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Understanding the molecular mechanisms of CRISPR toolbox using single molecule approaches Digvijay Singh, and Taekjip Ha ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.7b00905 • Publication Date (Web): 02 Feb 2018 Downloaded from http://pubs.acs.org on February 4, 2018

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ACS Chemical Biology

Understanding the molecular mechanisms of the CRISPR toolbox using single molecule approaches Digvijay Singh1 and Taekjip Ha1-4, * 1

Department of Biophysics and Biophysical Chemistry, Johns Hopkins

University School of Medicine, Baltimore, Maryland 21205, USA. 2

Department of Biophysics, Johns Hopkins University, Baltimore, MD 21218, USA.

3

Department of Biomedical Engineering, Johns Hopkins University, Baltimore, Maryland 21205,

USA. 4

Howard Hughes Medical Institute, Baltimore, Maryland 21205, USA.

ACS Paragon Plus Environment

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ABSTRACT Adaptive immunity against foreign genetic elements conferred by the CRISPR systems in microbial species has been repurposed as a revolutionary technology for wide-ranging biological applications - chiefly genome engineering. Biochemical, structural, genetic and genomics studies have revealed important insights into their function and mechanisms, but most ensemble studies cannot observe structural changes of these molecules during their function and are often blind to key reaction intermediates. Here, we review the use of single molecule approaches such as fluorescent particle tracking, FRET, magnetic tweezers and atomic force microscopy imaging in improving our understanding of the CRISPR toolbox.


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INTRODUCTION In certain bacteria and archaea, CRISPR (clustered regularly interspaced short palindromic repeats)–Cas systems form an adaptive defense against attacks by foreign genetic elements such as viruses1. The system functions by storing memory of attacks by acquiring sequences of the foreign genetic elements into host genome in regions known as CRISPR loci2. Short RNA transcript (Guide-RNA) from the CRISPR loci forms a complex with CRISPR protein (CRISPRRNA)3. During future invasions, CRISPR-RNA is directed by the guide-RNA to target foreign genetic elements for its nucleolytic impairment, chiefly by the virtue of base-pairing between the guide-RNA and nucleic-acid sequences (target) in foreign genetic elements4, 5. The region in the target spanning the canonical base-pairing between the guide-RNA and the target is called the protospacer. The single most important requirement of such targeting is that the protospacer be followed by a special motif called PAM (protospacer adjacent motif)5, 6. Sequences in the protospacer closest to PAM are referred to as being PAM-proximal whereas those farthest away from PAM are PAM-distal. Throughout this review, the base pairs in the protospacer that are complementary to the guide-RNA of CRISPR-RNA are referred to as matches whereas the others are called mismatches. nPD (the number of PAM-distal mismatches) and nPP (the number of PAM-proximal mismatches) are used to denote the location and extent of mismatches. Abilities to program a nuclease to induce a cut at a desired genomic site and to program a nucleolytically dead CRISPR fused with a marker or an effector to bind any genomic site has begun to revolutionize biology7, 8. Minimalistic versions of the CRISPR systems are employed for such applications because they provide the modularity and ease of programming. Class II of



CRISPR is one example as it uses a single CRISPR endonuclease in complex with guide-RNA to target DNA9. The most commonly used type is SpCas9 (Cas9 from Streptococcus Pyogenes, also known as SpyCas9), followed by SaCas9 from Staphylococcus Aureus10 and Cpf1 variants; AsCpf1 from Acidaminococcus sp and LbCpf1 from Lachnospiraceae bacterium11. In many other CRISPR systems, multiple CRISPR proteins form a complex with the guide-RNA, known as Cascade, to target DNA and then recruit Cas3 for DNA degradation9, 12, 13. The protospacer length of Cas9 and Cpf1 ranges from 20-24 base-pair (bp) and the PAM is 3-6 bp long. Cascades have ~33 bp protospacer and 2-3 bp PAM. SpCas9 is the most widely used Cas9 and is thus the default Cas9 referenced here unless stated otherwise7, 8. Understanding the molecular details of DNA binding and DNA cleavage of the CRISPR systems will help minimize Cas9/Cpf1’s off-target effects for accurate genome engineering14, 15, improve Cas9/Cpf1’s efficiency, help evaluate chemical regulators for CRISPR16 and engineered CRISPR enzymes17-19. Single molecule (sm) methods are suited for such investigations because they allow us to probe and manipulate individual biological reactions, detect transient interactions and also identify multiple intermediates states 20. Here we review the use of sm approaches that have been employed in the last ~3 years to study the CRISPR systems.

