Target DNA Strand Research Articles

Structural mechanism of bridge RNA-guided recombination

Insertion sequence (IS) elements are the simplest autonomous transposable elements found in prokaryotic genomes1. We recently discovered that IS110 family elements encode a recombinase and a non-coding bridge RNA (bRNA) that confers modular specificity for target DNA and donor DNA through two programmable loops2. Here we report the cryo-electron microscopy structures of the IS110 recombinase in complex with its bRNA, target DNA and donor DNA in three different stages of the recombination reaction cycle. The IS110 synaptic complex comprises two recombinase dimers, one of which houses the target-binding loop of the bRNA and binds to target DNA, whereas the other coordinates the bRNA donor-binding loop and donor DNA. We uncovered the formation of a composite RuvC–Tnp active site that spans the two dimers, positioning the catalytic serine residues adjacent to the recombination sites in both target and donor DNA. A comparison of the three structures revealed that (1) the top strands of target and donor DNA are cleaved at the composite active sites to form covalent 5′-phosphoserine intermediates, (2) the cleaved DNA strands are exchanged and religated to create a Holliday junction intermediate, and (3) this intermediate is subsequently resolved by cleavage of the bottom strands. Overall, this study reveals the mechanism by which a bispecific RNA confers target and donor DNA specificity to IS110 recombinases for programmable DNA recombination.

Ultrasensitive detection of Salmonella typhi using a PAM-free Cas14a-based biosensor

The effectiveness of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas14a1, widely utilized for pathogenic microorganism detection, has been limited by the requirement of a protospacer adjacent motif (PAM) on the target DNA strands. To overcome this limitation, this study developed a Single Primer isothermal amplification integrated-Cas14a1 biosensor (SPCas) for detecting Salmonella typhi that does not rely on a PAM sequence. The SPCas biosensor utilizes a novel primer design featuring an RNA-DNA primer and a 3′-biotin-modified primer capable of binding to the same single-stranded DNA (ssDNA) in the presence of the target gene. The RNA-DNA primer undergoes amplification and is blocked at the biotin-modified end. Subsequently, strand replacement is initiated to generate ssDNA assisted by RNase H and Bst enzymes, which activate the trans-cleavage activity of Cas14a1 even in the absence of a PAM sequence. Leveraging both cyclic chain replacement reaction amplification and Cas14a1 trans-cleavage activity, the SPCas biosensor exhibits a remarkable diagnostic sensitivity of 5 CFU/mL. Additionally, in the assessment of 20 milk samples, the SPCas platform demonstrated 100% diagnostic accuracy, which is consistent with the gold standard qPCR. This platform introduces a novel approach for developing innovative CRISPR-Cas-dependent biosensors without a PAM sequence.

Engineering of Cas12a nuclease variants with enhanced genome-editing specificity.

The clustered regularly interspaced short palindromic repeat (CRISPR)-Cas12a system is a powerful tool in gene editing; however, crRNA-DNA mismatches might induce unwanted cleavage events, especially at the distal end of the PAM. To minimize this limitation, we engineered a hyper fidelity AsCas12a variant carrying the mutations S186A/R301A/T315A/Q1014A/K414A (termed HyperFi-As) by modifying amino acid residues interacting with the target DNA and crRNA strand. HyperFi-As retains on-target activities comparable to wild-type AsCas12a (AsCas12aWT) in human cells. We demonstrated that HyperFi-As has dramatically reduced off-target effects in human cells, and HyperFi-As possessed notably a lower tolerance to mismatch at the position of the PAM-distal region compared with the wild type. Further, a modified single-molecule DNA unzipping assay at proper constant force was applied to evaluate the stability and transient stages of the CRISPR/Cas ribonucleoprotein (RNP) complex. Multiple states were sensitively detected during the disassembly of the DNA-Cas12a-crRNA complexes. On off-target DNA substrates, the HyperFi-As-crRNA was harder to maintain the R-loop complex state compared to the AsCas12aWT, which could explain exactly why the HyperFi-As has low off-targeting effects in human cells. Our findings provide a novel version of AsCas12a variant with low off-target effects, especially capable of dealing with the high off-targeting in the distal region from the PAM. An insight into how the AsCas12a variant behaves at off-target sites was also revealed at the single-molecule level and the unzipping assay to evaluate multiple states of CRISPR/Cas RNP complexes might be greatly helpful for a deep understanding of how CRISPR/Cas behaves and how to engineer it in future.

