Zfns are chimeric proteins composed of dnabinding cys 2his 2 zinc fingers fused to the nonspecific nuclease domain of the restriction enzyme foki 1. The benefit of building a nuclease using zinc fingers is that this. Each finger makes contact primarily with a separate dna triplet 2, 3. It is one of three methods of changing the genome with engineered nucleases they have two parts. Knockout rats via embryo microinjection of zincfinger. Engineered zinc finger proteins methods and protocols joel p. Pdf methods of modifying the human genome precisely and efficiently hold. Transcribed audio engineered nucleases are what researchers use to edit genomes.
The high levels of ccr5 disruption that we achieved in human hsc were possible because of an efficient gene editing technology based on zinc finger nucleases. Zinc finger nucleases zfns are artificial restriction enzymes generated by fusing a zinc finger dnabinding domain to a dna cleavage domain porteus and carroll, 2005. First in vivo human genome editing to be tested in new clinical trial abby olena may 18, 2017. Enhancing gene targeting with designed zinc finger nucleases. The dsb is induced by a zinc finger nuclease zfn4,5, which is a designed, sequence specific endonuclease that can be customized to cleave a userchosen dna target. The 44yearold patient has hunter syndrome, which doctors hope to treat using zinc finger nucleases. Two histidines from the helix and two cysteines from the beta sheet coordinately bind a zinc. Each zinc finger nuclease zfn consists of twofunctional domains.
Zinc finger domains can be engineered to target specific desired dna sequences and this enables zincfinger nucleases. Gene targeting using zinc finger nucleases nature biotechnology. Engineered zinc finger nickases induce homologydirected repair with reduced mutagenic effects cherie l. Targeted genome editing in human cells with zinc finger nucleases. We have been developing chimeric zinc finger nucleases zfns as potential stimulators of gene targeting 7, 8. These are based on zinc finger proteins, a family of naturally occurring transcription factors, fused on an endonuclease foki 1. Two finger modules are stitched together to form azinc finger. The zinc finger nuclease monopoly nature biotechnology.
Comparison between zfn, talen and crispr nuclineers. Zincfinger nucleases zfns are enzymes got by fusing a zinc finger. Contextdependent assembly coda of zinc finger nucleases is reported. Zincfinger nucleases zfns are targetable dna cleavage reagents that have been adopted as genetargeting tools. Genome engineering with zincfinger nucleases genetics. Typically, a zfn contains about three to nine zinc finger repeats each identifying three bases. Zinc finger nucleasemediated ccr5 knockout hematopoietic. Chimeric antigen receptor t cell cart, autologous cell, antisense, rna interference rnai, zinc finger nuclease. Zinc finger nucleases zfns show promise in improving the efficiency. Zinc fingers are manmade molecules made of a protein and zinc. Zinc finger nuclease zfn license agreement sigmaaldrich. Engineering a zinc finger nuclease zfn to introduce a doublestrand break at a specific single chromosomal locus and induce homologydirected repair with an exogenously added donor dna sequence. We performed targeted mutagenesis of a transgene and nine endogenous soybean glycine max genes using zinc finger nucleases zfns. Selectionfree zincfinger nuclease engineering by contextdependent assembly coda.
Delivery of both the zinc finger nucleases and the donor dna was accomplished with adenoassociated viral aav vectors, and zinc finger nuclease coding sequences fit comfortably into. The binding specificity of the designed zinc finger domain points the zfn to a specific genomic site. Fusing the zinc finger module to the foki nuclease domain the zinc finger module normally the module is tripartite, that is, consisting of three zinc fingers is fused via its carboxylterminus to the foki nuclease domain. Zinc finger nucleases or zfn are a tool used to target genes and change dna. Genome editing zinc finger nuclease part 1 lecture by. Zinc finger proteins and zinc finger nucleases zinc finger nucleases are sequence specific dna binding proteins.
The binding specificity of the designed zinc finger domain directs the zfn to a specific genomic site. A longstanding goal of molecular biologists has been to construct dnabinding proteins for the control of gene expression. The classical cys 2 his 2 c 2 h 2 zinc finger design is ideally suited for such purposes. The binding specificity of the designed zinc finger. Zincfinger nucleases zfns are artificial restriction enzymes generated by fusing a zinc finger dnabinding domain to a dnacleavage domain. Despite the advantages of genome editing with zfn, there are several potential. We used r5 and x4 zinc finger proteins zfps from our previous studies. However, recent advances in generating customized zinc finger nucleases zfns that can create a dna doublestrand break dsb at preselected sites in the human genome have paved the way for hr. The zfns have a dna recognition domain composed of three cys 2 his 2 zinc.