DNA interrogation, target search, and binding as a function of mismatches Sternberg et al6 employed an approach called DNA curtains to visualize the target search process of single Cas9-RNA molecules in real time. They aligned an array of flow-stretched lambda phage DNA (~50 kb) molecules on the surface of a flow cell and added Cas9-RNA molecules. Cas9-RNA’s search for target sequences along the DNA was visualized by simultaneous total internal reflection fluorescence (TIRF) imaging of the DNA (stained with YOYO1) and Cas9-


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RNA molecules (labeled with quantum dots) (Fig. 1A-B). Cas9-RNA sampled the DNA by random collisions off 3D diffusion in solution and did not engage in 1D diffusion along the DNA within the resolution limits of their assay (~250 bp). In contrast, TALEN, another genome engineering enzyme, shows extensive 1D diffusion21, 22. Single molecule imaging also revealed several dynamic features, including long-lived binding only at sites with high sequence matches (Fig. 1C), short duration binding on non-target sites that is unaffected by the ionic strength of solution, and increased transient sampling of DNA regions with increasing density of PAM sequences. The target search process was investigated in live cells by Knight et al23. dCas9 (nucleolytically dead Cas9; D10A/H840A mutations)4 with HaloTag was expressed from a stably integrated locus in NIH 3T3 mouse fibroblasts cells. The HaloTag was labeled in live cells with a photostable and bright cell-permeable fluorophore and imaged under epi-illumination for single particle tracking analysis (Fig. 1D). Single particle trajectories of dCas9-RNA had predominantly two diffusion components. A faster component was dominant when a guide-RNA with almost no matching sequence in the genome was used, and therefore was attributed to target search processes in 3D. A slower or immobile component was dominant for a guide-RNA with matching sequences in the genome. As expected, dCas9-RNA forays into the most densely packed heterochromatin region was far less frequently compared to other nuclear regions with lower diffusivity. Nevertheless, dCas9-RNA was still able to localize to specific sequences in heterochromatin regions. Knight et al, however, could not determine how long it takes for dCas9-RNA to find its target because in mammalian cells, there is currently no method to synchronize the cells with unoccupied target sites. Jones et al24 developed a method to block a target sequence using



transcription factor binding and then expose the target sequence at a defined moment by adding an inducer that causes the transcription factor to dissociate. A subsequent accumulation of single dCas9-RNA molecules to the target site could be imaged in live bacterial cells, and they estimated that it takes about 6 hours for a single dCas9-RNA to find a target. Why is the target search process so slow even for a small bacterial genome? Jones et al attributed the slow search process to dCas9-RNA’s association with nonspecific sites. Although the association with nonspecific sites were transient, about 30 ms lifetime, consistent with in vitro observation reported earlier by Singh et al25, the large number of such sites in the genome slows down the search process. The above studies helped uncover biophysical basis of target search processes of Cas9-RNA on large regions of DNA. To understand the molecular events that lead to the binding of Cas9-RNA to DNA regions comparable in size to the protospacer size, single molecule fluorescence resonance energy transfer (smFRET) was applied. smFRET can observe dynamic interactions between proteins and nucleic acids at a higher spatiotemporal resolution than single molecule tracking20, 26. Typically, a molecule of interest is immobilized to a passivated surface through a specific linkage and its interaction partner molecule is added. Their interaction and the associated changes in molecular conformations can be visualized through FRET between two fluorophores attached to the molecules (Fig. 2A-B). Singh et al25, 27 employed smFRET to study the interrogation of donor labeled DNA targets by an acceptor labeled Cas9-RNA. The donor was attached very close to, but just upstream of PAM so that smFRET restricts the detection window of interrogation events to within ~10-15 bp of PAM, and as a result, even very transient binding events lasting less than 0.2 s could be confidently detected (Fig. 2C). Cas9-RNA binding was very stable (bound state lifetime tavg > 1 hour) if the number of PAM-distal mismatches nPD is


