Kinetic dissection of pre-crRNA binding and processing by CRISPR-Cas12a.

CRISPR-Cas12a binds and processes a single pre-crRNA during maturation, providing a simple tool for genome editing applications. Here, we constructed a kinetic and thermodynamic framework for pre-crRNA processing by Cas12a in vitro, and we measured the contributions of distinct regions of the pre-crRNA to this reaction. We find that the pre-crRNA binds rapidly and extraordinarily tightly to Cas12a (K d = 0.6 pM), such that pre-crRNA binding is fully rate limiting for processing and therefore determines the specificity of Cas12a for different pre-crRNAs. The guide sequence contributes 10-fold to the binding affinity of the pre-crRNA, while deletion of an upstream sequence has no significant effect. After processing, the mature crRNA remains very tightly bound to Cas12a with a comparable affinity. Strikingly, the affinity contribution of the guide region increases to 600-fold after processing, suggesting that additional contacts are formed and may preorder the crRNA for efficient DNA target recognition. Using a direct competition assay, we find that pre-crRNA-binding specificity is robust to changes in the guide sequence, addition of a 3' extension, and secondary structure within the guide region. However, stable secondary structure in the guide region can strongly inhibit DNA targeting, indicating that care should be taken in crRNA design. Together, our results provide a quantitative framework for pre-crRNA binding and processing by Cas12a and suggest strategies for optimizing crRNA design in genome editing applications.

Cas12a domain flexibility guides R-loop formation and forces RuvC resetting.

The specific nature of CRISPR-Cas12a makes it a desirable RNA-guided endonuclease for biotechnology and therapeutic applications. To understand how R-loop formation within the compact Cas12a enables target recognition and nuclease activation, we used cryo-electron microscopy to capture wild-type Acidaminococcus sp. Cas12a R-loop intermediates and DNA delivery into the RuvC active site. Stages of Cas12a R-loop formation-starting from a 5-bp seed-are marked by distinct REC domain arrangements. Dramatic domain flexibility limits contacts until nearly complete R-loop formation, when the non-target strand is pulled across the RuvC nuclease and coordinated domain docking promotes efficient cleavage. Next, substantial domain movements enable target strand repositioning into the RuvC active site. Between cleavage events, the RuvC lid conformationally resets to occlude the active site, requiring re-activation. These snapshots build a structural model depicting Cas12a DNA targeting that rationalizes observed specificity and highlights mechanistic comparisons to other class 2 effectors.

Tracking Native Tetrahymena Ribozyme Folding with Fluorescence.

Folding of the Tetrahymena group I intron ribozyme and other structured RNAs has been measured using a catalytic activity assay to monitor the native state formation by cleavage of a radiolabeled oligonucleotide substrate. While highly effective, the assay has inherent limitations present in any radioactivity- and gel-based assay. Administrative and safety considerations arise from the radioisotope, and data collection is laborious due to the use of polyacrylamide gels. Here we describe a fluorescence-based, solution assay that allows for more efficient data acquisition. The substrate is labeled with 6-carboxyfluorescein (6FAM) fluorophore and black hole quencher (BHQ1) at the 5' and 3' ends, respectively. Substrate cleavage results in release of the quencher, increasing the fluorescence signal by an average of 30-fold. A side-by-side comparison with the radioactivity-based assay shows good agreement in monitoring Tetrahymena ribozyme folding from a misfolded conformation to the native state, albeit with increased uncertainty. The lower precision of the fluorescence assay is compensated for by the relative ease and efficiency of the workflow. In addition, this assay will allow institutions that do not use radioactive materials to monitor native folding of the Tetrahymena ribozyme, and the same strategy should be amenable to native folding of other ribozymes.

Impact of Ion-Mixing Entropy on Orientational Preferences of DNA Helices: FRET Measurements and Computer Simulations.