An alpha-helical lid guides the target DNA toward catalysis in CRISPR-Cas12a

CRISPR-Cas12a is a powerful RNA-guided genome-editing system that generates double-strand DNA breaks using its single RuvC nuclease domain by a sequential mechanism in which initial cleavage of the non-target strand is followed by target strand cleavage. How the spatially distant DNA target strand traverses toward the RuvC catalytic core is presently not understood. Here, continuous tens of microsecond-long molecular dynamics and free-energy simulations reveal that an α-helical lid, located within the RuvC domain, plays a pivotal role in the traversal of the DNA target strand by anchoring the crRNA:target strand duplex and guiding the target strand toward the RuvC core, as also corroborated by DNA cleavage experiments. In this mechanism, the REC2 domain pushes the crRNA:target strand duplex toward the core of the enzyme, while the Nuc domain aids the bending and accommodation of the target strand within the RuvC core by bending inward. Understanding of this critical process underlying Cas12a activity will enrich fundamental knowledge and facilitate further engineering strategies for genome editing.

Leveraging QM/MM and Molecular Dynamics Simulations to Decipher the Reaction Mechanism of the Cas9 HNH Domain to Investigate Off-Target Effects.

The clustered regularly interspaced short palindromic repeats (CRISPR) technology is an RNA-guided targeted genome-editing tool using Cas family proteins. Two magnesium-dependent nuclease domains of the Cas9 enzyme, termed HNH and RuvC, are responsible for cleaving the target DNA (t-DNA) and nontarget DNA strands, respectively. The HNH domain is believed to determine the DNA cleavage activity of both endonuclease domains and is sensitive to complementary RNA-DNA base pairing. However, the underlying molecular mechanisms of CRISPR-Cas9, by which it rebukes or accepts mismatches, are poorly understood. Thus, investigation of the structure and dynamics of the catalytic state of Cas9 with either matched or mismatched t-DNA can provide insights into improving its specificity by reducing off-target cleavages. Here, we focus on a recently discovered catalytic-active form of the Streptococcus pyogenes Cas9 (SpCas9) and employ classical molecular dynamics and coupled quantum mechanics/molecular mechanics simulations to study two possible mechanisms of t-DNA cleavage reaction catalyzed by the HNH domain. Moreover, by designing a mismatched t-DNA structure called MM5 (C to G at the fifth position from the protospacer adjacent motif region), the impact of single-guide RNA (sgRNA) and t-DNA complementarity on the catalysis process was investigated. Based on these simulations, our calculated binding affinities, minimum energy paths, and analysis of catalytically important residues provide atomic-level details of the differences between matched and mismatched cleavage reactions. In addition, several residues exhibit significant differences in their catalytic roles for the two studied systems, including K253, K263, R820, K896, and K913.

Furan-based (photo)oxidation reactions and their application in nucleic acid and protein targeting.

Oligonucleotides (ODNs) find applications as diagnostic and therapeutic tools due to their unique ability to interact, thanks to Watson-Crick base pairing, with a specific DNA or RNA target strand. Although most of the tools available today rely on mere hydrogen bond formation, chemical modifications to enable covalent interstrand-crosslinking (ICL) have been reported, and are gaining a place under the spotlight as they potentially offer a series of advantages over the state of the art, including a higher potency and selectivity. This methodological paper focuses on the use of a pro-reactive furan moiety and its subsequent oxidation for applications in ODN targeting. The design of effective capture and targeting probes to ensure high ICL yields is discussed and the mechanisms underlying the (photo)chemical oxidation of furan are explained. Furthermore, examples of furan-containing DNAs designed for different applications, including DNA-DNA or DNA-RNA ICL and DNA-peptide/protein targeting, are provided. The paper highlights the advantages of using different oxidative chemical triggers, such as N-bromosuccinimide or singlet oxygen, to offer additional selectivity control over the ICL reaction.

The energy landscape for R-loop formation by the CRISPR-Cas Cascade complex.

Clustered regularly interspaced short palindromic repeats (CRISPR) sequences and CRISPR-associated (Cas) genes comprise CIRSPR-Cas effector complexes, which have revolutionized gene editing with their ability to target specific genomic loci using CRISPR RNA (crRNA) complementarity. Recognition of double-stranded DNA targets proceeds via DNA unwinding and base pairing between crRNA and the DNA target strand, forming an R-loop structure. Full R-loop extension is a prerequisite for subsequent DNA cleavage. However, the recognition of unintended sequences with multiple mismatches has limited therapeutic applications and is still poorly understood on a mechanistic level. Here we set up ultrafast DNA unwinding experiments on the basis of plasmonic DNA origami nanorotors to study R-loop formation by the Cascade effector complex in real time, close to base-pair resolution. We resolve a weak global downhill bias of the forming R-loop, followed by a steep uphill bias for the final base pairs. We also show that the energy landscape is modulated by base flips and mismatches. These findings suggest that Cascade-mediated R-loop formation occurs on short timescales in submillisecond single base-pair steps, but on longer timescales in six base-pair intermediate steps, in agreement with the structural periodicity of the crRNA-DNA hybrid.

