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 transcription activator-like effectors nucleases (TALENs) with broadly improved DNA cleavage specificity, establishing DB-PACE as a versatile approach for improving the accuracy of genome-editing agents.

Small molecule-triggered Cas9 protein with improved genome-editing specificity.

Directly modulating the activity of genome-editing proteins has the potential to increase their specificity by reducing activity following target locus modification. We developed Cas9 nucleases that are activated by the presence of a cell-permeable small molecule by inserting an evolved 4-hydroxytamoxifen-responsive intein at specific positions in Cas9. In human cells, conditionally active Cas9s modify target genomic sites with up to 25-fold higher specificity than wild-type Cas9.

Chemical Biology Approaches to Genome Editing: Understanding, Controlling, and Delivering Programmable Nucleases.

Programmable DNA nucleases have provided scientists with the unprecedented ability to probe, regulate, and manipulate the human genome. Zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and the clustered regularly interspaced short palindromic repeat-Cas9 system (CRISPR-Cas9) represent a powerful array of tools that can bind to and cleave a specified DNA sequence. In their canonical forms, these nucleases induce double-strand breaks at a DNA locus of interest that can trigger cellular DNA repair processes that disrupt or replace genes. The fusion of these programmable nucleases with a variety of other protein domains has led to a rapidly growing suite of tools for activating, repressing, visualizing, and modifying loci of interest. Maximizing the usefulness and therapeutic relevance of these tools, however, requires precisely controlling their activity and specificity to minimize potentially toxic side effects arising from off-target activities. This need has motivated the application of chemical biology principles and methods to genome-editing proteins, including the engineering of variants of these proteins with improved or altered specificities, and the development of genetic, chemical, optical, and protein delivery methods that control the activity of these agents in cells. Advancing the capabilities, safety, effectiveness, and therapeutic relevance of genome-engineering proteins will continue to rely on chemical biology strategies that manipulate their activity, specificity, and localization.

From designer clusters to synthetic crystalline nanoassemblies.

Clusters have the potential to serve as building blocks of materials, enabling the tailoring of materials with novel electronic or magnetic properties. Historically, there has been a disconnect between magic clusters found in the gas phase and the synthetic assembly of cluster materials. We approach this challenge through a proposed protocol that combines gas-phase investigations to examine feasible units, theoretical investigations of energetic compositional diagrams and geometrical shapes to identify potential motifs, and synthetic chemical approaches to identify and characterize cluster assemblies in the solid state. Through this approach, we established As7(3-) as a potential stable species via gas-phase molecular beam experiments consistent with its known existence in molecular crystals with As to K ratios of 7:3. Our protocol also suggests another variant of this material. We report the synthesis of a cluster compound, As7K1.5(crypt222-K)1.5, composed of a lattice of As7 clusters stabilized by charge donation from cryptated K atoms and bound by sharing K atoms. The bond dimensions of this supercluster assembled material deduced by X-ray analysis are found to be in excellent agreement with the theoretical calculations. The new compound has a significantly larger band gap than the hitherto known solid. Thus, our approach allows the tuning of the electronic properties of solid cluster assemblies.