Biological processes require DNA and RNA helices to pack together in specific interhelical orientations. While electrostatic repulsion between backbone charges is expected to be maximized when helices are in parallel alignment, such orientations are commonplace in nature. To better understand how the repulsion is overcome, we used experimental and computational approaches to investigate how the orientational preferences of DNA helices depend on the concentration and valence of mobile cations. We used Förster resonance energy transfer (FRET) to probe the relative orientations of two 24-bp helices held together via a freely rotating PEG linker. At low cation concentrations, the helices preferred more "cross"-like orientations over those closer to parallel, and this preference was reduced with increasing salt concentrations. The results were in good quantitative agreement with Poisson-Boltzmann (PB) calculations for monovalent salt (Na+). However, PB underestimated the ability of mixtures of monovalent and divalent ions (Mg2+) to reduce the conformational preference. As a complementary approach, we performed all-atom molecular dynamics (MD) simulations and found better agreement with the experimental results. While MD and PB predict similar electrostatic forces, MD predicts a greater accumulation of Mg2+ in the ion atmosphere surrounding the DNA. Mg2+ occupancy is predicted to be greater in conformations close to the parallel orientation than in conformations close to the crossed orientation, enabling a greater release of Na+ ions and providing an entropic gain (one bound ion for two released). MD predicts an entropy gain larger than that of PB because of the increased Mg2+ occupancy. The entropy changes have a negligible effect at low Mg2+ concentrations because the free energies are dominated by electrostatic repulsion. However, as the Mg2+ concentration increases, charge screening is more effective and the mixing entropy produces readily detectable changes in packing preferences. Our results underline the importance of mixing entropy of counterions in nucleic acid interactions and provide a new understanding on the impact of a mixed ion atmosphere on the packing of DNA helices.

Kinetic dissection of pre-crRNA binding and processing by CRISPR-Cas12a.

CRISPR-Cas12a binds and processes a single pre-crRNA during maturation, providing a simple tool for genome editing applications. Here, we constructed a kinetic and thermodynamic framework for pre-crRNA processing by Cas12a in vitro, and we measured the contributions of distinct regions of the pre-crRNA to this reaction. We find that the pre-crRNA binds rapidly and extraordinarily tightly to Cas12a (Kd = 0.6 pM), such that pre-crRNA binding is fully rate limiting for processing and therefore determines the specificity of Cas12a for different pre-crRNAs. The guide sequence contributes 10-fold to the affinities of both the precursor and mature forms of the crRNA, while deletion of an upstream sequence had no significant effect on affinity of the pre-crRNA. After processing, the mature crRNA remains very tightly bound to Cas12a, with a half-life of ~1 day and a Kd value of 60 pM. Addition of a 5'-phosphoryl group, which is normally lost during the processing reaction as the scissile phosphate, tightens binding of the mature crRNA by ~10-fold by accelerating binding and slowing dissociation. Using a direct competition assay, we found that pre-crRNA binding specificity is robust to other changes in RNA sequence, including tested changes in the guide sequence, addition of a 3' extension, and secondary structure within the guide region. Together our results provide a quantitative framework for pre-crRNA binding and processing by Cas12a and suggest strategies for optimizing crRNA design in some genome editing applications.

Group II intron-like reverse transcriptases function in double-strand break repair.

Bacteria encode reverse transcriptases (RTs) of unknown function that are closely related to group II intron-encoded RTs. We found that a Pseudomonas aeruginosa group II intron-like RT (G2L4 RT) with YIDD instead of YADD at its active site functions in DNA repair in its native host and when expressed in Escherichia coli. G2L4 RT has biochemical activities strikingly similar to those of human DNA repair polymerase Î¸ and uses them for translesion DNA synthesis and double-strand break repair (DSBR) via microhomology-mediated end-joining (MMEJ). We also found that a group II intron RT can function similarly in DNA repair, with reciprocal active-site substitutions showing isoleucine favors MMEJ and alanine favors primer extension in both enzymes. These DNA repair functions utilize conserved structural features of non-LTR-retroelement RTs, including human LINE-1 and other eukaryotic non-LTR-retrotransposon RTs, suggesting such enzymes may have inherent ability to function in DSBR in a wide range of organisms.

