A DNA unwinding equilibrium serves as a checkpoint for CRISPR-Cas12a target discrimination.

CRISPR-associated proteins such as Cas9 and Cas12a are programable RNA-guided nucleases that have emerged as powerful tools for genome manipulation and molecular diagnostics. However, these enzymes are prone to cleaving off-target sequences that contain mismatches between the RNA guide and DNA protospacer. In comparison to Cas9, Cas12a has demonstrated distinct sensitivity to protospacer-adjacent-motif (PAM) distal mismatches, and the molecular basis of Cas12a's enhanced target discrimination is of great interest. In this study, we investigated the mechanism of Cas12a target recognition using a combination of site-directed spin labeling, fluorescent spectroscopy, and enzyme kinetics. With a fully matched RNA guide, the data revealed an inherent equilibrium between a DNA unwound state and a DNA-paired duplex-like state. Experiments with off-target RNA guides and pre-nicked DNA substrates identified the PAM-distal DNA unwinding equilibrium as a mismatch sensing checkpoint prior to the first step of DNA cleavage. The finding sheds light on the distinct targeting mechanism of Cas12a and may better inform CRISPR based biotechnology developments.

Generation of tamoxifen-inducible Tfap2b-CreERT2 mice using CRISPR-Cas9.

Tfap2b, a pivotal transcription factor, plays critical roles within neural crest cells and their derived lineage. To unravel the intricate lineage dynamics and contribution of these Tfap2b+ cells during craniofacial development, we established a Tfap2b-CreERT2 knock-in transgenic mouse line using the CRISPR-Cas9-mediated homologous direct repair. By breeding with tdTomato reporter mice and initiating Cre activity through tamoxifen induction at distinct developmental time points, we show the Tfap2b lineage within the key neural crest-derived domains, such as the facial mesenchyme, midbrain, cerebellum, spinal cord, and limbs. Notably, the migratory neurons stemming from the dorsal root ganglia are visible subsequent to Cre activity initiated at E8.5. Intriguingly, Tfap2b+ cells, serving as the progenitors for limb development, show activity predominantly commencing at E10.5. Across the mouse craniofacial landscape, Tfap2b exhibits a widespread presence throughout the facial organs. Here we validate its role as a marker of progenitors in tooth development and have confirmed that this process initiates from E12.5. Our study not only validates the Tfap2b-CreERT2 transgenic line, but also provides a powerful tool for lineage tracing and genetic targeting of Tfap2b-expressing cells and their progenitor in a temporally and spatially regulated manner during the intricate process of development and organogenesis.

CRISPR-Cas9 Activities with Truncated 16-Nucleotide RNA Guides Are Tuned by Target Duplex Stability Beyond the RNA/DNA Hybrid.

CRISPR-Cas9 has been adapted as a readily programmable genome manipulation agent, and continuing technological advances rely on an in-depth mechanistic understanding of Cas9 target discrimination. Cas9 interrogates a target by unwinding the DNA duplex to form an R-loop, where the RNA guide hybridizes with one of the DNA strands. It has been shown that RNA guides shorter than the normal length of 20-nucleotide (-nt) support Cas9 cleavage activity by enabling partial unwinding beyond the RNA/DNA hybrid. To investigate whether DNA segment beyond the RNA/DNA hybrid can impact Cas9 target discrimination with truncated guides, Cas9 double-stranded DNA cleavage rates (kcat) were measured with 16-nt guides on targets with varying sequences at +17 to +20 positions distal to the protospacer-adjacent-motif (PAM). The data reveal a log-linear inverse correlation between kcat and the PAM+(17-20) DNA duplex dissociation free energy (ΔGNN(17-20)0), with sequences having smaller ΔGNN(17-20)0 showing faster cleavage and a higher degree of unwinding. The results indicate that, with a 16-nt guide, "peripheral" DNA sequences beyond the RNA/DNA hybrid contribute to target discrimination by tuning the cleavage reaction transition state through the modulation of PAM-distal unwinding. The finding provides mechanistic insights for the further development of strategies that use RNA guide truncation to enhance Cas9 specificity.

Coordinated Actions of Cas9 HNH and RuvC Nuclease Domains Are Regulated by the Bridge Helix and the Target DNA Sequence.

