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.

Generation of tamoxifen-inducible Tfap2b-CreER 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-CreER 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.

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.

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.

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.

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.

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 Cas9.

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.

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 (FnoCas12a ) to test mechanistic conservation.

Reduced Expression of VEGF-A in Human Retinal Pigment Epithelial Cells and Human Muller Cells Following CRISPR-Cas9 Ribonucleoprotein-Mediated Gene Disruption.

Purpose: To evaluate the effects of vascular endothelial growth factor-A (VEGF-A) gene editing in human retinal pigment epithelial (RPE) cells and human Muller cells, which are the main VEGF-A producing cells in the eye.

Methods: CRISPR-Cas9 ribonucleoprotein was used to target exon 1 in VEGF-A gene. Lipofectamine CRISPRMAX was used as a vehicle.

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.

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.

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.

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.

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)] L}(NO ) , 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.

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.

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.

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.

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 (PtL) 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 PtL and hTel GQ.

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.

Computer Modeling of Spin Labels: NASNOX, PRONOX, and ALLNOX.

Measurement of distances between spin labels using electron paramagnetic resonance with the double electron-electron resonance (DEER) technique is an important method for evaluation of biomolecular structures. Computation of interlabel distances is of value for experimental planning, validation of known structures using DEER-measured distances, and determination of theoretical data for comparison with experiment. This requires steps of building labels at two defined sites on proteins, DNA or RNA; calculation of allowable label conformers based on rotation around torsional angles; computation of pairwise interlabel distances on a per conformer basis; and calculation of an average distance between the two label ensembles.