Measurement of ATP utilization in RNA unwinding and RNA chaperone activities by DEAD-box helicase proteins.

RNA helicase proteins perform coupled reactions in which cycles of ATP binding and hydrolysis are used to drive local unwinding of double-stranded RNA (dsRNA). For some helicases in the ubiquitous DEAD-box family, these local unwinding events are integral to folding transitions in structured RNAs, and thus these helicases function as RNA chaperones. An important measure of the efficiency of the helicase-catalyzed reaction is the ATP utilization value, which represents the average number of ATP molecules hydrolyzed during RNA unwinding or a chaperone-assisted RNA structural rearrangement. Here we outline procedures that can be used to measure the ATP utilization value in RNA unwinding or folding transitions. As an example of an RNA folding transition, we focus on the refolding of the Tetrahymena thermophila group I intron ribozyme from a long-lived misfolded structure to its native structure, and we outline strategies for adapting this assay to other RNA folding transitions. For a simple dsRNA unwinding event, the ATP utilization value provides a measure of the coupling between the ATPase and RNA unwinding activities, and for a complex RNA structural transition it can give insight into the scope of the rearrangement and the efficiency with which the helicase uses the energy from ATPase cycles to promote the rearrangement.

Kinetics measurements of G-quadruplex binding and unfolding by helicases.

G-quadruplex structures (G4s) form readily in DNA and RNA and play diverse roles in gene expression and other processes, and their inappropriate formation and stabilization are linked to human diseases. G4s are inherently long-lived, such that their timely unfolding depends on a suite of DNA and RNA helicase proteins. Biochemical analysis of G4 binding and unfolding by individual helicase proteins is important for establishing their levels of activity, affinity, and specificity for G4s, including individual G4s of varying sequence and structure. Here we describe a set of simple, accessible methods in which electrophoretic mobility shift assays (EMSA) are used to measure the kinetics of G4 binding, dissociation, and unfolding by helicase proteins. We focus on practical considerations and the pitfalls that are most likely to arise when these methods are used to study the activities of helicases on G4s.

Direct Measurement of Interhelical DNA Repulsion and Attraction by Quantitative Cross-Linking.

To better understand the forces that mediate nucleic acid compaction in biology, we developed the disulfide cross-linking approach xHEED (X-linking of Helices to measure Electrostatic Effects at Distance) to measure the distance-dependent encounter frequency of two DNA helices in solution. Using xHEED, we determined the distance that the electrostatic potential extends from DNA helices, the dependence of this distance on ionic conditions, and the magnitude of repulsion when two helices approach one another. Across all conditions tested, the potential falls to that of the bulk solution within 15 Å of the major groove surface. For separations of ∼30 Å, we measured a repulsion of 1.8 kcal/mol in low monovalent ion concentration (30 mM Na+), with higher Na+ concentrations ameliorating this repulsion, and 2 M Na+ or 100 mM Mg2+ eliminating it. Strikingly, we found full screening at very low Co3+ concentrations and net attraction at higher concentrations, without the higher-order DNA condensation that typically complicates studies of helical attraction. Our measurements define the relevant distances for electrostatic interactions of nucleic-acid helices in biology and introduce a new method to propel further understanding of how these forces impact biological processes.

How to Kinetically Dissect an RNA Machine.

RNA-based machines are ubiquitous in Nature and increasingly important for medicines. They fold into complex, dynamic structures that process information and catalyze reactions, including reactions that generate new RNAs and proteins across biology. What are the experimental strategies and steps that are necessary to understand how these complex machines work? Two 1990 papers from Herschlag and Cech on "Catalysis of RNA Cleavage by the Tetrahymena thermophila Ribozyme" provide a master class in dissecting an RNA machine through kinetics approaches. By showing how to propose a kinetic framework, fill in the numbers, do cross-checks, and make comparisons across mutants and different RNA systems, the papers illustrate how to take a mechanistic approach and distill the results into general insights that are difficult to attain through other means.