Induction and repair of zincfinger nucleasetargeted. Genome editing with engineered zinc finger nucleases. Pdf the zinc finger nuclease monopoly researchgate. Selectionfree zincfingernuclease engineering by context. A suite of zfns were engineered by the recently described contextdependent assembly platforma rapid, opensource method for generating zinc finger.
Zinc finger nucleases zfns are synthetic restriction enzymes evolved by fusing zinc finger. Originally coined to describe the finger like appearance of a hypothesized structure from xenopus laevis transcription factor iiia, the zinc finger. Zinc finger domains can be engineered to target specific desired dna sequences and this enables zincfinger nucleases to target unique sequences within complex genomes. Discriminating between closely related dna sequences both in vitro and in vivo, this naturally occurring design was adopted for engineering zinc finger. Here we describe contextdependent assembly coda, a platform for engineering zfns using only standard cloning techniques or custom dna synthesis. Each zinc finger contacts primarily 3 bp of dna through interactions in the major groove, and fingers that recognize many of the 64 triplets have been isolated s egal et al. Zinc finger nuclease and homing endonucleasemediated. Here we describe contextdependent assembly coda, a platform for engineering zfns using only standard cloning. The toolbox of rat genetics currently lacks the ability to introduce sitedirected, heritable mutations into the genome to create knockout animals. The first endonucleases were zinc finger nucleases zfns. Zinc finger nucleases zfns are versatile reagents that. With the advent of zinc finger nucleases zfns to the world of genetic research, the theoretical ideal became a reality. Since other nucleases with a similar function to zfn are considered in this opinion the term sitedirected nuclease.
Zfn dimers induce targeted dna dsbs that stimulate dna damage response pathways. Engineering a zinc finger nuclease zfn to introduce a doublestrand break at a specific single chromosomal locus and induce homologydirected repair with an exogenously added donor. 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. Selectionfree zincfinger nuclease engineering by context. Zinc finger nucleases zfnsproteins customdesigned to cut at specific dna sequenceswere originally developed in our lab for this purpose of delivering a targeted genomic dsb.
Zinc finger domains can recognize a trinucleotide dna sequence. The foki restriction enzyme is a dimer, with each of the two nuclease. Starting from an archive of zinc finger modules known to function well together, effective multifinger arrays can be. Engineered zinc finger nucleases zfns can be used to introduce targeted alterations into genomes of model organisms, plants, and human cells. Simultaneous zincfinger nuclease editing of the hiv. Selection free zinc finger nuclease engineering by contextdependent assembly coda.
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. A dsb is created in gene b, which is then processed to form free 3. Engineered zinc finger nucleases zfns enable targeted genome modification. Pdf selectionfree zincfingernuclease engineering by. Zinc finger proteins engineered to recognize a unique chromosomal site can be fused to a nuclease domain, and a doublestrand break induced by the resulting zinc finger nuclease can create. Zinc finger nucleases zfns are chimeric engineered nucleases designed to target userspecified sequences within the genome durai et al. Genome editing zinc finger nuclease part 1 lecture by vikas mangal. Dna cleavage domain contains nucleases like fok1 from flavobacterium. Recently, a new technology based on the use of genetargeted zinc finger nucleases zfns was developed and used for the generation of organisms with genetargeted deletions andor. Genome engineering with zinc finger nuclease and talens. Zinc finger nucleases zfns are artificial dna restriction enzymes that. Figure 1zincfinger nuclease zfnmediated genome editing. Generally, zfns are made up of two modular domains, dna binding domain and dna cleavage domain that collectively mediate site directed mutagenesis porteus, 2008.
Efficient gene targeting in drosophila with zincfinger. Zinc finger simple english wikipedia, the free encyclopedia. Our zinc finger nuclease zfn technology is based on a naturally occurring class of proteins called zinc finger dnabinding proteins zfps, which recognize and bind to specific sequences of dna. Breakthrough technologies zinc finger nuclease and homing endonucleasemediated assembly of multigene plant transformation vectors1oa vardit zeevi 2, zhuobin liang2, uri arieli, and tzvi. Zinc finger nucleases zfns and transcription activatorlike effector nucleases talens comprise a powerful class of tools that are redefining the boundaries of biological research. Engineered zinc finger nickases induce homologydirected. The first family of these nucleases to be used was the zfns. Each zinc finger nuclease zfn consists of two functional domains. Targeted mutagenesis of duplicated genes in soybean with.