Chromatin regulation at the frontier of synthetic biology

As synthetic biology approaches are extended to diverse applications throughout medicine, biotechnology and basic biological research, there is an increasing need to engineer yeast, plant and mammalian cells. Eukaryotic genomes are regulated by the diverse biochemical and biophysical states of chromatin, which brings distinct challenges, as well as opportunities, over applications in bacteria. Recent synthetic approaches, including 'epigenome editing', have allowed the direct and functional dissection of many aspects of physiological chromatin regulation. These studies lay the foundation for biomedical and biotechnological engineering applications that could take advantage of the unique combinatorial and spatiotemporal layers of chromatin regulation to create synthetic systems of unprecedented sophistication.National Institute of General Medical Sciences (U.S.) (Ruth L. Kirschstein Postdoctoral Fellowship )United States. Defense Advanced Research Projects AgencyNational Institutes of Health (U.S.) (R24 Grant)Wyss Institute for Biologically Inspired EngineeringHoward Hughes Medical Institut

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

Zinc Finger Targeter (ZiFiT): an engineered zinc finger/target site design tool

Zinc Finger Targeter (ZiFiT) is a simple and intuitive web-based tool that facilitates the design of zinc finger proteins (ZFPs) that can bind to specific DNA sequences. The current version of ZiFiT is based on a widely employed method of ZFP design, the ‘modular assembly’ approach, in which pre-existing individual zinc fingers are linked together to recognize desired target DNA sequences. Several research groups have described experimentally characterized zinc finger modules that bind many of the 64 possible DNA triplets. ZiFiT leverages the combined capabilities of three of the largest and best characterized module archives by enabling users to select fingers from any of these sets. ZiFiT searches a query DNA sequence for target sites for which a ZFP can be designed using modules available in one or more of the three archives. In addition, ZiFiT output facilitates identification of specific zinc finger modules that are publicly available from the Zinc Finger Consortium. ZiFiT is freely available at http://bindr.gdcb.iastate.edu/ZiFiT/

Counter-selectable marker for bacterial-based interaction trap systems

Counter-selectable markers can be used in two-hybrid systems to search libraries for a protein or compound that interferes with a macromolecular interaction or to identify macromolecules from a population that cannot mediate a particular interaction. In this report, we describe the adaptation of the yeast URA3/5-FOA counter-selection system for use in bacterial interaction trap experiments. Two different URA3 reporter systems were developed that allow robust counter-selection: (i) a single copy F\u27 episome reporter and (ii) a co-cistronic HIS3-URA3 reporter vector. The HIS3-URA3 reporter can be used for either positive or negative selections in appropriate bacterial strains. These reagents extend the utility of the bacterial two-hybrid system as an alternative to its yeast-based counterpart

CRISPR RNA-guided activation of endogenous human genes

Catalytically inactive CRISPR-associated 9 nuclease (dCas9) can be directed by short guide RNAs (gRNAs) to repress endogenous genes in bacteria and human cells. Here we show that a dCas9-VP64 transcriptional activation domain fusion protein can be directed by single or multiple gRNAs to increase expression of specific endogenous human genes. These results provide an important proof-of-principle that CRISPR-Cas systems can be used to target heterologous effector domains in human cells

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

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

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