Structural basis for template switching by a group II intron-encoded non-LTR-retroelement reverse transcriptase.

Reverse transcriptases (RTs) can switch template strands during complementary DNA synthesis, enabling them to join discontinuous nucleic acid sequences. Template switching (TS) plays crucial roles in retroviral replication and recombination, is used for adapter addition in RNA-Seq, and may contribute to retroelement fitness by increasing evolutionary diversity and enabling continuous complementary DNA synthesis on damaged templates. Here, we determined an X-ray crystal structure of a TS complex of a group II intron RT bound simultaneously to an acceptor RNA and donor RNA template-DNA primer heteroduplex with a 1-nt 3'-DNA overhang. The structure showed that the 3' end of the acceptor RNA binds in a pocket formed by an N-terminal extension present in non-long terminal repeat-retroelement RTs and the RT fingertips loop, with the 3' nucleotide of the acceptor base paired to the 1-nt 3'-DNA overhang and its penultimate nucleotide base paired to the incoming dNTP at the RT active site. Analysis of structure-guided mutations identified amino acids that contribute to acceptor RNA binding and a phenylalanine residue near the RT active site that mediates nontemplated nucleotide addition. Mutation of the latter residue decreased multiple sequential template switches in RNA-Seq. Our results provide new insights into the mechanisms of TS and nontemplated nucleotide addition by RTs, suggest how these reactions could be improved for RNA-Seq, and reveal common structural features for TS by non-long terminal repeat-retroelement RTs and viral RNA-dependent RNA polymerases.

The DHX36-specific-motif (DSM) enhances specificity by accelerating recruitment of DNA G-quadruplex structures.

DHX36 is a eukaryotic DEAH/RHA family helicase that disrupts G-quadruplex structures (G4s) with high specificity, contributing to regulatory roles of G4s. Here we used a DHX36 truncation to examine the roles of the 13-amino acid DHX36-specific motif (DSM) in DNA G4 recognition and disruption. We found that the DSM promotes G4 recognition and specificity by increasing the G4 binding rate of DHX36 without affecting the dissociation rate. Further, for most of the G4s measured, the DSM has little or no effect on the G4 disruption step by DHX36, implying that contacts with the G4 are maintained through the transition state for G4 disruption. This result suggests that partial disruption of the G4 from the 3' end is sufficient to reach the overall transition state for G4 disruption, while the DSM remains unperturbed at the 5' end. Interestingly, the DSM does not contribute to G4 binding kinetics or thermodynamics at low temperature, indicating a highly modular function. Together, our results animate recent DHX36 crystal structures, suggesting a model in which the DSM recruits G4s in a modular and flexible manner by contacting the 5' face early in binding, prior to rate-limiting capture and disruption of the G4 by the helicase core.

Inhibition of CRISPR-Cas12a DNA targeting by nucleosomes and chromatin.

Genome engineering nucleases must access chromatinized DNA. Here, we investigate how AsCas12a cleaves DNA within human nucleosomes and phase-condensed nucleosome arrays. Using quantitative kinetics approaches, we show that dynamic nucleosome unwrapping regulates target accessibility to Cas12a and determines the extent to which both steps of binding-PAM recognition and R-loop formation-are inhibited by the nucleosome. Relaxing DNA wrapping within the nucleosome by reducing DNA bendability, adding histone modifications, or introducing target-proximal dCas9 enhances DNA cleavage rates over 10-fold. Unexpectedly, Cas12a readily cleaves internucleosomal linker DNA within chromatin-like, phase-separated nucleosome arrays. DNA targeting is reduced only ~5-fold due to neighboring nucleosomes and chromatin compaction. This work explains the observation that on-target cleavage within nucleosomes occurs less often than off-target cleavage within nucleosome-depleted genomic regions in cells. We conclude that nucleosome unwrapping regulates accessibility to CRISPR-Cas nucleases and propose that increasing nucleosome breathing dynamics will improve DNA targeting in eukaryotic cells.

