Chimeric crRNAs with 19 DNA residues in the guide region show the retained DNA cleavage activity of Cas9 with potential to improve the specificity.

We demonstrated that 19 out of 20 RNA residues in the guide region of crRNA can be replaced with DNA residues with high GC-contents. The cellular activity of the chimeric crRNAs to disrupt the target gene was comparable to that of the native crRNA.

Direct observation of DNA target searching and cleavage by CRISPR-Cas12a.

Cas12a (also called Cpf1) is a representative type V-A CRISPR effector RNA-guided DNA endonuclease, which provides an alternative to type II CRISPR-Cas9 for genome editing. Previous studies have revealed that Cas12a has unique features distinct from Cas9, but the detailed mechanisms of target searching and DNA cleavage by Cas12a are still unclear. Here, we directly observe this entire process by using single-molecule fluorescence assays to study Cas12a from Acidaminococcus sp. (AsCas12a). We determine that AsCas12a ribonucleoproteins search for their on-target site by a one-dimensional diffusion along elongated DNA molecules and induce cleavage in the two DNA strands in a well-defined order, beginning with the non-target strand. Furthermore, the protospacer-adjacent motif (PAM) for AsCas12a makes only a limited contribution of DNA unwinding during R-loop formation and shows a negligible role in the process of DNA cleavage, in contrast to the Cas9 PAM.

Dynamics of Proofreading by the E. coli Pol III Replicase.

The αɛθ core of Escherichia coli DNA polymerase III (Pol III) associates with the ß2 sliding clamp to processively synthesize DNA and remove misincorporated nucleotides. The α subunit is the polymerase while ɛ is the 3' to 5' proofreading exonuclease. In contrast to the polymerase activity of Pol III, dynamic features of proofreading are poorly understood. We used single-molecule assays to determine the excision rate and processivity of the ß2-associated Pol III core, and observed that both properties are enhanced by mutational strengthening of the interaction between ɛ and ß2. Thus, the ɛ-ß2 contact is maintained in both the synthesis and proofreading modes. Remarkably, single-molecule real-time fluorescence imaging revealed the dynamics of transfer of primer-template DNA between the polymerase and proofreading sites, showing that it does not involve breaking of the physical interaction between ɛ and ß2.

Light-Induced Fluorescence Modulation of Quantum Dot-Crystal Violet Conjugates: Stochastic Off-On-Off Cycles for Multicolor Patterning and Super-Resolution.

Photoswitching or modulation of quantum dots (QDs) can be promising for many fields that include display, memory, and super-resolution imaging. However, such modulations have mostly relied on photomodulations of conjugated molecules in QD vicinity, which typically require high power of high energy photons at UV. We report a visible light-induced facile modulation route for QD-dye conjugates. QD crystal violets conjugates (QD-CVs) were prepared and the crystal violet (CV) molecules on QD quenched the fluorescence efficiently. The fluorescence of QD-CVs showed a single cycle of emission burst as they go through three stages of (i) initially quenched "off" to (ii) photoactivated "on" as the result of chemical change of CVs induced by photoelectrons from QD and (iii) back to photodarkened "off" by radical-associated reactions. Multicolor on-demand photopatterning was demonstrated using QD-CV solid films. QD-CVs were introduced into cells, and excitation with visible light yielded photomodulation from "off" to "on" and "off" by nearly ten fold. Individual photoluminescence dynamics of QD-CVs was investigated using fluorescence correlation spectroscopy and single QD emission analysis, which revealed temporally stochastic photoactivations and photodarkenings. Exploiting the stochastic fluorescence burst of QD-CVs, simultaneous multicolor super-resolution localizations were demonstrated.

Dynamic control of strand excision during human DNA mismatch repair.