CRISPR-Cas systems are RNA-guided nucleases that provide adaptive immune protection in bacteria and archaea against intruding genomic materials. Cas9, a type-II CRISPR effector protein, is widely used for gene editing applications since a single guide RNA can direct Cas9 to cleave specific genomic targets. The conformational changes associated with RNA/DNA binding are being modulated to develop Cas9 variants with reduced off-target cleavage. Previously, we showed that proline substitutions in the arginine-rich bridge helix (BH) of Streptococcus pyogenes Cas9 (SpyCas9-L64P-K65P, SpyCas92Pro) improve target DNA cleavage selectivity. In this study, we establish that kinetic analysis of the cleavage of supercoiled plasmid substrates provides a facile means to analyze the use of two parallel routes for DNA linearization by SpyCas9: (i) nicking by HNH followed by RuvC cleavage (the TS (target strand) pathway) and (ii) nicking by RuvC followed by HNH cleavage (the NTS (nontarget strand) pathway). BH substitutions and DNA mismatches alter the individual rate constants, resulting in changes in the relative use of the two pathways and the production of nicked and linear species within a given pathway. The results reveal coordinated actions between HNH and RuvC to linearize DNA, which is modulated by the integrity of the BH and the position of the mismatch in the substrate, with each condition producing distinct conformational energy landscapes as observed by molecular dynamics simulations. Overall, our results indicate that BH interactions with RNA/DNA enable target DNA discrimination through the differential use of the parallel sequential pathways driven by HNH/RuvC coordination.

Publisher Correction: Single-DNA electron spin resonance spectroscopy in aqueous solutions.

In the version of this paper originally published online, the ORCID ID for Peter Z. Qin was incorrectly assigned to Zhuoyang Qin. In addition, the ORCID for Fazhan Shi was omitted. These errors have been corrected in the print, PDF, and HTML versions of the paper.

Single-DNA electron spin resonance spectroscopy in aqueous solutions.

Magnetic resonance spectroscopy of single biomolecules under near-physiological conditions could substantially advance understanding of their biological function, but this approach remains very challenging. Here we used nitrogen-vacancy centers in diamonds to detect electron spin resonance spectra of individual, tethered DNA duplexes labeled with a nitroxide spin label in aqueous buffer solutions at ambient temperatures. This work paves the way for magnetic resonance studies on single biomolecules and their intermolecular interactions in native-like environments.

Bridge Helix of Cas9 Modulates Target DNA Cleavage and Mismatch Tolerance.

CRISPR-Cas systems are RNA-guided nucleases that provide adaptive immune protection for bacteria and archaea against intruding genomic materials. The programmable nature of CRISPR-targeting mechanisms has enabled their adaptation as powerful genome engineering tools. Cas9, a type II CRISPR effector protein, has been widely used for gene-editing applications owing to the fact that a single-guide RNA can direct Cas9 to cleave desired genomic targets. An understanding of the role of different domains of the protein and guide RNA-induced conformational changes of Cas9 in selecting target DNA has been and continues to enable development of Cas9 variants with reduced off-targeting effects. It has been previously established that an arginine-rich bridge helix (BH) present in Cas9 is critical for its activity. In the present study, we show that two proline substitutions within a loop region of the BH of Streptococcus pyogenes Cas9 impair the DNA cleavage activity by accumulating nicked products and reducing target DNA linearization. This in turn imparts a higher selectivity in DNA targeting. We discuss the probable mechanisms by which the BH-loop contributes to target DNA recognition.

Experimental Validation of the ALLNOX Program for Studying Protein-Nucleic Acid Complexes.

Measurement of distances between spectroscopic labels (e.g., spin labels, fluorophores) attached to specific sites of biomolecules is an important method for studying biomolecular complexes. ALLNOX (Addition of Labels and Linkers) has been developed as a program to model interlabel distances based on an input macromolecule structure. Here, we report validation of ALLNOX using measured distances between nitroxide spin labels attached to specific sites of a protein-DNA complex. The results demonstrate that ALLNOX predicts average interspin distances that matched with values measured with pairs of labels attached at the protein and/or DNA. This establishes a solid foundation for using spin labeling in conjunction with ALLNOX to investigate complexes without high-resolution structures. With its high degree of flexibility for the label or the target biomolecule, ALLNOX provides a useful tool for investigating the structure-function relationship in a large variety of biological molecules.

A Platinum(II)-based Photosensitive Tripod as an Effective Photodynamic Anticancer Agent through DNA Damage.

Herein, two photosensitive platinum(II)-based tripods were designed and synthesized. Notably, complex 1 ({[Pt(dien)]3 L}(NO3 )6 , L=tri(4-pyridylphenyl)amine and dien=diethylenetriamine), which mainly accumulated in the cell nucleus, exhibited very low cytotoxicity in the absence of light irradiation, but displayed a remarkable increase in cytotoxicity upon visible light irradiation. Mechanistic investigations revealed that the tripod interacted with DNA in the nucleus, induced ROS generation upon light irradiation, and consequently elicited rapid DNA damage response; thereby triggering cancer cell apoptosis.

