Synthetic protein–protein interaction domains created by shuffling Cys(2)His(2) zinc-fingers

Cys(2)His(2) zinc-fingers (C2H2 ZFs) mediate a wide variety of protein–DNA and protein–protein interactions. DNA-binding C2H2 ZFs can be shuffled to yield artificial proteins with different DNA-binding specificities. Here we demonstrate that shuffling of C2H2 ZFs from transcription factor dimerization zinc-finger (DZF) domains can also yield two-finger DZFs with novel protein–protein interaction specificities. We show that these synthetic protein–protein interaction domains can be used to mediate activation of a single-copy reporter gene in bacterial cells and of an endogenous gene in human cells. In addition, the synthetic two-finger domains we constructed can also be linked together to create more extended, four-finger interfaces. Our results demonstrate that shuffling of C2H2 ZFs can yield artificial protein-interaction components that should be useful for applications in synthetic biology

A Note on B-observables in Ponzano-Regge 3d Quantum Gravity

We study the insertion and value of metric observables in the (discrete) path integral formulation of the Ponzano-Regge spinfoam model for 3d quantum gravity. In particular, we discuss the length spectrum and the relation between insertion of such B-observables and gauge fixing in the path integral.Comment: 17 page

Robust, synergistic regulation of human gene expression using TALE activators

Artificial transcription activator-like (TAL) effector-based activators (TALE activators) have broad utility but previous studies suggest that these monomeric proteins often possess low activities. Here we demonstrate that TALE activators can robustly function individually or in synergistic combinations to increase expression of endogenous human genes over wide dynamic ranges. These findings will encourage applications of TALE activators for research and therapy and guide design of novel monomeric TAL effector-based fusion proteins

High frequency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells

CRISPR RNA-guided endonucleases (RGENs) have rapidly emerged as a facile and efficient platform for genome editing. Here, we use a human cell-based reporter assay to characterize off-target cleavage of Cas9-based RGENs. We find that single and double mismatches are tolerated to varying degrees depending on their position along the guide RNA (gRNA)-DNA interface. We readily detected off-target alterations induced by four out of six RGENs targeted to endogenous loci in human cells by examination of partially mismatched sites. The off-target sites we identified harbor up to five mismatches and many are mutagenized with frequencies comparable to (or higher than) those observed at the intended on-target site. Our work demonstrates that RGENs are highly active even with imperfectly matched RNA-DNA interfaces in human cells, a finding that might confound their use in research and therapeutic applications

ZiFiT (Zinc Finger Targeter): an updated zinc finger engineering tool

ZiFiT (Zinc Finger Targeter) is a simple and intuitive web-based tool that provides an interface to identify potential binding sites for engineered zinc finger proteins (ZFPs) in user-supplied DNA sequences. In this updated version, ZiFiT identifies potential sites for ZFPs made by both the modular assembly and OPEN engineering methods. In addition, ZiFiT now integrates additional tools and resources including scoring schemes for modular assembly, an interface with the Zinc Finger Database (ZiFDB) of engineered ZFPs, and direct querying of NCBI BLAST servers for identifying potential off-target sites within a host genome. Taken together, these features facilitate design of ZFPs using reagents made available to the academic research community by the Zinc Finger Consortium. ZiFiT is freely available on the web without registration at http://bindr.gdcb.iastate.edu/ZiFiT/

Broad Specificity Profiling of TALENs Results in Engineered Nucleases With Improved DNA Cleavage Specificity

Although transcription activator-like effector nucleases (TALENs) can be designed to cleave chosen DNA sequences, TALENs have been shown to have activity against related off-target sequences. To better understand TALEN specificity and engineer TALENs with improved specificity, we profiled 30 unique TALENs with varying target sites, array length, and domain sequences for their ability to cleave any of 1012 potential off-target DNA sequences using in vitro selection and high-throughput sequencing. Computational analysis of the selection results predicted 76 off-target substrates in the human genome, 16 of which were accessible and modified by TALENs in human cells. The results collectively suggest that (i) TALE repeats bind DNA relatively independently; (ii) longer TALENs are more tolerant of mismatches, yet are more specific in a genomic context; and (iii) excessive DNA-binding energy can lead to reduced TALEN specificity in cells. Based on these findings, we engineered a TALEN variant, Q3, that exhibits equal on-target cleavage activity but 10-fold lower average off-target activity in human cells. Our results demonstrate that identifying and mutating residues that contribute to non-specific DNA-binding can yield genome editing reagents with improved DNA specificities

