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.

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.

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.

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.

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.

Phosphorothioate-Based Site-Specific Labeling of Large RNAs for Structural and Dynamic Studies.

Pulsed electron-electron double resonance (PELDOR) spectroscopy, X-ray scattering interferometry (XSI), and single-molecule Förster resonance energy transfer (smFRET) are molecular rulers that provide inter- or intramolecular pair-wise distance distributions in the nanometer range, thus being ideally suitable for structural and dynamic studies of biomolecules including RNAs. The prerequisite for such applications requires site-specific labeling of biomolecules with spin labels, gold nanoparticles, and fluorescent tags, respectively. Recently, site-specific labeling of large RNAs has been achieved by a combination of transcription of an expanded genetic alphabet containing A-T/G-C base pairs and NaM-TPT3 unnatural base pair (UBP) with post-transcriptional modifications at UBP bases by click chemistry or amine-NHS ester reactions. However, due to the bulky sizes of functional groups or labeling probes used, such strategies might cause structural perturbation and decrease the accuracy of distance measurements. Here, we synthesize an α-thiophosphorylated variant of rTPT3TP (rTPT3αS), which allows for post-transcriptional site-specific labeling of large RNAs at the internal α-phosphate backbone via maleimide-modified probes. Subsequent PELDOR, XSI, and smFRET measurements result in narrower distance distributions than labeling at the TPT3 base. The presented strategy provides a new route to empower the molecular rulers for structural and dynamic studies of large RNA and its complex.

Site-Specific Labeling Reveals Cas9 Induces Partial Unwinding Without RNA/DNA Pairing in Sequences Distal to the PAM.

CRISPR-Cas9 is an RNA-guided nuclease that has been widely adapted for genome engineering. A key determinant in Cas9 target selection is DNA duplex unwinding to form an R-loop, in which the single-stranded RNA guide hybridizes with one of the DNA strands. To advance understanding on DNA unwinding by Cas9, we combined two types of spectroscopic label, 2-aminopurine and nitroxide spin-label, to investigate unwinding at a specific DNA base pair induced by Streptococcus pyogenes Cas9. Data obtained with RNA guide lengths varying from 13 to 20 nucleotide revealed that the DNA segment distal to the protospacer adjacent motif can adopt a "partial unwinding" state, in which a mixture of DNA-paired and DNA-unwound populations exist in equilibrium. Significant unwinding can occur at positions not supported by RNA/DNA pairing, and the degree of unwinding depends on RNA guide length and modulates DNA cleavage activity. The results shed light on Cas9 target selection and may inform developments of genome-engineering strategies.

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.

The bridge helix of Cas12a imparts selectivity in cis-DNA cleavage and regulates trans-DNA cleavage.

Cas12a is an RNA-guided DNA endonuclease of the type V-A CRISPR-Cas system that has evolved convergently with the type II Cas9 protein. We previously showed that proline substitutions in the bridge helix (BH) impart target DNA cleavage selectivity in Streptococcus pyogenes (Spy) Cas9. Here, we examined a BH variant of Cas12a from Francisella novicida (FnoCas12aKD2P ) to test mechanistic conservation. Our results show that for RNA-guided DNA cleavage (cis-activity), FnoCas12aKD2P accumulates nicked products while cleaving supercoiled DNA substrates with mismatches, with certain mismatch positions being more detrimental for linearization. FnoCas12aKD2P also possess reduced trans-single-stranded DNA cleavage activity. These results implicate the BH in substrate selectivity in both cis- and trans-cleavages and show its conserved role in target discrimination among Cas nucleases.

Posttranscriptional site-directed spin labeling of large RNAs with an unnatural base pair system under non-denaturing conditions.

Site-directed spin labeling (SDSL) of large RNAs for electron paramagnetic resonance (EPR) spectroscopy has remained challenging to date. We here demonstrate an efficient and generally applicable posttranscriptional SDSL method for large RNAs using an expanded genetic alphabet containing the NaM-TPT3 unnatural base pair (UBP). An alkyne-modified TPT3 ribonucleotide triphosphate (rTPT3COTP) is synthesized and site-specifically incorporated into large RNAs by in vitro transcription, which allows attachment of the azide-containing nitroxide through click chemistry. We validate this strategy by SDSL of a 419-nucleotide ribonuclease P (RNase P) RNA from Bacillus stearothermophilus under non-denaturing conditions. The effects of site-directed UBP incorporation and subsequent spin labeling on the global structure and function of RNase P are marginal as evaluated by Circular Dichroism spectroscopy, Small Angle X-ray Scattering, Sedimentation Velocity Analytical Ultracentrifugation and enzymatic assay. Continuous-Wave EPR analyses reveal that the labeling reaction is efficient and specific, and Pulsed Electron-Electron Double Resonance measurements yield an inter-spin distance distribution that agrees with the crystal structure. The labeling strategy as presented overcomes the size constraint of RNA labeling, opening new avenues of spin labeling and EPR spectroscopy for investigating the structure and dynamics of large RNAs.

CRISPR-Cas12a Nucleases Bind Flexible DNA Duplexes without RNA/DNA Complementarity.