Conformations of Human Telomeric G-Quadruplex Studied Using a Nucleotide-Independent Nitroxide Label.

Guanine-rich oligonucleotides can form a unique G-quadruplex (GQ) structure with stacking units of four guanine bases organized in a plane through Hoogsteen bonding. GQ structures have been detected in vivo and shown to exert their roles in maintaining genome integrity and regulating gene expression. Understanding GQ conformation is important for understanding its inherent biological role and for devising strategies to control and manipulate functions based on targeting GQ. Although a number of biophysical methods have been used to investigate structure and dynamics of GQs, our understanding is far from complete. As such, this work explores the use of the site-directed spin labeling technique, complemented by molecular dynamics simulations, for investigating GQ conformations. A nucleotide-independent nitroxide label (R5), which has been previously applied for probing conformations of noncoding RNA and DNA duplexes, is attached to multiple sites in a 22-nucleotide DNA strand derived from the human telomeric sequence (hTel-22) that is known to form GQ. The R5 labels are shown to minimally impact GQ folding, and inter-R5 distances measured using double electron-electron resonance spectroscopy are shown to adequately distinguish the different topological conformations of hTel-22 and report variations in their occupancies in response to changes of the environment variables such as salt, crowding agent, and small molecule ligand. The work demonstrates that the R5 label is able to probe GQ conformation and establishes the base for using R5 to study more complex sequences, such as those that may potentially form multimeric GQs in long telomeric repeats.

A Nitroxide-Tagged Platinum(II) Complex Enables the Identification of DNA G-Quadruplex Binding Mode.

We reported a novel strategy for investigating small molecule binding to G-quadruplexes (GQs). A newly synthesized dinuclear platinum(II) complex (Pt2L) containing a nitroxide radical was shown to selectively bind a GQ-forming sequence derived from human telomere (hTel). Using the nitroxide moiety as a spin label, electron paramagnetic resonance (EPR) spectroscopy was carried out to investigate binding between Pt2L and hTel GQ. Measurements indicated that two molecules of Pt2L bind with one molecule of hTel GQ. The inter-spin distance measured between the two bound Pt2L, together with molecular docking analyses, revealed that Pt2L predominately binds to the neighboring narrow and wide grooves of the G-tetrads as hTel adopts the antiparallel conformation. The design and synthesis of nitroxide tagged GQ binders, and the use of spin-labeling/EPR to investigate their interactions with GQs, will aid the development of small molecules for manipulating GQs involved in crucial biological processes.

Experimental mapping of DNA duplex shape enabled by global lineshape analyses of a nucleotide-independent nitroxide probe.

Sequence-dependent variation in structure and dynamics of a DNA duplex, collectively referred to as 'DNA shape', critically impacts interactions between DNA and proteins. Here, a method based on the technique of site-directed spin labeling was developed to experimentally map shapes of two DNA duplexes that contain response elements of the p53 tumor suppressor. An R5a nitroxide spin label, which was covalently attached at a specific phosphate group, was scanned consecutively through the DNA duplex. X-band continuous-wave electron paramagnetic resonance spectroscopy was used to monitor rotational motions of R5a, which report on DNA structure and dynamics at the labeling site. An approach based on Pearson's coefficient analysis was developed to collectively examine the degree of similarity among the ensemble of R5a spectra. The resulting Pearson's coefficients were used to generate maps representing variation of R5a mobility along the DNA duplex. The R5a mobility maps were found to correlate with maps of certain DNA helical parameters, and were capable of revealing similarity and deviation in the shape of the two closely related DNA duplexes. Collectively, the R5a probe and the Pearson's coefficient-based lineshape analysis scheme yielded a generalizable method for examining sequence-dependent DNA shapes.

Conformations of p53 response elements in solution deduced using site-directed spin labeling and Monte Carlo sampling.

The tumor suppressor protein p53 regulates numerous signaling pathways by specifically recognizing diverse p53 response elements (REs). Understanding the mechanisms of p53-DNA interaction requires structural information on p53 REs. However, such information is limited as a 3D structure of any RE in the unbound form is not available yet. Here, site-directed spin labeling was used to probe the solution structures of REs involved in p53 regulation of the p21 and Bax genes. Multiple nanometer distances in the p21-RE and BAX-RE, measured using a nucleotide-independent nitroxide probe and double-electron-electron-resonance spectroscopy, were used to derive molecular models of unbound REs from pools of all-atom structures generated by Monte-Carlo simulations, thus enabling analyses to reveal sequence-dependent DNA shape features of unbound REs in solution. The data revealed distinct RE conformational changes on binding to the p53 core domain, and support the hypothesis that sequence-dependent properties encoded in REs are exploited by p53 to achieve the energetically most favorable mode of deformation, consequently enhancing binding specificity. This work reveals mechanisms of p53-DNA recognition, and establishes a new experimental/computational approach for studying DNA shape in solution that has far-reaching implications for studying protein-DNA interactions.