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less than 12 but tavg dropped to ~0.1-15 s with nPD ≥12. PAM-proximal mismatches are far more deleterious of stable binding, with tavg dropping precipitously even with two PAM-proximal mismatches (nPP=2) (Fig. 2D). DNA targets that precluded stable binding due to mismatches showed reversible binding events with mid FRET and high FRET (Fig. 2C). The lifetime of the high FRET state follows a pattern of mismatch-dependence similar to that of τavg, but mid FRET lifetime was short (< 0.2 s) for all DNA targets (Fig. 2D). The bimolecular association rate constant kon was sequence-independent (Fig. 2D). These observations further support the bimodal binding behavior proposed in earlier studies6. Cas9-RNA first binds any DNA sequence (even those without PAM) for PAM surveillance, giving rise to the mid FRET species. It dissociates within less than a second if PAM is not detected. Upon PAM detection, RNA-DNA heteroduplex formation ensues resulting in a longerlived high FRET event. If the number of PAM-proximal matches exceeds 9, the bound complex becomes ultrastable. Singh et al performed similar experiments with three major Cpf1 orthologues (AsCpf1, LbCpf1 and FnCpf1)11, 28-30 and Engineered Cas9 (EngCas9; eCas918 and Cas9-HF117). EngCas9s were highly similar to WT Cas9 in their sequence-dependent binding properties that a similar binding stability requires one additional bp match. In contrast, Cpf1 was far more binding specific than Cas9, requiring 17 bp PAM-proximal matches for ultrastable binding, compared to 9 bp for Cas9, and was also more sensitive to PAM-proximal mismatches30. Nevertheless, all of the Cpf1 and EngCas9 appeared to employ the bimodal mechanism, suggesting a generalizable target search and recognition mechanism.



Boyle et al29 employed a high throughput ensemble biophysical method to assess the effects of mismatches on the association and dissociation kinetics of Cas9. These methods use the Illumina sequencing platform to sequence a large number of DNA clusters, followed by profiling of their interaction with Cas9 via fluorescence imaging. After clusters of DNA molecules were sequenced and their spatial coordinates recorded, fluorescently labeled Cas9-RNA was added (or rinsed away) to observe their association (or dissociation) via changes in fluorescence intensities of each cluster (Fig. 2E). Because the DNA sequence in each cluster is known, this approach can investigate the combinatorial effects of mismatches and PAM mutations in a high throughput manner. They reported that the PAM and PAM-proximal seed region (~8-10 bp) determine the rate of association, in apparent conflict with the sequence-independent initial association deduced from smFRET measurements25 . But this discrepancy can be reconciled by the fact that Boyle et al’s ensemble measurement cannot detect transient binding events that occur even without PAM or PAM-proximal sequence matches.

The Cascade counterparts of single molecule tracking on stretched DNA, smFRET and high throughput sequencing-based experiments were performed by Redding et al31, Blosser et al32 , and Jung et al33, respectively. Redding et al31 applied DNA curtains to E. coli Cascade and reported that Cascade also employs 3D diffusion for target search but a small population was observed making 1D diffusion. Cascade stably localized to the protospacer even without PAM, consistent with an earlier report based on smFRET analysis32, but the presence of PAM increases affinity (Fig. 1E). Cascade bound to targets with PAM, and then recruits Cas3 nuclease which makes a nick and translocates away in the 3’ to 5’ direction (Fig. 1F). The first phase of translocation leads to the degradation of one of the DNA strands resulting in a ssDNA patch,


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followed by the second phase of translocation without degradation. Cascade bound to targets without PAM can also recruit Cas3, but only if Cas1-Cas2 are also present. Cas3-Cas1-Cas2 then creates a nick in the DNA without accompanying degradation, followed by their translocation in either direction. In Blosser et al32 experiments, E. Coli Cascade complex was immobilized on a surface and target DNA labeled with FRET probes on two opposite strands was added to observe DNA binding and unwinding in real time (Fig. 2F). E values and their dwell times showed two different binding behaviors: (1) PAM-dependent ultrastable binding attributed to Cascade interference mode for nucleolytic impairment and (2) PAM-independent binding to the protospacer (~25 s lifetime) that is needed for the priming function of acquiring DNA sequences into the CRISPR loci as a memory of infection34. Ensemble fluorescence assays for binding of E. Coli Cascade and Cas3 to DNA clusters whose identities are determined by on-chip sequencing gave rise to several interesting observations. First, there is a significant effect of sequences beyond the conventional 3 bp PAM well into 6th bp. Second, every 6th bp in the protospacer has the least effect on the Cascade binding affinity, consistent with the structures of Cascade-DNA complex13 where every 6th bp is flipped out to ease torsional strain within the RNA-DNA hybrid. A sequence preference was observed even for flipped bases, which is consistent with the structural studies where Cascade residues were found interacting with the flipped bases35. Finally, Cas3 recruitment only occurs on Cascade bound sites and is greatly perturbed by single base substitutions within the PAM but is only mildly perturbed by substitutions in the protospacer.



