Gene targeting in nod mouse embryos using zincfinger nucleases studies in nod mice have provided important insight into the genetics and pathogenesis of type 1 diabetes t1d. Zfns can be designed to target a chosen locus with high speci. Their utility has been limited, however, by the significant failure rate of new designs, reflecting the complexity of dna recognition by zinc fingers. Knockout of wasp on k562 cells k562wasko cells resulted in several megakaryocyticrelated defects such as morphological alterations, lower expression of cd41. Enhancing gene targeting with designed zinc finger nucleases marinabibikova,kellybeumer,jonathank.
A general method for improving the efficiency of gene targeting would be valuable in many circumstances, as would the extension of this genetic tool to a broader range of organisms. Targeted transgene integration in plant cells using designed. Increased induction of zfn expression led to higher frequencies of gene targeting, demonstrating the beneficial effect of activating the target. Zincfinger nucleases zfns are artificial restriction enzymes generated by fusing a zinc finger dnabinding domain to a dnacleavage domain. Efficient gene targeting in drosophila with zincfinger. Gene targeting has been successfully applied to cattle, sheep, swine and many fungi. Qqr is an artificial zinc finger dna binding domain that recognizes the sequence 5. In the case of genome editing using donor constructs, drug selection is often also not required, and the mutations created can be as small as a singlebasepair change a process referred to as gene correction. Testing zinc finger nucleases using the gfp gene targeting system. It was expected that, in zf37 cells, the designed cop3. Gallb,2, and dana carrolla,2 adepartment of biochemistry, university of utah school of medicine, 15 north medical drive east, salt lake city, ut 841125650.
Figure 1zincfinger nuclease zfnmediated genome editing. Hermann m, maeder ml, rector k, ruiz j, becher b, et al. Abstract zinc finger nucleases zfns enable precise genome modification in a variety of organisms and cell types. We tested the feasibility of using zincfinger nucleases zfns to knock out a gene directly in a pure nod background, bypassing the need of embryonic stem cells. Selectionindependent generation of gene knockout mouse. Efficient gene targeting in drosophila with zincfinger nucleases. The present invention relates to a vector comprising a a nucleic acid encoding a chimeric nuclease, wherein the chimeric nuclease comprises. We modified qqr chimeric nucleases qqrcns 7, 9 and tested whether they stimulated gene targeting. Here it is reported that designed zinc finger nucleases zfns can drive sitedirected dna integration into transgenic and native gene loci. Recent results have shown that zfns can be used to create targeting frequencies of up to 20% in a human diseasecausing gene. In addition, the impact on biomedical research of classical gene targeting.
We have previously shown that the zifzfn stimulates gene targeting as a heterodimer when the heterodimer target binding sites are oriented as a zfn full site. Gene editing using zinc finger nucleases encoded within lentiviral vectors zinc finger nucleases zfns have the remarkable ability to a bind to a specific location in the doublestranded dna, break the doublestranded dna at that specific location and, if an endogenous repair template is provided, initiate homologydirected repair, restoring. Studies in nod mice have provided important insight into the genetics and pathogenesis of type 1 diabetes t1d. Zinc finger nuclease an overview sciencedirect topics. Effective gene targeting in rabbits using rnaguided cas9. Gene targeting by homologous recombination in mouse zygotes mediated by zincfinger nucleases. Targeted genetic modification of the porcine genome using. Targeted mutagenesis using zincfinger nucleases in. The jagged arrow on the left indicates an unspecified mutagenic agent targeted to one. Nuclear gene targeting in chlamydomonas using engineered zinc.