Cas12a (also known as "Cpf1") is a class 2 type V-A CRISPR-associated nuclease that can cleave double-stranded DNA at specific sites. The Cas12a effector enzyme comprises a single protein and a CRISPR-encoded small RNA (crRNA) and has been used for genome editing and manipulation. Work reported here examined in vitro interactions between the Cas12a effector enzyme and DNA duplexes with varying states of base-pairing between the two strands. The data revealed that in the absence of complementarity between the crRNA guide and the DNA target-strand, Cas12a binds duplexes with unpaired segments. These off-target duplexes were bound at the Cas12a site responsible for RNA-guided double-stranded DNA binding but were not cleaved due to the lack of RNA/DNA hybrid formation. Such promiscuous binding was attributed to increased DNA flexibility induced by the unpaired segment present next to the protospacer-adjacent-motif. The results suggest that target discrimination of Cas12a can be influenced by flexibility of the DNA. As such, in addition to the linear sequence, flexibility and other physical properties of the DNA should be considered in Cas12a-based genome engineering applications.

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.

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.

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.

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.

CRISPR-Cas9 Mediated DNA Unwinding Detected Using Site-Directed Spin Labeling.

The RNA-guided CRISPR-Cas9 nuclease has revolutionized genome engineering, yet its mechanism for DNA target selection is not fully understood. A crucial step in Cas9 target recognition involves unwinding of the DNA duplex to form a three-stranded R-loop structure. Work reported here demonstrates direct detection of Cas9-mediated DNA unwinding by a combination of site-directed spin labeling and molecular dynamics simulations. The results support a model in which the unwound nontarget strand is stabilized by a positively charged patch located between the two nuclease domains of Cas9 and reveal uneven increases in flexibility along the unwound nontarget strand upon scissions of the DNA backbone. This work establishes the synergistic combination of spin-labeling and molecular dynamics to directly monitor Cas9-mediated DNA conformational changes and yields information on the target DNA in different stages of Cas9 function, thus advancing mechanistic understanding of CRISPR-Cas9 and aiding future technological development.

Nucleic Acid-Dependent Conformational Changes in CRISPR-Cas9 Revealed by Site-Directed Spin Labeling.

In a type II clustered regularly interspaced short palindromic repeats (CRISPR) system, RNAs that are encoded at the CRISPR locus complex with the CRISPR-associated (Cas) protein Cas9 to form an RNA-guided nuclease that cleaves double-stranded DNAs at specific sites. In recent years, the CRISPR-Cas9 system has been successfully adapted for genome engineering in a wide range of organisms. Studies have indicated that a series of conformational changes in Cas9, coordinated by the RNA and the target DNA, direct the protein into its active conformation, yet details on these conformational changes, as well as their roles in the mechanism of function of Cas9, remain to be elucidated. Here, nucleic acid-dependent conformational changes in Streptococcus pyogenes Cas9 (SpyCas9) were investigated using the method of site-directed spin labeling (SDSL). Single nitroxide spin labels were attached, one at a time, at one of the two native cysteine residues (Cys80 and Cys574) of SpyCas9, and the spin-labeled proteins were shown to maintain their function. X-band continuous-wave electron paramagnetic resonance spectra of the nitroxide attached at Cys80 revealed conformational changes of SpyCas9 that are consistent with a large-scale domain re-arrangement upon binding to its RNA partner. The results demonstrate the use of SDSL to monitor conformational changes in CRISPR-Cas9, which will provide key information for understanding the mechanism of CRISPR function.

Theranostic TEMPO-functionalized Ru(ii) complexes as photosensitizers and oxidative stress indicators.

New TEMPO-functionalized Ru(ii) polypyridyl complexes were synthesized as efficient theranostic photosensitizers for cancer treatment. Interestingly, due to the presence of a redox sensitive TEMPO moiety, an enhancement in the intracellular fluorescence of TEMPO-functionalized Ru(ii) complexes was observed during photodynamic treatment in both confocal microscopy and flow cytometry. This can be explained by the conversion of the TEMPO radical moiety to diamagnetic non-radical species in cells upon PDT-induced oxidative stress. To the best of our knowledge this is the first ruthenium complex capable of simultaneously inducing and monitoring the oxidative stress. The tethered TEMPO moiety decreased the inherent dark-cytotoxicity and increased the photo-toxicity simultaneously, both of which contributed to the greatly improved photodynamic therapy (PDT) efficacy, ultimately resulting in cancer cell apoptosis. The phototoxicity index value for TEMPO-functionalized Ru(ii) complexes was selective towards cancer cell lines (280.5 for HeLa cells vs. 30.2 for LO2 cells) and ca. 40-fold higher than that for TEMPO-free Ru(ii) analogues (6.7 for HeLa cells). The main contributor for such a greatly enhanced PDT efficacy was the effect of the TEMPO moiety on the cellular uptake and intracellular ROS levels. We therefore demonstrate that the combination of TEMPO with the photosensitizers may be an emerging strategy to develop novel photosensitizer-based theranostic platforms, which can induce and monitor the PDT response simultaneously.

Investigating Functional DNA Grafted on Nanodiamond Surface Using Site-Directed Spin Labeling and Electron Paramagnetic Resonance Spectroscopy.

Nanodiamonds (NDs) are a new and attractive class of materials for sensing and delivery in biological systems. Methods for functionalizing ND surfaces are highly valuable in these applications, yet reported approaches for covalent modification with biological macromolecules are still limited, and characterizing behaviors of ND-tethered biomolecules is difficult. Here we demonstrated the use of copper-free click chemistry to covalently attach DNA strands at ND surfaces. Using site-directed spin labeling and electron paramagnetic resonance spectroscopy, we demonstrated that the tethered DNA strands maintain the ability to undergo repetitive hybridizations and behave similarly to those in solutions, maintaining a large degree of mobility with respect to the ND. The work established a method to prepare and characterize an easily addressable identity tag for NDs. This will open up future applications such as targeted ND delivery and developing sensors for investigating biomolecules.