Single molecule fluorescence experiments are typically performed using TIRF or epifluorescence imaging of the surface of flow cells, thus only molecular entity near the surface can be visualized. Therefore, dissociation of any fluorescently labeled component, for example, the release of DNA products cleaved by CRISPR-RNA enzymes, can be visualized in real time as an abrupt disappearance of fluorescence. DNA curtain experiments by Sternberg et al6 and other sm experiments25 observed that Cas9-RNA remains irreversibly bound to the DNA even after cleavage. Singh et al performed sm experiments to investigate the fate of DNA molecules cleaved by Cpf130 and EngCas927. While EngCas9s did not release any cleavage products under physiological conditions, Cpf1 released the PAM-distal cleavage product, but not the PAMproximal product, in the time scale ranging from ~30 s to 30 min depending on nPD and Cpf1 variants. On one hand, Cas9 tightly holding on to the cleavage products by Cas9 can restrict the access to the cleaved DNA ends. On the other hand, holding onto both cleaved strands can transmit positive torsional strain generated by transcription machinery36, 37into the DNA, restoring the parental DNA duplex and rapidly ejecting CRISPR-RNA38. A careful investigation is needed to determine the advantages and disadvantages of product release properties in genome engineering applications.

Many of these biophysical experiments were supplemented with biochemical experiments to measure DNA cleavage. Cas9 cleaved DNA targets only with more than 16 bp PAM-proximal matches, indicating that the cleavage specificity is far higher that of binding, which requires 9 bp PAM-proximal matches for stable binding4, 25. In contrast, Cpf1 has the same threshold (≥ 17 bp PAM-proximal matches) for both stable binding and DNA cleavage. Ultrastable binding of Cas9-RNA onto off-target DNA can sequester Cas9, thus increasing Cas9 concentration required


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for genome editing25. Higher Cas9 concentration required in turn would increase off-target cleavage15. The much higher binding specificity by Cpf1 could provide an added security against such off-target DNA cleavage. Collectively, these studies have uncovered valuable insight into the target search process, binding specificity and handling of DNA product after cleavage. To summarize the main findings: (a) Cas9-RNA can perform 3D target search in varied ionic conditions and cellular environments (Fig. 3A). (b) Cas9-RNA’s target search is primarily driven by its PAM surveillance process. It can sample any DNA sequence25, which may be the reason why a vast number of non-specific sequences appeared in Cas9’s footprint in ChIP-seq studies39 (Fig. 3B). (c) Cas9-RNA and Cpf1-RNA share bimodal DNA binding characteristics but Cpf1 is much more sequence-specific in forming stable complexes with DNA (Fig. 3B). (d) PAM-proximal mismatches are far more deleterious for stable binding compared to PAM-distal mismatches. (e) the RNA-DNA heteroduplex extends in unidirectional manner from PAM-proximal to PAMdistal end (Fig. 3B). Cas9-RNA can sometimes bypass 2 PAM-proximal mismatches25, likely because of DNA bending it causes near PAM to create a kink which acts as a seed for heteroduplex extension40-43. (f) Cas9-RNA and Cpf1-RNA are both single turnover enzymes in vitro because they stay stably bound to one or both of their products, but Cpf1-RNA does release one end of cleaved DNA. (g) Cascade-RNA can bind protospacers both with and without PAM. PAM-dependent binding recruits Cas3 for nucleolytic degradation of DNA target whereas PAMindependent binding recruits Cas1, Cas2 and Cas3 for priming function (Fig. 3C).

