CRISPR systems have been broadly adopted for basic science, biotechnology, and gene and cell therapy. In some cases, these bacterial nucleases have demonstrated off-target activity. This creates a potential hazard for therapeutic applications and could confound results in biological research. Therefore, improving the precision of these nucleases is of broad interest. Here we show that engineering a hairpin secondary structure onto the spacer region of single guide RNAs (hp-sgRNAs) can increase specificity by several orders of magnitude when combined with various CRISPR effectors. We first demonstrate that designed hp-sgRNAs can tune the activity of a transactivator based on Cas9 from S. pyogenes (SpCas9). We then show that hp-sgRNAs increase the specificity of gene editing using five different Cas9 or Cas12a variants. Our results demonstrate that RNA secondary structure is a fundamental parameter that can tune the activity of diverse CRISPR systems.

CRISPR-associated endonuclease Cas9 cuts DNA at variable target sites designated by a Cas9-bound RNA molecule. Cas9's ability to be directed by single ‘guide RNA’ molecules to target nearly any sequence has been recently exploited for a number of emerging biological and medical applications. Therefore, understanding the nature of Cas9's off-target activity is of paramount importance for its practical use. Using atomic force microscopy (AFM), we directly resolve individual Cas9 and nuclease-inactive dCas9 proteins as they bind along engineered DNA substrates. High-resolution imaging allows us to determine their relative propensities to bind with different guide RNA variants to targeted or off-target sequences. Mapping the structural properties of Cas9 and dCas9 to their respective binding sites reveals a progressive conformational transformation at DNA sites with increasing sequence similarity to its target. With kinetic Monte Carlo (KMC) simulations, these results provide evidence of a ‘conformational gating’ mechanism driven by the interactions between the guide RNA and the 14th–17th nucleotide region of the targeted DNA, the stabilities of which we find correlate significantly with reported off-target cleavage rates. KMC simulations also reveal potential methodologies to engineer guide RNA sequences with improved specificity by considering the invasion of guide RNAs into targeted DNA duplex.

The nanoscale arrangement of the DNA probe molecules on sensor surfaces has a profound impact on molecular recognition and signaling reactions on DNA biosensors and microarrays. Using electrochemical atomic force microscopy, we have directly determined the nanoscale spatial distribution of thiolated DNA that are attached to gold via different methods. We discovered significant heterogeneity in the probe density and limited stability for DNA monolayers prepared by the backfilling method, that is, first exposing the surface to thiolated DNA then "backfilling" with a passivating alkanethiol. On the other hand, the monolayers prepared by "inserting" thiolated DNA into a preformed alkanethiol monolayer lead to a more uniformly distributed layer of DNA. With high-resolution images of single DNA molecules on the surface, we have introduced spatial statistics to characterize the nanoscale arrangement of DNA probes. The randomness of the spatial distribution has been characterized. By determining the local densities surrounding individual molecules, we observed subpopulations of probes with dramatically different levels of "probe crowding". We anticipate that the novel application of spatial statistics to DNA monolayers can enable a framework to understand heterogeneity in probe spatial distributions, interprobe interactions, and ultimately probe activity on sensor surfaces.

Surfaces that can actively regulate binding affinities or catalytic properties in response to external stimuli are a powerful means to probe and control the dynamic interactions between the cell and its microenvironment. Active surfaces also enable novel functionalities in biosensors and biomolecular separation technologies. Although electrical stimuli are often appealing due to their speed and localization, the operation of these electrically activated surfaces has mostly been characterized with techniques averaging over many molecules. Without a molecular-scale understanding of how biomolecules respond to electric fields, achieving the ultimate detection sensitivity or localized biological perturbation with the ultimate resolution would be difficult. Using electrochemical atomic force microscopy, we are able to follow the conformational changes of individual, short DNA molecules tethered to a gold electrode in response to an applied potential. Our study reveals conformations and dynamics that are difficult to infer from ensemble measurements: defects in the self-assembled monolayer (SAM) significantly perturb conformations and adsorption/desorption kinetics of surface-tethered DNA; on the other hand, the SAM may be actively molded by the DNA at different potentials. These results underscore the importance of characterizing the systems at the relevant length scale in the development of electrically switchable biofunctional surfaces.

Here we describe a novel surface that enables direct visualization of the hybridization of single DNA molecules with an unprecedented resolution using atomic force microscopy. The surface consists of single-stranded DNA probes that are covalently anchored to a self-assembled monolayer. The surface satisfies the contradictory requirements for high-resolution imaging and hybridization by switching the DNA-surface interaction between a strong state and a weak state. Our approach opens up unique opportunities in elucidating hybridization at the molecular scale.

Despite the variety of nucleic acid sensors developed, we still do not have definite answers to some questions that are important to the molecular binding and, ultimately, the sensitivity and reliability of the sensors. How do the DNA probes distribute on the surface at the nanoscale? As the functionalized surfaces are highly heterogeneous, how are the conformations affected when the probe molecules interact with defects? How do DNA molecules respond to electric fields on the surface, which are applied in a variety of detection methods? With in situ electrochemical atomic force microscopy and careful tailoring of nanoscale surface interactions, we are able to observe the nanoscale conformations of individual DNA molecules on a model biosensor surface: thiolated DNA on a gold surface passivated with a hydroxyl-terminated alkanethiol self-assembled monolayer. We find that under applied electric fields, the conformations are highly sensitive to the choice of the alkanethiol molecule. Depending on the monolayer and the nature of the defects, the DNA molecules may either adopt a highly linear or a highly curved conformation. These unusual structures are difficult to observe through existing "ensemble" characterizations of nucleic acid sensors. These findings provide a step toward correlating target-binding affinity, selectivity, and kinetics to the nanoscale chemical structure of and around the probe molecules in practical nucleic acid devices.

Here we report a method to assemble nanoscale DNA structures with single-molecule precision. This assembly is accomplished by performing nanografting in the presence of short, thiolated DNA strands that have been diluted by a positively charged alkanethiol. The expected number of DNA molecules per patch can be modulated by the application of an electric potential to the surface during patterning. Our ability to position individual DNA within a controlled nanoscale environment and observe these molecules in situ will allow us to understand and potentially decouple the heterogeneity caused by the local environment from the intrinsic properties in single-molecule biophysical measurements. Additionally, our approach can potentially be extended to the molecule-by-molecule assembly of larger artificial test structures of nucleic acids or proteins.

To generate nanoscale biochemical patterns for fundamental biophysical studies as well as practical biosensors, there remains a need for a high quality and versatile substrate. We show that chemically synthesized gold microplates on indium tin oxide are an ideal substrate that combines several desirable characteristics, including low cost, single crystallinity, optical transparency, electrical conductivity, and ease in chemical functionalization. We have developed a convenient one-pot method that allows us to synthesize plates of desired dimensions and surface coverage directly on indium tin oxide. We have used electrochemical desorption to strip the capping agents, allowing reliable functionalization with alkanethiol self-assembled monolayers. These plates can serve as nanoscale "lab benches" that allow high-resolution scanning probe lithography, high-resolution imaging, and electrical manipulation. Two applications are demonstrated here: nanoshaved self-assembled monolayers (SAMs) on the single crystalline microplates serve as a high-resolution etching resist; AFM nanografting on the plates generates SAM patterns with tailored terminal chemical functionalities.