Rapid Mutation of Endogenous Zebrafish Genes Using Zinc Finger Nucleases Made by Oligomerized Pool ENgineering (OPEN)

Background: Customized zinc finger nucleases (ZFNs) form the basis of a broadly applicable tool for highly efficient genome modification. ZFNs are artificial restriction endonucleases consisting of a non-specific nuclease domain fused to a zinc finger array which can be engineered to recognize specific DNA sequences of interest. Recent proof-of-principle experiments have shown that targeted knockout mutations can be efficiently generated in endogenous zebrafish genes via non-homologous end-joining-mediated repair of ZFN-induced DNA double-stranded breaks. The Zinc Finger Consortium, a group of academic laboratories committed to the development of engineered zinc finger technology, recently described the first rapid, highly effective, and publicly available method for engineering zinc finger arrays. The Consortium has previously used this new method (known as OPEN for Oligomerized Pool ENgineering) to generate high quality ZFN pairs that function in human and plant cells. Methodology/Principal Findings: Here we show that OPEN can also be used to generate ZFNs that function efficiently in zebrafish. Using OPEN, we successfully engineered ZFN pairs for five endogenous zebrafish genes: tfr2, dopamine transporter, telomerase, hif1aa, and gridlock. Each of these ZFN pairs induces targeted insertions and deletions with high efficiency at its endogenous gene target in somatic zebrafish cells. In addition, these mutations are transmitted through th

Zinc Finger Database (ZiFDB): a repository for information on C2H2 zinc fingers and engineered zinc-finger arrays

Zinc fingers are the most abundant DNA-binding motifs encoded by eukaryotic genomes and one of the best understood DNA-recognition domains. Each zinc finger typically binds a 3-nt target sequence, and it is possible to engineer zinc-finger arrays (ZFAs) that recognize extended DNA sequences by linking together individual zinc fingers. Engineered zinc-finger proteins have proven to be valuable tools for gene regulation and genome modification because they target specific sites in a genome. Here we describe ZiFDB (Zinc Finger Database; http://bindr.gdcb.iastate.edu/ZiFDB), a web-accessible resource that compiles information on individual zinc fingers and engineered ZFAs. To enhance its utility, ZiFDB is linked to the output from ZiFiT—a software package that assists biologists in finding sites within target genes for engineering zinc-finger proteins. For many molecular biologists, ZiFDB will be particularly valuable for determining if a given ZFA (or portion thereof) has previously been constructed and whether or not it has the requisite DNA-binding activity for their experiments. ZiFDB will also be a valuable resource for those scientists interested in better understanding how zinc-finger proteins recognize target DNA

Targeted Deletion and Inversion of Tandemly Arrayed Genes in Arabidopsis thaliana Using Zinc Finger Nucleases

Tandemly arrayed genes (TAGs) or gene clusters are prevalent in higher eukaryotic genomes. For example, approximately 17% of genes are organized in tandem in the model plant Arabidopsis thaliana. The genetic redundancy created by TAGs presents a challenge for reverse genetics. As molecular scissors, engineered zinc finger nucleases (ZFNs) make DNA double-strand breaks in a sequence-specific manner. ZFNs thus provide a means to delete TAGs by creating two double-strand breaks in the gene cluster. Using engineered ZFNs, we successfully targeted seven genes from three TAGs on two Arabidopsis chromosomes, including the well-known RPP4 gene cluster, which contains eight resistance (R) genes. The resulting gene cluster deletions ranged from a few kb to 55 kb with frequencies approximating 1% in somatic cells. We also obtained large chromosomal deletions of ~9 Mb at approximately one tenth the frequency, and gene cluster inversions and duplications also were achieved. This study demonstrates the ability to use sequence-specific nucleases in plants to make targeted chromosome rearrangements and create novel chimeric genes for reverse genetics and biotechnology