An optimized two-finger archive for ZFN-mediated gene targeting

Gupta, Ankit; Christensen, Ryan; Rayla, Amy L.; Lakshmanan, Abirami; Stormo, Gary D.; Wolfe, Scot A.

doi:10.1038/nmeth.1994

Cited by 114 publications

(120 citation statements)

References 21 publications

(48 reference statements)

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“…We tested the activity of each TALEN at its target site upon injecting its mRNAs into zebrafish embryos and verified that the lesion rates for each nuclease were in excess of 20% in normal embryos 24 h post-fertilization (hpf ) (Supplemental Fig. 1; Meng et al 2008;Gupta et al 2012). We then examined whether coinjecting mRNAs encoding the two most proximal TALENs would delete the intervening genomic fragment, which spanned ;500 bp (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…15). The 59 ZFA was assembled from our archive of single finger modules , whereas the 39 ZFA was created using a combination of single finger modules ) and selection of a two-finger module for the AACAGA subsite (Gupta et al 2012). These ZFAs were coupled to the DD/RR versions of FokI endonuclease (Miller et al 2007;Szczepek et al 2007) in a pCS2 backbone (Meng et al 2008).…”

Section: Slc24a5 Locus Zinc Finger Nuclease (Zfn) Constructionmentioning

confidence: 99%

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Targeted chromosomal deletions and inversions in zebrafish

et al. 2013

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Zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) provide powerful platforms for genome editing in plants and animals. Typically, a single nuclease is sufficient to disrupt the function of protein-coding genes through the introduction of microdeletions or insertions that cause frameshifts within an early coding exon. However, interrogating the function of cis-regulatory modules or noncoding RNAs in many instances requires the excision of this element from the genome. In human cell lines and invertebrates, two nucleases targeting the same chromosome can promote the deletion of intervening genomic segments with modest efficiencies. We have examined the feasibility of using this approach to delete chromosomal segments within the zebrafish genome, which would facilitate the functional study of large noncoding sequences in a vertebrate model of development. Herein, we demonstrate that segmental deletions within the zebrafish genome can be generated at multiple loci and are efficiently transmitted through the germline. Using two nucleases, we have successfully generated deletions of up to 69 kb at rates sufficient for germline transmission (1%–15%) and have excised an entire lincRNA gene and enhancer element. Larger deletions (5.5 Mb) can be generated in somatic cells, but at lower frequency (0.7%). Segmental inversions have also been generated, but the efficiency of these events is lower than the corresponding deletions. The ability to efficiently delete genomic segments in a vertebrate developmental system will facilitate the study of functional noncoding elements on an organismic level.

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Section: Resultsmentioning

confidence: 99%

Section: Slc24a5 Locus Zinc Finger Nuclease (Zfn) Constructionmentioning

confidence: 99%

Targeted chromosomal deletions and inversions in zebrafish

et al. 2013

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“…Each zinc finger cluster was displayed as a C-terminal fusion to the omega subunit of Escherichia coli RNA polymerase without an accessory DNA-binding domain (Noyes et al 2008b). Complementary binding sites for each ZFP were identified through a single round of selection from a 28-bp randomized library with the recovered sequences characterized by both Sanger and Illumina sequencing (Zhu et al 2011a;Gupta et al 2012). Recognition motifs were identified as overrepresented sequence motifs within the recovered sequences (Zhu et al 2011a;Christensen et al 2012).…”

Section: Determining the Dna-binding Specificities Of Drosophila Zfpsmentioning

confidence: 99%

“…The recognition properties of individual zinc fingers can be influenced by their position in an array and the recognition determinants of their immediate neighbors (Desjarlais and Berg 1993;Wolfe et al 1999;Dreier et al 2001;Sander et al 2009;Zhu et al 2011a), but in some cases, in particular for subsets of specificity determinants with well-defined recognition properties, individual fingers can be assembled in novel combinations to create new recognition modalities (Desjarlais and Berg 1993;Segal et al 1999;Dreier et al 2000Dreier et al , 2001Dreier et al , 2005Liu et al 2002;Bae et al 2003;Kim et al 2009;Zhu et al 2011a). Although some principles that govern the recognition properties of zinc fingers have been developed through the analysis of natural Pabo 1991, 1993;Fairall et al 1993;Laity et al 2000;Bae et al 2003) and artificial (Rebar and Pabo 1994;Segal et al 1999;Dreier et al 2000Dreier et al , 2001Dreier et al , 2005Liu et al 2002;Bae et al 2003;Maeder et al 2008;Kim et al 2009;Sander et al 2011;Zhu et al 2011a;Gupta et al 2012) ZFPs, the ability to accurately predict the DNA-binding specificity of naturally occurring zinc finger assemblies remains suboptimal.…”