R-loops and internal dynamics of Cas9/Cpf1-RNA-DNA complex and its relation to cleavage



Cas9/Cpf1-RNA targets DNA and unwinds the dsDNA and verifies the correct sequence by annealing one of the DNA strands (target strand) to the guide RNA while displacing the other strand (non-target strand). This three-stranded nucleic acid structure is known as the R-loop. Szczelkun et al38 and Rutsuakas et al44 employed magnetic tweezers to investigate the formation, dissolution and stability of the R-loop by St (Streptococcus Thermophilus) Cascade and StCas9. 2.2 Kbp long DNA molecules containing PAM and protospacer were immobilized on a surface. One end of the DNA was conjugated to a magnetic bead. Using a pair of magnets, the DNA molecules can be subjected to stretching and twisting. At different levels of stretching and twisting, abrupt changes in the end to end DNA extension were recorded. R-loop formation/dissolution changes DNA supercoiling which in turn is detected as a change in DNA extension45 (Fig. 3D). Dwell times between these abrupt changes were used to calculate the rate of R-loop formation (kRon) and dissolution (kRoff) (Fig. 3E). The presence of PAM influenced only kRon not kRoff, and PAM-distal mismatches/truncations did not affect kRon but increased kRoff . The R-loop was much more stable for StCascade than for StCas9, likely because the R-loop induced by StCascade has two modes that differ in stability such that locking in to the ultrastable mode requires nearly complete sequence match. Cas3 recruitment and degradation of DNA were observed only if the R-loop locks into the ultrastable mode, which occurred if nPD eCas9 > Cas9-HF1. These studies show that that EngCas9 achieve improved cleavage specificity by disrupting the maximally unwound state and by reducing kc,int. Mismatches or EngCas9 mutations prevent the Cas9-RNADNA from accessing the unwound state, which serve as a trigger for nuclease activation after being proofread by REC3 and HNH movement (Fig. 4E). Similar to Cas9, the Cpf1 rate of cleavage also decreases with increasing nPD. In fact, even the release kinetics of one of the cleavage products by Cpf1 depended on nPD, indicating that PAM-distal mismatches may influence post-cleavage steps30. Taken together, the studies looking at the internal dynamics of Cas9-RNA-DNA complex have provided valuable insights into the molecular events within these complex that determine the cleavage reaction and handling of the product.

CONCLUSIONS AND OUTLOOK There are many outstanding questions about the CRISPR system which can be addressed using sm approaches. For example, it should be possible to employ 3-4 color smFRET51, 52 for



simultaneous observation of the DNA unwinding and nuclease and proofreading domain dynamics, helping us understand the molecular details of allosteric communication between the RNA-DNA heteroduplex, DNA unwinding and cleavage action. While the majority of sm investigations have focused on CRISPR-RNA’s DNA binding and its conformational changes upon DNA binding, a largely unexplored territory has been the assembly of CRISPR-RNA complex. For example, the canonical guide-RNA for Cas9 consists of crRNA (with a segment complementary to the protospacer) and trans-activating crRNA (tracrRNA). Their hybridization results in formation of two hairpin motifs that leads to binding of Cas9 resulting in Cas9-RNA53. smFRET is a powerful technique to probe the folding pathways of RNA54 and thus could be used to probe the structural rearrangements within guide-RNA and also within CRISPR enzymes that ensure efficient complexation of guide-RNA with CRISPR enzyme. The information from such experiments will aid in the rational design of efficient guide-RNAs and explain the variations in activity of different guide-RNA sequences55.

Another unexplored territory is the use of high resolution force (optical traps56 & nanopore sequencer57) or correlative force-fluorescence spectroscopy. A suitable geometry of force application, with and without fluorescence visualization, can be used to extract additional information about transitions states between different dominant intermediates and also probe mechanical stability of CRISPR-RNA-DNA complex58. Cas9-RNA-DNA is an ultrastable complex that persists even post DNA cleavage, thus masking the cleaved DNA sites from genome editing machinery. Chromatin in vivo is likely to be under tension and torsional stress, which would be altered during the process of transcription and replication59, 60. DNA stretching and twisting may help in quick dissociation of CRISPR-RNA-DNA complex and help expose


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cleaved DNA sites for genome editing machinery. Therefore, probing the mechanical stability of the CRISPR-RNA-DNA complex may be useful in this regard.

Dead CRISPR-RNA fused with transcriptional effectors are being increasingly used to achieve in vivo site-specific transcriptional control as the fused transcriptional effectors recruit additional effectors at targeted sites61, but the molecular details of their recruitment are unknown. This recruitment assembly can be studied using another sm technique called Co-localization Single Molecule Spectroscopy 62, wherein entry and exit of fluorescently labeled biomolecules of interest (additional effectors in this case) can be studied at a particular site or a molecule (CRISPR targeted site in a DNA molecule).

