specificity of such endonucleases (36)(37)(38) to meet the high requirements of therapeutic applications. The scaffold of I-DmoI could be a very good starting point to engineer very specific endonucleases for such purposes.…”

Section: Discussionmentioning

confidence: 99%

Crystal structure of I-DmoI in complex with its target DNA provides new insights into meganuclease engineering

Marcaida

Prieto

Redondo

et al. 2008

Self Cite

Homing endonucleases, also known as meganucleases, are sequencespecific enzymes with large DNA recognition sites. These enzymes can be used to induce efficient homologous gene targeting in cells and plants, opening perspectives for genome engineering with applications in a wide series of fields, ranging from biotechnology to gene therapy. Here, we report the crystal structures at 2.0 and 2.1 Å resolution of the I-DmoI meganuclease in complex with its substrate DNA before and after cleavage, providing snapshots of the catalytic process. Our study suggests that I-DmoI requires only 2 cations instead of 3 for DNA cleavage. The structure sheds light onto the basis of DNA binding, indicating key residues responsible for nonpalindromic target DNA recognition. In silico and in vivo analysis of the I-DmoI DNA cleavage specificity suggests that despite the relatively few protein-base contacts, I-DmoI is highly specific when compared with other meganucleases. Our data open the door toward the generation of custom endonucleases for targeted genome engineering using the monomeric I-DmoI scaffold.gene targeting ͉ genetics ͉ protein-DNA interactions ͉ X-ray crystallography M eganucleases are sequence-specific enzymes that recognize large (12-45 bp) DNA target sites. These enzymes are often encoded by introns or inteins behaving as mobile genetic elements. They produce double strand breaks (DSB) that get repaired by homologous recombination with an intron-or intein-containing gene, resulting in the insertion of the intron or intein in that particular site (1). Meganucleases are being used to stimulate homologous gene targeting in the vicinity of their target sequences with the aim of improving current genome engineering approaches, alleviating the risks due to the randomly inserted transgenes (2, 3).The use of meganuclease-induced recombination has long been limited by the repertoire of natural meganucleases. In nature, meganucleases are essentially represented by homing endonucleases, a family of enzymes encoded by mobile genetic elements whose function is to initiate DSB induced recombination events in a process referred to as homing (4). The probability of finding a homing endonuclease cleavage site in a chosen gene is extremely low. Thus, making artificial meganucleases with custom-made substrate specificity is an intense area of research.Sequence homology has been used to classify homing endonucleases into 5 families, the largest one having the conserved LAGLI-DADG sequence motif. Homing endonucleases containing one such motif function as homodimers. In contrast, homing endonucleases containing 2 motifs are single chain proteins (5, 6). Structural information for several members of the LAGLIDADG endonuclease family indicate that these proteins adopt a similar active conformation as homodimers or as monomers with 2 separate domains (7-9). The LAGLIDADG motifs form structurally conserved ␣-helices tightly packed at the center of the interdomain or intermonomer interface. The last acidic residue of this LAGLI-DADG motif part...