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confidence: 99%

Global analysis of Drosophila Cys₂-His₂ zinc finger proteins reveals a multitude of novel recognition motifs and binding determinants

et al. 2013

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Cys 2 -His 2 zinc finger proteins (ZFPs) are the largest group of transcription factors in higher metazoans. A complete characterization of these ZFPs and their associated target sequences is pivotal to fully annotate transcriptional regulatory networks in metazoan genomes. As a first step in this process, we have characterized the DNA-binding specificities of 129 zinc finger sets from Drosophila using a bacterial one-hybrid system. This data set contains the DNA-binding specificities for at least one encoded ZFP from 70 unique genes and 23 alternate splice isoforms representing the largest set of characterized ZFPs from any organism described to date. These recognition motifs can be used to predict genomic binding sites for these factors within the fruit fly genome. Subsets of fingers from these ZFPs were characterized to define their orientation and register on their recognition sequences, thereby allowing us to define the recognition diversity within this finger set. We find that the characterized fingers can specify 47 of the 64 possible DNA triplets. To confirm the utility of our finger recognition models, we employed subsets of Drosophila fingers in combination with an existing archive of artificial zinc finger modules to create ZFPs with novel DNA-binding specificity. These hybrids of natural and artificial fingers can be used to create functional zinc finger nucleases for editing vertebrate genomes.

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“…In addition to this "modular assembly" approach to zinc-finger construction, selection-based methods for constructing zinc-finger proteins have also been reported (Greisman and Pabo 1997;Isalan et al 2001;Hurt et al 2003;Magnenat et al 2004), including those that consider context-dependent interactions between adjacent zinc-finger domains, such as oligomerized pool engineering (OPEN) (Maeder et al 2008). In addition, specialized sets of validated two-finger, zinc-finger modules have been used to assemble zincfinger arrays (Kim et al 2009;Bhakta et al 2013), including those that take context-dependent effects into account (Sander et al 2011b;Gupta et al 2012).…”

Section: Zinc-finger Nucleasesmentioning

confidence: 99%

Genome-Editing Technologies: Principles and Applications

Gaj

Sirk

Shui

et al. 2016

Cold Spring Harb Perspect Biol

299

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Targeted nucleases have provided researchers with the ability to manipulate virtually any genomic sequence, enabling the facile creation of isogenic cell lines and animal models for the study of human disease, and promoting exciting new possibilities for human gene therapy. Here we review three foundational technologies-clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9), transcription activator-like effector nucleases (TALENs), and zinc-finger nucleases (ZFNs). We discuss the engineering advances that facilitated their development and highlight several achievements in genome engineering that were made possible by these tools. We also consider artificial transcription factors, illustrating how this technology can complement targeted nucleases for synthetic biology and gene therapy.

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An optimized two-finger archive for ZFN-mediated gene targeting

Cited by 114 publications

References 21 publications

Targeted chromosomal deletions and inversions in zebrafish

Targeted chromosomal deletions and inversions in zebrafish

Global analysis of Drosophila Cys₂-His₂ zinc finger proteins reveals a multitude of novel recognition motifs and binding determinants

Genome-Editing Technologies: Principles and Applications

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An optimized two-finger archive for ZFN-mediated gene targeting

Cited by 114 publications

References 21 publications

Targeted chromosomal deletions and inversions in zebrafish

Targeted chromosomal deletions and inversions in zebrafish

Global analysis of Drosophila Cys2-His2 zinc finger proteins reveals a multitude of novel recognition motifs and binding determinants

Genome-Editing Technologies: Principles and Applications

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Global analysis of Drosophila Cys₂-His₂ zinc finger proteins reveals a multitude of novel recognition motifs and binding determinants