Identification of critical steps in various stages of DNA targeting will not only help in designing strategies to improve efficiency and reduce off-target effects, but will also be useful for evaluating the rational design of new CRISPR enzymes and their chemical and enzymatic regulators, which are being increasingly pursued for additional control over CRISPR enzymes16, 63, 64

. For example, a chemical regulator may be rationally designed to target an important

intermediate of the CRISPR enzyme. sm approaches allow for precise characterization of differences between different CRISPR enzymes. This characterization of differences can be used to explain the variation in activity of different CRISPR enzymes in different conditions and organisms. CRISPR enzymes with different biophysical and biochemical parameters may be employed for different applications, expanding the functionalities of the CRISPR toolbox.



AUTHOR INFORMATION Corresponding Author *E-mail: [email protected] Notes The authors declare no competing financial interest. ACKNOWLEDGMENTS The project was supported by grants from National Science Foundation (PHY-1430124 to T.H.) and National Institutes of Health (GM065367; GM112659 to T.H); T.H. is an investigator with the Howard Hughes Medical Institute.


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KEYWORDS CRISPR: Acronym for Clustered regularly interspaced short palindromic repeats, which are segments of short, repetitive base sequences found in genome of certain microbes and impart adaptive immunity. Genome engineering and Genome editing: Process of employing strategies and techniques for modification of the genetic information at specific target sites in the genome. Cas9, Cpf1, Cascade, Cas3: Some of the most commonly known proteins and enzymes of the CRISPR system which are being widely used for genome engineering applications. Single molecule experiments: An experimental scheme allowing the observation of properties of individual molecules. Fluorescence: Phenomenon of light emission by molecules or substances upon its excitation by another light. FRET: Acronym for Fluorescence resonance energy transfer which is a phenomenon of energy transfer between two light-sensitive molecules (FRET pairs). By labeling biomolecular complexes with FRET pairs at judiciously selected sites, their conformational changes or interactions can be studied. Molecular Mechanism: A model or a system describing how various parts and processes of a biological system interact amongst each other at molecular level to produce an effect (for e.g. How does domains in Cas9 enzyme move to cleave DNA?) Force Spectroscopy: Investigation of the behavior of molecules under the application of stretching or torsional tension.



FIGURES

Graphical Abstract


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Figure 1. Target search process of the CRISPR proteins. (A) Schematic overview of DNA curtains. (B) Cas9 or Cascade is complexed with guide-RNA that targets a site in lambda DNA (λ1 – λ6 or λ1- λ3) that contains both PAM and protospacer sequence. (C) A fluorescence image showing Cas9-RNA targeting λ2. Magenta for the quantum dot attached to Cas9-RNA and green for YOYO stained DNA. Bottom panel shows Cas9-RNA binding position distribution along the lambda DNA. (D) Schematic of experimental design for in vivo tracking of single dCas9-RNA-HaloTag molecules (left), an image of dCas9-RNA-HaloTag molecules in live 3T3 nuclei (middle) and 2D projections of single-particle trajectories of dCas9RNA movement with two different guide-RNAs (right). The colors of the tracks indicate the log of diffusion coefficient based (color scale shown on the bottom). 0/0 is for the guide RNA



against Short interspersed element (SINE) repeat sequences. The number of PAM-distal (nPD) and PAM-proximal (nPP) mismatches are shown in cyan and orange, respectively. (E) Binding position distributions of Cascade to two sites sharing the same protospacer, λ3 and mutλ3. Mutλ3 has a mutated PAM and requires higher Cascade concentration for binding. (F) Kymograph showing the translocation of Cas3 (green), recruited by Cascade (magenta) to the λ3 site. Some panels of this figure have been taken/adapted from previous publications6, 23, 65 and reproduced here with permission.


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Figure 2. Single molecule investigation of DNA interrogation and product release by CRISPR complexes. (A) Schematic of smFRET assay for DNA interrogation. (B) DNA targets with mismatches in the protospacer region against the guide-RNA. The number of mismatches PAM-distal (nPD) and PAM-proximal (nPP) are shown in cyan and orange, respectively. (C) Single molecule intensity time traces of donor (green) and acceptor (red) are shown (left), along with FRET efficiency E