Key Points to Consider When Studying RNA Remodeling by Proteins.

Cellular RNAs depend on proteins for efficient folding to specific functional structures and for transitions between functional structures. This dependence arises from intrinsic properties of RNA structure. Specifically, RNAs possess stable local structure, largely in the form of helices, and there are abundant opportunities for RNAs to form alternative helices and tertiary contacts and therefore to populate alternative structures. Proteins with RNA chaperone activity, either ATP-dependent or ATP-independent, can promote structural transitions by interacting with single-stranded RNA (ssRNA) to compete away partner interactions and then release ssRNA so that it can form new interactions. In this chapter we review the basic properties of RNA and the proteins that function as chaperones and remodelers. We then use these properties as a foundation to explore key points for the design and interpretation of experiments that probe RNA rearrangements and their acceleration by proteins.

ATP utilization by a DEAD-box protein during refolding of a misfolded group I intron ribozyme.

DEAD-box helicase proteins perform ATP-dependent rearrangements of structured RNAs throughout RNA biology. Short RNA helices are unwound in a single ATPase cycle, but the ATP requirement for more complex RNA structural rearrangements is unknown. Here we measure the amount of ATP used for native refolding of a misfolded group I intron ribozyme by CYT-19, a Neurospora crassa DEAD-box protein that functions as a general chaperone for mitochondrial group I introns. By comparing the rates of ATP hydrolysis and ribozyme refolding, we find that several hundred ATP molecules are hydrolyzed during refolding of each ribozyme molecule. After subtracting nonproductive ATP hydrolysis that occurs in the absence of ribozyme refolding, we find that approximately 100 ATPs are hydrolyzed per refolded RNA as a consequence of interactions specific to the misfolded ribozyme. This value is insensitive to changes in ATP and CYT-19 concentration and decreases with decreasing ribozyme stability. Because of earlier findings that ∼90% of global ribozyme unfolding cycles lead back to the kinetically preferred misfolded conformation and are not observed, we estimate that each global unfolding cycle consumes ∼10 ATPs. Our results indicate that CYT-19 functions as a general RNA chaperone by using a stochastic, energy-intensive mechanism to promote RNA unfolding and refolding, suggesting an evolutionary convergence with protein chaperones.

Template-switching mechanism of a group II intron-encoded reverse transcriptase and its implications for biological function and RNA-Seq.

The reverse transcriptases (RTs) encoded by mobile group II introns and other non-LTR retroelements differ from retroviral RTs in being able to template-switch efficiently from the 5' end of one template to the 3' end of another with little or no complementarity between the donor and acceptor templates. Here, to establish a complete kinetic framework for the reaction and to identify conditions that more efficiently capture acceptor RNAs or DNAs, we used a thermostable group II intron RT (TGIRT; GsI-IIC RT) that can template switch directly from synthetic RNA template/DNA primer duplexes having either a blunt end or a 3'-DNA overhang end. We found that the rate and amplitude of template switching are optimal from starter duplexes with a single nucleotide 3'-DNA overhang complementary to the 3' nucleotide of the acceptor RNA, suggesting a role for nontemplated nucleotide addition of a complementary nucleotide to the 3' end of cDNAs synthesized from natural templates. Longer 3'-DNA overhangs progressively decreased the template-switching rate, even when complementary to the 3' end of the acceptor template. The reliance on only a single bp with the 3' nucleotide of the acceptor together with discrimination against mismatches and the high processivity of group II intron RTs enable synthesis of full-length DNA copies of nucleic acids beginning directly at their 3' end. We discuss the possible biological functions of the template-switching activity of group II intron- and other non-LTR retroelement-encoded RTs, as well as the optimization of this activity for adapter addition in RNA- and DNA-Seq protocols.

