The bacterial adaptive immune system CRISPR-Cas9 has been appropriated as a versatile tool for editing genomes, controlling gene expression, and visualizing genetic loci. To analyze Cas9's ability to bind DNA rapidly and specifically, we generated multiple libraries of potential binding partners for measuring the kinetics of nucleasedead Cas9 (dCas9) interactions. Using a massively parallel method to quantify protein-DNA interactions on a high-throughput sequencing flow cell, we comprehensively assess the effects of combinatorial mismatches between guide RNA (gRNA) and target nucleotides, both in the seed and in more distal nucleotides, plus disruption of the protospacer adjacent motif (PAM). We report two consequences of PAM-distal mismatches: reversal of dCas9 binding at long time scales, and synergistic changes in association kinetics when other gRNA-target mismatches are present. Together, these observations support a model for Cas9 specificity wherein gRNA-DNA mismatches at PAM-distal bases modulate different biophysical parameters that determine association and dissociation rates. The methods we present decouple aspects of kinetic and thermodynamic properties of the Cas9-DNA interaction and broaden the toolkit for investigating off-target binding behavior.DNA | molecular biophysics | kinetics | sequencing | CRISPR C RISPR-associated protein 9 (Cas9) is programmed to bind its target DNA by a guide RNA (gRNA) that, once loaded, forms a ribonucleoprotein (RNP) complex. The Streptococcus pyogenes CRISPR system, the most extensively studied and applied system to date, targets a 23-bp DNA sequence containing (i) an "NGG" protospacer adjacent motif (PAM) element downstream of the single-guide RNA (sgRNA) target DNA (1) and (ii) a 20-bp sequence upstream of the PAM bearing complementarity to the gRNA (2). Genome engineering applications leverage the nuclease activity of the Cas9 RNP, but Cas9 engineered to lack the residues required for cleavage [dCas9 (nuclease-dead Cas9)] has proven valuable by enabling the creation of customizable and programmable DNA binding elements that can activate and repress gene expression with high precision (CRISPRa and CRISPRi) (3).The biophysical underpinnings of the Cas9 target search have been investigated both by directed biochemical assays (4, 5) and through measurements of off-target Cas9 activity (6-11). These studies have led to a model for binding wherein Cas9 proceeds through a series of steps starting with PAM recognition, followed by DNA melting, RNA strand invasion, and heteroduplex formation dependent on complementarity with a 5-10-bp seed. Structural data have further suggested that conformational changes in the HNH domain reposition catalytic residues and permit allosteric regulation of the RuvC domain. This conformational gating ensures that cleavage occurs only in the context of substantial homology between gRNA and target (12, 13).The specificity of Cas9 DNA binding is crucial for all potential applications of Cas9's RNA-programmable targeting. Localization of dCas9...

Mechanically interlocked molecules that change their conformation in response to stimuli have been developed by synthetic chemists as building blocks for molecular machines. Here we describe a natural product, the lasso peptide benenodin-1, which exhibits conformational switching between two distinct threaded conformers upon actuation by heat. We have determined the structures of both conformers and have characterized the kinetics and energetics of the conformational switch. Single amino acid substitutions to benenodin-1 generate peptides that are biased to a single conformer, showing that the switching behavior is potentially an evolvable trait in these peptides. Lasso peptides such as benenodin-1 can be recognized and cleaved by enzymes called lasso peptide isopeptidases. We show that only the native conformer of benenodin-1 is cleaved by its cognate isopeptidase. Thus thermally-induced conformational switching of benenodin-1 may also be relevant to the biological function of these molecules.

Designing RNAs that form specific secondary structures is enabling better understanding and control of living systems through RNA-guided silencing, genome editing and protein organization. Little is known, however, about which RNA secondary structures might be tractable for downstream sequence design, increasing the time and expense of design efforts due to inefficient secondary structure choices. Here, we present insights into specific structural features that increase the difficulty of finding sequences that fold into a target RNA secondary structure, summarizing the design efforts of tens of thousands of human participants and three automated algorithms (RNAInverse, INFO-RNA and RNA-SSD) in the Eterna massive open laboratory. Subsequent tests through three independent RNA design algorithms (NUPACK, DSS-Opt, MODENA) confirmed the hypothesized importance of several features in determining design difficulty, including sequence length, mean stem length, symmetry, and specific difficult-to-design motifs like zig-zags. Based on these results, we have compiled an Eterna100 benchmark of 100 secondary structure design challenges that span a large range in design difficulty to help test future efforts. Our in silico results suggest new routes for improving computational RNA design methods and for extending these insights to assessing “designability” of single RNA structures as well as of switches for in vitro and in vivo applications.

Riboswitches that couple binding of ligands to conformational changes offer sensors and control elements for RNA synthetic biology and medical biotechnology. However, design of these riboswitches has required expert intuition or software specialized to transcription or translation outputs; design has been particularly challenging for applications in which the riboswitch output cannot be amplified by other molecular machinery. We present a fully automated design method called RiboLogic for such “stand-alone” riboswitches and test it via high-throughput experiments on 2875 molecules using RNA-MaP (RNA on a massively parallel array) technology. These molecules consistently modulate their affinity to the MS2 bacteriophage coat protein upon binding of flavin mononucleotide, tryptophan, theophylline, and microRNA miR-208a, achieving activation ratios of up to 20 and significantly better performance than control designs. By encompassing a wide diversity of stand-alone switches and highly quantitative data, the resulting ribologic-solves experimental data set provides a rich resource for further improvement of riboswitch models and design methods.

Neuropsychiatric disorders are the leading cause of disability worldwide and there is no gold standard currently available for the measurement of mental health. This issue is exacerbated by the fact that the information physicians use to diagnose these disorders is episodic and often subjective. Current methods to monitor mental health involve the use of subjective DSM-5 guidelines, and advances in EEG and video monitoring technologies have not been widely adopted due to invasiveness and inconvenience. Wearable technologies have surfaced as a ubiquitous and unobtrusive method for providing continuous, quantitative data about a patient. Here, we introduce PRISM — Passive, Real-time Information for Sensing Mental Health. This platform integrates motion, light and heart rate data from a smart watch application with user interactions and text insights from a web application. We have demonstrated a proof of concept by collecting preliminary data through a pilot study of 13 subjects. We have engineered appropriate features and applied both unsupervised and supervised learning to develop models that can recapitulate user-reported ratings of their emotional state. This demonstrates that the data has the potential to be useful for evaluating mental health. This platform will allow us to leverage continuous streams of passive data for early and accurate diagnosis as well as constant monitoring of patients suffering from mental disorders.