Mismatch repair (MMR) is activated by evolutionarily conserved MutS homologs (MSH) and MutL homologs (MLH/PMS). MSH recognizes mismatched nucleotides and form extremely stable sliding clamps that may be bound by MLH/PMS to ultimately authorize strand-specific excision starting at a distant 3'- or 5'-DNA scission. The mechanical processes associated with a complete MMR reaction remain enigmatic. The purified human (Homo sapien or Hs) 5'-MMR excision reaction requires the HsMSH2-HsMSH6 heterodimer, the 5' → 3' exonuclease HsEXOI, and the single-stranded binding heterotrimer HsRPA. The HsMLH1-HsPMS2 heterodimer substantially influences 5'-MMR excision in cell extracts but is not required in the purified system. Using real-time single-molecule imaging, we show that HsRPA or Escherichia coli EcSSB restricts HsEXOI excision activity on nicked or gapped DNA. HsMSH2-HsMSH6 activates HsEXOI by overcoming HsRPA/EcSSB inhibition and exploits multiple dynamic sliding clamps to increase tract length. Conversely, HsMLH1-HsPMS2 regulates tract length by controlling the number of excision complexes, providing a link to 5' MMR.

Single-molecule views of MutS on mismatched DNA.

Base-pair mismatches that occur during DNA replication or recombination can reduce genetic stability or conversely increase genetic diversity. The genetics and biophysical mechanism of mismatch repair (MMR) has been extensively studied since its discovery nearly 50 years ago. MMR is a strand-specific excision-resynthesis reaction that is initiated by MutS homolog (MSH) binding to the mismatched nucleotides. The MSH mismatch-binding signal is then transmitted to the immediate downstream MutL homolog (MLH/PMS) MMR components and ultimately to a distant strand scission site where excision begins. The mechanism of signal transmission has been controversial for decades. We have utilized single molecule Forster Resonance Energy Transfer (smFRET), Fluorescence Tracking (smFT) and Polarization Total Internal Reflection Fluorescence (smP-TIRF) to examine the interactions and dynamic behaviors of single Thermus aquaticus MutS (TaqMutS) particles on mismatched DNA. We determined that TaqMutS forms an incipient clamp to search for a mismatch in ~1 s intervals by 1-dimensional (1D) thermal fluctuation-driven rotational diffusion while in continuous contact with the helical duplex DNA. When MutS encounters a mismatch it lingers for ~3 s to exchange bound ADP for ATP (ADP→ATP exchange). ATP binding by TaqMutS induces an extremely stable clamp conformation (~10 min) that slides off the mismatch and moves along the adjacent duplex DNA driven simply by 1D thermal diffusion. The ATP-bound sliding clamps rotate freely while in discontinuous contact with the DNA. The visualization of a train of MSH proteins suggests that dissociation of ATP-bound sliding clamps from the mismatch permits multiple mismatch-dependent loading events. These direct observations have provided critical clues into understanding the molecular mechanism of MSH proteins during MMR.

Assessment of radiation damage in single-shot coherent diffraction of DNA molecules by an extreme-ultraviolet free-electron laser.

We have investigated the progress of structural distortions in DNA molecules by single-shot coherent diffraction using extreme-ultraviolet radiation from a free-electron laser. A speckle pattern of DNA molecules was successfully acquired using photons in a single pulse with a 100 fs pulse width. The radiation damage was assessed by a cross correlation, revealing that the first exposure has significantly deformed most of the original structures. Molecules were not completely destroyed by the first single-shot exposure and underwent subsequent distortions through continued exposure, until eventually deforming into a radiation-hard structure.

Single-molecule analysis reveals the kinetics and physiological relevance of MutL-ssDNA binding.

DNA binding by MutL homologs (MLH/PMS) during mismatch repair (MMR) has been considered based on biochemical and genetic studies. Bulk studies with MutL and its yeast homologs Mlh1-Pms1 have suggested an integral role for a single-stranded DNA (ssDNA) binding activity during MMR. We have developed single-molecule Förster resonance energy transfer (smFRET) and a single-molecule DNA flow-extension assays to examine MutL interaction with ssDNA in real time. The smFRET assay allowed us to observe MutL-ssDNA association and dissociation. We determined that MutL-ssDNA binding required ATP and was the greatest at ionic strength below 25 mM (K(D) = 29 nM) while it dramatically decreases above 100 mM (K(D)>2 µM). Single-molecule DNA flow-extension analysis suggests that multiple MutL proteins may bind ssDNA at low ionic strength but this activity does not enhance stability at elevated ionic strengths. These studies are consistent with the conclusion that a stable MutL-ssDNA interaction is unlikely to occur at physiological salt eliminating a number of MMR models. However, the activity may infer some related dynamic DNA transaction process during MMR.