In situations in which zinc finger nucleases are used to drive targeted integration, selection is. Heritable targeted inactivation of myostatin gene in yellow. A suite of zfns were engineered by the recently described contextdependent assembly platforma rapid, opensource method for generating zincfinger arrays. Gene targeting in nod mouse embryos using zincfinger nucleases. We performed targeted mutagenesis of a transgene and nine endogenous soybean glycine max genes using zincfinger nucleases zfns. The frequency of gene targeting can be significantly enhanced through the use of engineered endonucleases such as zinc finger nucleases, engineered homing endonucleases, and nucleases based on engineered tal effectors. Targeted transgene integration in plants remains a significant technical challenge for both basic and applied research. The underlying technology is known as gene targeting and is based on the cellular homologous recombination hr pathway, which has. Zinc finger domains can be engineered to target specific desired dna sequences and this enables zincfinger nucleases to target unique sequences within complex genomes. Targeted gene editing in the sea urchin embryo using zinc. Targeting of photoreceptor genes in chlamydomonas reinhardtii via zincfinger nucleases and crisprcas9 andre greiner,a,1,2 simon kelterborn,a,1 heide evers,a georg kreimer,b irina sizova,a,2,3 and peter hegemanna a institute of biology, experimental biophysics, humboldt university of berlin, 10099 berlin, germany b department of biology, friedrichalexander university, 91058 erlangen, germany. All four of these systems have been shown to function in crop plants and have been used for sitespecific gene targeting and gene editing. Efficient gene targeting in drosophilaby direct embryo injection with zinc finger nucleases kelly j. Dec 27, 2008 targeted transgene integration in plants remains a significant technical challenge for both basic and applied research.
In tissue culture systems, a chromosomal integrated reporter inactivated gfp gene that harbors the zfn target site is commonly used to test designed zfns zou et al. Zfns that form pairs, which target the dhfr locus, were designed and screened in vitro for dna binding to. Efficient methods for targeted mutagenesis in zebrafish using zinc finger nucleases. Targeted chromosomal cleavage and mutagenesis in drosophila using zincfinger nucleases. Design of zinc finger nuclease targeting myostatin gene in yellow catfish. Doublestrand breaks are important for sitespecific mutagenesis in that they stimulate the cells natural dnarepair processes, namely homologous recombination and nonhomologous end joining nhej.
Zinc finger domains can be engineered to target specific desired dna sequences and this enables zinc finger nucleases to target unique sequences within complex genomes. Gene targeting using zinc finger nucleases nature biotechnology. We knocked out the was gene in k562 cells using a zincfinger nuclease zfn pair targeting the was intron 1 and a homologous donor dna that disrupted wasp expression. Trautman, danacarroll gene targetingthe process of gene replacement by homologous recombinationis a very useful but typically inefficient technique 1. Presentations of the thesis these topics have been presented at national or international conferences as poster or oral presentations. Transcription activatorlike effector tale dnabinding domains depend on a simple.
Here we report that gene targeting is achieved in 1. Trautman a, ana bozas a, jilong liu b,1, jared rutter a, joseph g. For the first time researchers have the opportunity to manipulate essentially any gene in a plethora of cells and organisms, using targeted nucleases that were designed for sequencespecific. Zfns are also used to create a new generation of genetic disease models called isogenic human disease models. Zincfinger nucleases zfns are artificial restriction enzymes generated by fusing a zincfinger dnabinding domain to a dna cleavage domain porteus and carroll, 2005. Efficient gene targeting of the rosa26 locus in mouse zygotes using tale nucleases. Gene targeting by homologous recombination in mouse. The procedure that generates these high frequencies relies on cleavage of the target by designed zincfinger nucleases zfns and production of a linear donor in situ. Targeted mutagenesis using zincfinger nucleases in arabidopsis.
Results design of zinc finger nucleases targeting the dhfr gene. Targeted chromosomal cleavage and mutagenesis in drosophila using zinc finger nucleases. Gene targeting the process of gene replacement by homologous recombinationis a very useful but typically inefficient technique 11. Targeted mutagenesis using zinc finger nucleases in arabidopsis. Enhancing gene targeting with designed zinc finger nucleases. Apr 15, 2008 design of zinc finger nucleases targeting the dhfr gene. Targeted genome modification in mice using zincfinger nucleases. Upon transient expression of these nucleases the target gene is. Nuclear gene targeting in chlamydomonas using engineered. Chimeric nucleases stimulate gene targeting in human cells. Although targeted mutagenesis is routine in several model organisms including yeast and mouse, efficient and widely usable methods to generate targeted modifications in plant genes are.