Anomalously Rapid Hydration Water Diffusion Dynamics Near DNA Surfaces.

The emerging Overhauser effect dynamic nuclear polarization (ODNP) technique measures the translational mobility of water within the vicinity (5-15 Å) of preselected sites. The work presented here expands the capabilities of the ODNP technique and illuminates an important, previously unseen, property of the translational diffusion dynamics of water at the surface of DNA duplexes. We attach nitroxide radicals (i.e., spin labels) to multiple phosphate backbone positions of DNA duplexes, allowing ODNP to measure the hydration dynamics at select positions along the DNA surface. With a novel approach to ODNP analysis, we isolate the contributions of water molecules at these sites that undergo free translational diffusion from water molecules that either loosely bind to or exchange protons with the DNA. The results reveal that a significant population of water in a localized volume adjacent to the DNA surface exhibits fast, bulk-like characteristics and moves unusually rapidly compared to water found in similar probe volumes near protein and membrane surfaces. Control studies show that the observation of these characteristics are upheld even when the DNA duplex is tethered to streptavidin or the mobility of the nitroxides is altered. This implies that, as compared to protein or lipid surfaces, it is an intrinsic feature of the DNA duplex surface that it interacts only weakly with a significant fraction of the surface hydration water network. The displacement of this translationally mobile water is energetically less costly than that of more strongly bound water by up to several kBT and thus can lower the activation barrier for interactions involving the DNA surface.

Structure of p53 binding to the BAX response element reveals DNA unwinding and compression to accommodate base-pair insertion.

The p53 core domain binds to response elements (REs) that contain two continuous half-sites as a cooperative tetramer, but how p53 recognizes discontinuous REs is not well understood. Here we describe the crystal structure of the p53 core domain bound to a naturally occurring RE located at the promoter of the Bcl-2-associated X protein (BAX) gene, which contains a one base-pair insertion between the two half-sites. Surprisingly, p53 forms a tetramer on the BAX-RE that is nearly identical to what has been reported on other REs with a 0-bp spacer. Each p53 dimer of the tetramer binds in register to a half-site and maintains the same protein-DNA interactions as previously observed, and the two dimers retain all the protein-protein contacts without undergoing rotation or translation. To accommodate the additional base pair, the DNA is deformed and partially disordered around the spacer region, resulting in an apparent unwinding and compression, such that the interactions between the dimers are maintained. Furthermore, DNA deformation within the p53-bound BAX-RE is confirmed in solution by site-directed spin labeling measurements. Our results provide a structural insight into the mechanism by which p53 binds to discontinuous sites with one base-pair spacer.

A single-stranded junction modulates nanosecond motional ordering of the substrate recognition duplex of a group I ribozyme.

Rigid spinning: Site-directed spin-labeling studies using a rigid nitroxide spin label (Ç) reveal that both length and sequence of a single-stranded junction (J1/2) modulate nanosecond motional ordering of the substrate-recognition duplex (P1) of the 120 kD group I ribozyme. The studies demonstrate an approach for experimental measurements of nanosecond dynamics in high-molecular-weight RNA complexes.

A DNA Unwinding Equilibrium Serves as a Checkpoint for CRISPR-Cas12a Target Discrimination.

CRISPR-associated proteins such as Cas9 and Cas12a are programable RNA-guided nucleases that have emerged as powerful tools for genome manipulation and molecular diagnostics. However, these enzymes are prone to cleaving off-target sequences that contain mismatches between the RNA guide and DNA protospacer. In comparison to Cas9, Cas12a has demonstrated distinct sensitivity to protospacer-adjacent-motif (PAM) distal mismatches, and the molecular basis of Cas12a's enhanced target discrimination is of great interest. In this study, we investigated the mechanism of Cas12a target recognition using a combination of site-directed spin labeling, fluorescent spectroscopy, and enzyme kinetics. With a fully matched RNA guide, the data revealed an inherent equilibrium between a DNA unwound state and a DNA-paired duplex-like state. Experiments with off-target RNA guides and pre-nicked DNA substrates identified the PAM-distal DNA unwinding equilibrium as a mismatch sensing checkpoint prior to the first step of DNA cleavage. The data sheds light on the distinct targeting mechanism of Cas12a and may better inform CRISPR based biotechnology developments.

