Continuous directed evolution of DNA-binding proteins to improve TALEN specificity

Nucleases containing programmable DNA-binding domains can alter the genomes of model organisms and have the potential to become human therapeutics. Here we present DNA-binding phage-assisted continuous evolution (DB-PACE) as a general approach for the laboratory evolution of DNA-binding activity and specificity. We used this system to generate TALE nucleases with broadly improved DNA cleavage specificity, establishing DB-PACE as a versatile approach for improving the accuracy of genome-editing agents

Engineering the Redox Potential over a Wide Range within a New Class of FeS Proteins

Abstract: MitoNEET is a newly discovered mitochondrial protein and a target of the TZD class of antidiabetes drugs. MitoNEET is homodimeric with each protomer binding a [2Fe-2S] center through a rare 3-Cys and 1-His coordination geometry. Both the fold and the coordination of the [2Fe-2S] centers suggest that it could have novel properties compared to other known [2Fe-2S] proteins. We tested the robustness of mitoNEET to mutation and the range over which the redox potential (EM) could be tuned. We found that the protein could tolerate an array of mutations that modified the EM of the [2Fe-2S] center over a range of ∼700 mV, which is the largest EM range engineered in an FeS protein and, importantly, spans the cellular redox range (+200 to-300 mV). These properties make mitoNEET potentially useful for both physiological studies and industrial applications as a stable, water-soluble, redox agent

Structure and Molecular Evolution of CDGSH Iron-Sulfur Domains

The recently discovered CDGSH iron-sulfur domains (CISDs) are classified into seven major types with a wide distribution throughout the three domains of life. The type 1 protein mitoNEET has been shown to fold into a dimer with the signature CDGSH motif binding to a [2Fe-2S] cluster. However, the structures of all other types of CISDs were unknown. Here we report the crystal structures of type 3, 4, and 6 CISDs determined at 1.5 Å, 1.8 Å and 1.15 Å resolution, respectively. The type 3 and 4 CISD each contain one CDGSH motif and adopt a dimeric structure. Although similar to each other, the two structures have permutated topologies, and both are distinct from the type 1 structure. The type 6 CISD contains tandem CDGSH motifs and adopts a monomeric structure with an internal pseudo dyad symmetry. All currently known CISD structures share dual iron-sulfur binding modules and a β-sandwich for either intermolecular or intramolecular dimerization. The iron-sulfur binding module, the β-strand N-terminal to the module and a proline motif are conserved among different type structures, but the dimerization module and the interface and orientation between the two iron-sulfur binding modules are divergent. Sequence analysis further shows resemblance between CISD types 4 and 7 and between 1 and 2. Our findings suggest that all CISDs share common ancestry and diverged into three primary folds with a characteristic phylogenetic distribution: a eukaryote-specific fold adopted by types 1 and 2 proteins, a prokaryote-specific fold adopted by types 3, 4 and 7 proteins, and a tandem-motif fold adopted by types 5 and 6 proteins. Our comprehensive structural, sequential and phylogenetic analysis provides significant insight into the assembly principles and evolutionary relationship of CISDs

Applications of CRISPR–Cas systems in neuroscience

Genome-editing tools, and in particular those based on CRISPR-Cas (clustered regularly interspaced short palindromic repeat (CRISPR)-CRISPR-associated protein) systems, are accelerating the pace of biological research and enabling targeted genetic interrogation in almost any organism and cell type. These tools have opened the door to the development of new model systems for studying the complexity of the nervous system, including animal models and stem cell-derived in vitro models. Precise and efficient gene editing using CRISPR-Cas systems has the potential to advance both basic and translational neuroscience research.National Institute of Mental Health (U.S.) (Grant 5DP1-MH100706)National Institute of Mental Health (U.S.) (Grant 1R01-MH110049)National Institute of Diabetes and Digestive and Kidney Diseases (U.S.) (Grant 5R01DK097768-03

Efficient Delivery of Genome-Editing Proteins In Vitro and In Vivo

Efficient intracellular delivery of proteins is needed to fully realize the potential of protein therapeutics. Current methods of protein delivery commonly suffer from low tolerance for serum, poor endosomal escape, and limited in vivo efficacy. Here we report that common cationic lipid nucleic acid transfection reagents can potently deliver proteins that are fused to negatively supercharged proteins, that contain natural anionic domains, or that natively bind to anionic nucleic acids. This approach mediates the potent delivery of nM concentrations of Cre recombinase, TALE- and Cas9-based transcriptional activators, and Cas9:sgRNA nuclease complexes into cultured human cells in media containing 10% serum. Delivery of Cas9:sgRNA complexes resulted in up to 80% genome modification with substantially higher specificity compared to DNA transfection. This approach also mediated efficient delivery of Cre recombinase and Cas9:sgRNA complexes into the mouse inner ear in vivo, achieving 90% Cre-mediated recombination and 20% Cas9-mediated genome modification in hair cells