A dimer of designed 4finger zfns enabled intrachromosomal reconstitution of a disabled gfp reporter gene and sitespecific transgene. Reppert1,5 1department of neurobiology, university of massachusetts medical school, worcester, massachusetts 01605, usa. Our goal was to further explore novel methods of genetic manipulation in this mouse model. Drews proceedings of the national academy of sciences feb 2005, 102 6 22322237. Genome editing with engineered zinc finger nucleases. In other animal models in which a gene editing approach is not available, to analyze the function of a gene of interest. Zinc finger nucleases zfns show promise in improving the efficiency of gene targeting by introducing dna doublestrand breaks in target genes, which then stimulate the cells endogenous homologous. Genome editing in plant cells by zinc finger nucleases uf plant. Targeted gene knockout in mammalian cells using engineered zinc finger nucleases. Targeted mutagenesis is an essential tool of reverse genetics that could be used experimentally to investigate basic plant biology or modify crop plants for improvement of important agricultural traits. Zinc finger nucleases zfns show promise in improving the efficiency of gene targeting by introducing dna doublestrand breaks in target genes, which then stimulate the cells endogenous.
Zincfinger nucleases have proven to be successful as reagents for targeted genome manipulation in drosophila melanogaster and many other organisms. A dimer of designed 4 finger zfns enabled intrachromosomal reconstitution of a disabled gfp reporter gene and sitespecific transgene. Designer nucleases are molecules that can be engineered to cleave. Jan 22, 2015 four designer nuclease platforms are currently available. Pdf methods of modifying the human genome precisely and efficiently hold great promise for revolutionizing the gene therapy arena. Specific zfns targeting dicer like dcl genes and other genes involved in rna silencing were. Achieving the full potential of zincfinger nucleases zfns for genome engineering in human cells requires their efficient delivery to the relevant cell types. Specific zfns targeting dicer like dcl genes and other genes involved in rna silencing. Zinc finger nucleases zfns are artificial restriction enzymes generated by fusing a zinc finger dnabinding domain to a dnacleavage domain. Dear editor, recently, zinc finger nuclease, transcription activatorlike effector nuclease, and rnaguided cas9 endonuclease cas9 have emerged as powerful means for genome editing conklin, 20.
In the past, the application of gene targeting to mammalian cells has been limited by its low efficiency. Gene targeting is a process in which a dna molecule introduced into a cell replaces. To test the new zfns, 293 cell lines were made with singlecopy integrants of the gfp target gene. Mammalian gene targeting with designed zinc finger nucleases. B revia enhancing gene targeting with designed zinc finger. With increasing global food demands in the face of challenging biotic and abiotic pressures on crop production, there is a vital need for good crop improvement strategies. By taking advantage of endogenous dna repair machinery zfns can be designed to precisely alter the genomes of higher organisms, allowing almost any region of the genome to be targeted fig. Targeted mutagenesis of duplicated genes in soybean with zinc. Targeting of photoreceptor genes in chlamydomonas reinhardtii. We designed a test system for zfns in chlamydomonas that is based on repair of an inactivated marker gene that was stably integrated into the genome. Gene correction by homologous recombination with zinc finger. Request pdf nuclear gene targeting in chlamydomonas using engineered zincfinger nucleases the unicellular green alga chlamydomonas reinhardtii is a versatile model for fundamental and. Efficient gene targeting in drosophilaby direct embryo injection with zincfinger nucleases kelly j. Oct 28, 2007 achieving the full potential of zinc finger nucleases zfns for genome engineering in human cells requires their efficient delivery to the relevant cell types.
Targeting of photoreceptor genes in chlamydomonas reinhardtii via zinc finger nucleases and crisprcas9 andre greiner,a,1,2 simon kelterborn,a,1 heide evers,a georg kreimer,b irina sizova,a,2,3 and peter hegemanna a institute of biology, experimental biophysics, humboldt university of berlin, 10099 berlin, germany. Gene editing and gene targeting using designer nucleases are relatively new, sophisticated approaches that can be used for crop improvement. Method efficient targeted mutagenesis in the monarch butterfly using zincfinger nucleases christine merlin,1,5 lauren e. Zinc finger nucleases zfns are a class of engineered dnabinding proteins that facilitate targeted editing of the genome by creating doublestrand breaks in dna at userspecified locations. Efficient gene targeting in drosophilaby direct embryo. This study presents a powerful new technology for more ef. We report very high gene targeting frequencies in drosophila by direct embryo injection of mrnas encoding specific zincfinger nucleases zfns.