values idealized by hidden Markov modeling (right) (smFRET traces) for different DNA targets. Cognate DNA (nPD=0) stays bound stably (constant high E) whereas reversible binding to high and mid E states are seen with nPD > 12. (D) Average lifetimes of all bound states (τavg), high FRET state (τhigh) and mid FRET state (τmid) for various mismatches. Also shown is the bimolecular association rate kon calculated from the lifetime of the unbound state. (E) Schematic of high-throughput biophysical characterization of CRISPR-RNA binding and dissociation. Also accompanied are example images of Cy5 intensities of different DNA clusters (red spots on left), and example images of evolution and reduction of Cy3 intensities on DNA clusters with 12-hour incubation followed by removal of Cy3 labeled Cas9-RNA respectively. (F) Schematic of a smFRET assay to monitor binding of fluorescently labeled DNA to a surface-tethered Cascade complex. Representative smFRET traces of Mode I (top) and Mode II (bottom) binding. Mode I is PAM-dependent, long-lived. Mode II is PAM-independent and short-lived. The number of PAM-distal mismatches (nPD) and PAM-proximal mismatches (nPP) are shown in cyan and orange, respectively. Some panels of this figure have been taken/adapted from previous publications25, 32, 48 and reproduced here with permission.


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Figure 3. Mechanism of DNA interrogation by CRISPR-RNA as revealed from various single molecule investigations.



(A) A model describing the target search process where the lifetime of the Cas9-RNA complex with DNA increases processively as target is verified directionally starting from PAM. (B) Molecular mechanism of DNA interrogation and dual binding modes (transient PAM sampling and heteroduplex extension mode). (C) Model describing DNA recognition and processing by Cascade, Cas1, Cas2 and Cas3 as revealed by Redding et al.65 and Blosser et al.32. (D) Magnetic tweezers-based twisting assay to investigate CRISPR-RNA induced R-loop formation. Formation of R-loop on surface tethered supercoiled DNA molecules at fixed rotation causes local DNA untwisting which is compensated by over-twisting the DNA molecules. (E) This compensation changes the supercoiling, causing a change in the end to end distance of DNA when Cas9-RNA binds to a negatively supercoiled DNA (black arrows) and dissociates (red arrows) from a positively supercoiled DNA. Black and red stretches indicate −6 and +3 turns in the DNA respectively. (F) Ensemble average of AFM images of Cas9-RNA-DNA complex with low number of mismatches (left) and high number of mismatches (right). Bottom panel shows that Cas9-RNA-DNA complexes with various mismatches can be distinguished by the mean volume and mean height. The number of PAM-distal mismatches (nPD) and PAM-proximal mismatches (nPP) are shown in cyan and orange, respectively. Some panels of this figure have been taken/adapted from previous publications6, 25, 38, 46 and reproduced here with permission.


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Figure 4. smFRET assays to probe the internal dynamics of Cas9-RNA-DNA complex and final model. (A) Proposed model of HNH (in black) transition between inactive and active states using structure of Cas9-RNA (PDB ID :4ZT0) and Cas9-RNA-DNA (PDB ID:5F9R). Schematic of smFRET assay to study this HNH transition. Histograms of E values vs nPD (right). (B) Proposed model of REC2 (in black) transition between inactive and active states using structure of Cas9-RNA (PDB ID :4ZT0) and Cas9-RNA-DNA (PDB ID:5F9R). HNH domain has been omitted for clarity. This REC2 transition was measured using FRET probes shown. E histograms show an increasing conformational heterogeneity of REC2 states with increasing nPD, which is more pronounced for Cas9-HF1. (C) Schematic of smFRET assay to investigate the R-loop extension at the PAM-distal site for two different DNA targets. FRET histograms (middle) showing the decrease in fraction of fully extended R-loop states with PAM-distal mismatches. Also, accompanied (right) is correlation between high FRET fraction and relative cleavage activity for different DNA. (D) Schematic of smFRET assay to investigate the Cas9-RNA induced PAM-distal DNA unwinding. Formation of RNA-DNA heteroduplex between guideRNA and target strand accompanied with increase in separation (i.e. DNA unwinding) between FRET pair results in lower FRET value. (E) Model of allosteric communication between DNA unwinding, proofreading REC domain and nuclease HNH domain for DNA cleavage. Cas9RNA-DNA structure generated from PDB ID:5F9R. HNH, REC2, and REC3 domains in crystal structure has been omitted for clarity. Only inactive and active conformations of HNH, REC2, and REC3 are shown, their RNA-only conformation has been omitted for clarity. Number of PAM-distal (nPD) are shown in cyan digits. Some panels of this figure have been taken/adapted from previous publications19, 49, 50 and reproduced here with permission.


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