Kinetic Basis for DNA Target Specificity of CRISPR-Cas12a.

Class 2 CRISPR-Cas nucleases are programmable genome editing tools with promising applications in human health and disease. However, DNA cleavage at off-target sites that resemble the target sequence is a pervasive problem that remains poorly understood mechanistically. Here, we use quantitative kinetics to dissect the reaction steps of DNA targeting by Acidaminococcus sp Cas12a (also known as Cpf1). We show that Cas12a binds DNA tightly in two kinetically separable steps. Protospacer-adjacent motif (PAM) recognition is followed by rate-limiting R-loop propagation, leading to inevitable DNA cleavage of both strands. Despite functionally irreversible binding, Cas12a discriminates strongly against mismatches along most of the DNA target sequence. This result implies substantial reversibility during R-loop formation-a late transition state-and defies common descriptions of a "seed" region. Our results provide a quantitative basis for the DNA cleavage patterns measured in vivo and observations of greater reported target specificity for Cas12a than for the Cas9 nuclease.

Hidden Structural Modules in a Cooperative RNA Folding Transition.

Large-scale, cooperative rearrangements underlie the functions of RNA in RNA-protein machines and gene regulation. To understand how such rearrangements are orchestrated, we used high-throughput chemical footprinting to dissect a seemingly concerted rearrangement in P5abc RNA, a paradigm of RNA folding studies. With mutations that systematically disrupt or restore putative structural elements, we found that this transition reflects local folding of structural modules, with modest and incremental cooperativity that results in concerted behavior. First, two distant secondary structure changes are coupled through a bridging three-way junction and Mg2+-dependent tertiary structure. Second, long-range contacts are formed between modules, resulting in additional cooperativity. Given the sparseness of RNA tertiary contacts after secondary structure formation, we expect that modular folding and incremental cooperativity are generally important for specifying functional structures while also providing productive kinetic paths to these structures. Additionally, we expect our approach to be useful for uncovering modularity in other complex RNAs.

The G-quadruplex (G4) resolvase DHX36 efficiently and specifically disrupts DNA G4s via a translocation-based helicase mechanism.

Single-stranded DNA (ssDNA) and RNA regions that include at least four closely spaced runs of three or more consecutive guanosines strongly tend to fold into stable G-quadruplexes (G4s). G4s play key roles as DNA regulatory sites and as kinetic traps that can inhibit biological processes, but how G4s are regulated in cells remains largely unknown. Here, we developed a kinetic framework for G4 disruption by DEAH-box helicase 36 (DHX36), the dominant G4 resolvase in human cells. Using tetramolecular DNA and RNA G4s with four to six G-quartets, we found that DHX36-mediated disruption is highly efficient, with rates that depend on G4 length under saturating conditions (kcat) but not under subsaturating conditions (kcat/Km ). These results suggest that a step during G4 disruption limits the kcat value and that DHX36 binding limits kcat/Km Similar results were obtained for unimolecular DNA G4s. DHX36 activity depended on a 3' ssDNA extension and was blocked by a polyethylene glycol linker, indicating that DHX36 loads onto the extension and translocates 3'-5' toward the G4. DHX36 unwound dsDNA poorly compared with G4s of comparable intrinsic lifetime. Interestingly, we observed that DHX36 has striking 3'-extension sequence preferences that differ for G4 disruption and dsDNA unwinding, most likely arising from differences in the rate-limiting step for the two activities. Our results indicate that DHX36 disrupts G4s with a conventional helicase mechanism that is tuned for great efficiency and specificity for G4s. The dependence of DHX36 on the 3'-extension sequence suggests that the extent of formation of genomic G4s may not track directly with G4 stability.