Differential Divalent Metal Binding by SpyCas9's RuvC Active Site Contributes to Nonspecific DNA Cleavage.

To protect against mobile genetic elements (MGEs), some bacteria and archaea have clustered regularly interspaced short palindromic repeats-CRISPR associated (CRISPR-Cas) adaptive immune systems. CRISPR RNAs (crRNAs) bound to Cas nucleases hybridize to MGEs based on sequence complementarity to guide the nucleases to cleave the MGEs. This programmable DNA cleavage has been harnessed for gene editing. Safety concerns include off-target and guide RNA (gRNA)-free DNA cleavages, both of which are observed in the Cas nuclease commonly used for gene editing, Streptococcus pyogenes Cas9 (SpyCas9). We developed a SpyCas9 variant (SpyCas9H982A) devoid of gRNA-free DNA cleavage activity that is more selective for on-target cleavage. The H982A substitution in the metal-dependent RuvC active site reduces Mn2+-dependent gRNA-free DNA cleavage by ∼167-fold. Mechanistic molecular dynamics analysis shows that Mn2+, but not Mg2+, produces a gRNA-free DNA cleavage competent state that is disrupted by the H982A substitution. Our study demonstrates the feasibility of modulating cation:protein interactions to engineer safer gene editing tools.

Global structure of a three-way junction in a phi29 packaging RNA dimer determined using site-directed spin labeling.

The condensation of bacteriophage phi29 genomic DNA into its preformed procapsid requires the DNA packaging motor, which is the strongest known biological motor. The packaging motor is an intricate ring-shaped protein/RNA complex, and its function requires an RNA component called packaging RNA (pRNA). Current structural information on pRNA is limited, which hinders studies of motor function. Here, we used site-directed spin labeling to map the conformation of a pRNA three-way junction that bridges binding sites for the motor ATPase and the procapsid. The studies were carried out on a pRNA dimer, which is the simplest ring-shaped pRNA complex and serves as a functional intermediate during motor assembly. Using a nucleotide-independent labeling scheme, stable nitroxide radicals were attached to eight specific pRNA sites without perturbing RNA folding and dimer formation, and a total of 17 internitroxide distances spanning the three-way junction were measured using Double Electron-Electron Resonance spectroscopy. The measured distances, together with steric chemical constraints, were used to select 3662 viable three-way junction models from a pool of 65 billion. The results reveal a similar conformation among the viable models, with two of the helices (H(T) and H(L)) adopting an acute bend. This is in contrast to a recently reported pRNA tetramer crystal structure, in which H(T) and H(L) stack onto each other linearly. The studies establish a new method for mapping global structures of complex RNA molecules, and provide information on pRNA conformation that aids investigations of phi29 packaging motor and developments of pRNA-based nanomedicine and nanomaterial.

Conformational distributions at the N-peptide/boxB RNA interface studied using site-directed spin labeling.

In bacteriophage λ, interactions between a 22-amino acid peptide (called the N-peptide) and a stem-loop RNA element (called boxB) play a critical role in transcription anti-termination. The N-peptide/boxB complex has been extensively studied, and serves as a paradigm for understanding mechanisms of protein/RNA recognition. Particularly, ultrafast spectroscopy techniques have been applied to monitor picosecond fluorescence decay behaviors of 2-aminopurines embedded at various positions of the boxB RNA. The studies have led to a model in which the bound N-peptide exists in dynamic equilibrium between two states, with peptide C-terminal fragment either stacking on (i.e., the stacked state) or peeling away from (i.e., the unstacked state) the RNA loop. The function of the N-peptide/boxB complex seems to correlate with the fraction of the stacked state. Here, the N-peptide/boxB system is studied using the site-directed spin labeling technique, in which X-band electron paramagnetic resonance spectroscopy is applied to monitor nanosecond rotational behaviors of stable nitroxide radicals covalently attached to different positions of the N-peptide. The data reveal that in the nanosecond regime the C-terminal fragment of bound N-peptide adopts multiple discrete conformations within the complex. The characteristics of these conformations are consistent with the proposed stacked and unstacked states, and their distributions vary upon mutations within the N-peptide. These results suggest that the dynamic two-state model remains valid in the nanosecond regime, and represents a unique mode of function in the N-peptide/boxB RNA complex. It also demonstrates a connection between picosecond and nanosecond dynamics in a biological complex.