Targeted genome modifications with tailored nucleases. Method efficient targeted mutagenesis in the monarch. Gene editing in human stem cells using zinc finger. However, this situation changed with the availability of newly developed gene targeting technologies, called engineered nucleases or gene. Targeted gene knockout in mammalian cells by using engineered. Gene targetingthe process of gene replacement by homologous recombinationis a very useful but typically inefficient technique 11. One early example of successful zfnmediated genetic disruption in the rat includes targeting to endogenous immunoglobulin m igm and rab38 genes as well as to an integrated gfp reporter gene 33. Zinc finger nuclease technology zfn targeted genome. Design and in vitro validation of zfns targeting cry2 using a computer algorithm, we scanned the monarch cry2 gene for sequences targetable with our archive of zinc finger modules zhuetal. Use of zincfinger nucleases to knock out the was gene in. Request pdf nuclear gene targeting in chlamydomonas using engineered zinc finger nucleases the unicellular green alga chlamydomonas reinhardtii is a versatile model for fundamental and. Both local mutagenesis via nonhomologous end joining nhej and targeted gene replacement via homologous recombination hr have been achieved in up to 10% of all targets at a given locus. Efficient gene targeting of the rosa26 locus in mouse. Heritable targeted inactivation of myostatin gene in.
Exon 1 of the chos dhfr locus was chosen for targeting with pairs of zfns, using the same strategy as in ref. The zfns targeting yellow catfish mstn gene were designed using the modular assembly method. Here again, the use of a kanamycinresistance gene, flanked by regions of homology to the target locus, proved useful for producing transgenic plants with a. Targeted gene knockout in mammalian cells by using. Moreover, we devised a reporter assay to assess talens activity and.
Dec 16, 2008 we report very high gene targeting frequencies in drosophila by direct embryo injection of mrnas encoding specific zinc finger nucleases zfns. Homologous recombinationbased gene targeting using mus musculus embryonic stem cells has greatly impacted biomedical research. Commercial zfns were reported to enhance gene targeting directly in mouse zygotes, whereas similar approaches using publicly available resources have not yet been described. Efficient methods for targeted mutagenesis in zebrafish using zincfinger nucleases. A general method for improving the efficiency of gene targeting would be valuable in. In embryos that are wild type for dna repair, the products. Gene targeting by homologous recombination in mouse zygotes. Gene targeting in nod mouse embryos using zincfinger. Making directed genetic changes is often called gene targeting. Structural studies of human dhfr have shown this region to be critical for substrate and cofactor binding. Drews department of biology, university of utah, 257 south 1400 east, salt lake city, ut 841120840. Doublestranded breaks occur when two zfns bind to the target dna, bringing their nuclease domains together. Model experiments have demonstrated that a doublestrand break dsb.
Trautmana, ana bozasa, jilong liub,1, jared ruttera, joseph g. Targeted genome editing using engineered zinc finger. Download fulltext pdf download fulltext pdf gene targeting in nod mouse embryos using zincfinger nucleases article pdf available in diabetes 631 august 20 with 42 reads. We have been developing chimeric zinc finger nucleases zfns as potential stimulators of gene targeting 7, 8. Apr 01, 2006 the procedure that generates these high frequencies relies on cleavage of the target by designed zinc finger nucleases zfns and production of a linear donor in situ. Zinc finger nucleases zfns show promise in improving the efficiency of gene targeting by introducing dna doublestrand breaks in target genes. Thisanalysisidentifiedahighquality zfn site in exon 2 comprising two 12bp zinc finger recog. Ko animals other than mice using gene targeting technologies, as germlinecompetent es cells were available only for mice. Gene targeting in nod mouse embryos using zincfinger nucleases article pdf available in diabetes 631 august 20 with 42 reads how we measure reads. Targeted mutagenesis using zincfinger nucleases in arabidopsis alan lloyd, christopher l. It seems timely to consider the adoption of terminology that more accurately reflects what is being done. Gene editing in human stem cells using zinc finger nucleases.