Acyclic 2-Ethynylnaphthalene-Modified 8-Aza-3,7-dideaza-2′-deoxyadenosine Allows Thymine Discrimination by Probing the DNA Minor Groove Environment

AbstractTwo novel acyclic environment-sensitive fluorescent nucleosides named ac37zA and an37zA are synthesized and their photophysical properties are investigated. Both ac37zA and an37zA exhibit dual fluorescence emission depending upon molecular coplanarity. Oligodeoxynucleotide probes containing ac37zA clearly discriminate perfectly matched thymine in target DNA strands by strong charge transfer (CT) emission in the longer wavelength region and by strong quenching of emission from the twisted conformation in the mismatched case.

PASTE, Don't Cut: Genome Editing Tool Looks Beyond CRISPR and Prime

PASTE, Don't Cut: Genome Editing Tool Looks Beyond CRISPR and Prime

Prime editing with genuine Cas9 nickases minimizes unwanted indels

Unlike CRISPR-Cas9 nucleases, which yield DNA double-strand breaks (DSBs), Cas9 nickases (nCas9s), which are created by replacing key catalytic amino-acid residues in one of the two nuclease domains of S. pyogenesis Cas9 (SpCas9), produce nicks or single-strand breaks. Two SpCas9 variants, namely, nCas9 (D10A) and nCas9 (H840A), which cleave target (guide RNA-pairing) and non-target DNA strands, respectively, are widely used for various purposes, including paired nicking, homology-directed repair, base editing, and prime editing. In an effort to define the off-target nicks caused by these nickases, we perform Digenome-seq, a method based on whole genome sequencing of genomic DNA treated with a nuclease or nickase of interest, and find that nCas9 (H840A) but not nCas9 (D10A) can cleave both strands, producing unwanted DSBs, albeit less efficiently than wild-type Cas9. To inactivate the HNH nuclease domain further, we incorporate additional mutations into nCas9 (H840A). Double-mutant nCas9 (H840A + N863A) does not exhibit the DSB-inducing behavior in vitro and, either alone or in fusion with the M-MLV reverse transcriptase (prime editor, PE2 or PE3), induces a lower frequency of unwanted indels, compared to nCas9 (H840A), caused by error-prone repair of DSBs. When incorporated into prime editor and used with engineered pegRNAs (ePE3), we find that the nCas9 variant (H840A + N854A) dramatically increases the frequency of correct edits, but not unwanted indels, yielding the highest purity of editing outcomes compared to nCas9 (H840A).

Key role of an alpha-helical lid in driving the target DNA toward catalysis in CRISPR-Cas12a.

CRISPR-Cas12a is an emerging gene editing system that came to the forefront with innovative applications such as rapid and ultrasensitive nucleic acid detection. At the core of this molecular complex, the RNA-guided Cas12a enzyme cleaves double-stranded DNA using a single catalytic cleft in the RuvC domain. This opens an overarching question on how the spatially distant DNA target strand (TS) traverses toward the RuvC domain for its accommodation and subsequent cleavage in the catalytic core. Here, continuous tens of microsecond-long molecular dynamics and free-energy simulations reveal that an α-helical lid, located within the RuvC domain, plays a pivotal role in the traversal of the TS by anchoring the crRNA:TS hybrid and elegantly guiding the TS toward the RuvC core, as also corroborated by DNA cleavage experiments. In this mechanism, the Rec2 domain drives the DNA target strand toward the RuvC catalytic cleft, owing to concerted motions with the Nuc domain. While the REC2 domain pushes the TS inward into the core of the complex with its short alpha helices, the Nuc domain aids the bending and accommodation of the TS within the RuvC core by bending inward. The identified intermediates provide information on the critical residues involved in the biophysical process, holding promises for future engineering strategies aimed at improving the overall Cas12a activity.

The effect of DNA tension on CRISPR/Cas12a cleavage can be reversible.

CRISPR/Cas12a (known as Cpf1) is a class 2 type V endonuclease which offers a two-component system for double stranded DNA cleavage. Cas12a is the enzymatic component with an active nuclease domain (RuvC) for cleaving the non-target and target DNA strands in order. CRISPR RNA (crRNA) is the RNA component which is initially processed by Cas12a and later associates with the Cas12a enzyme for the target detection. After Cas12a and crRNA are associated, they form a stable Cas12a/crRNA complex to interrogate the DNA target. Once the complementarity of the guide RNA and the target DNA is maintained, DNA cleavage occurs. Like other CRISPR enzymes, Cas12a is mainly derived from bacteria. However, unlike prokaryotic DNA, eukaryotic DNA faces mechanical stress. For example, DNA stretching and torsional stress can occur during various cellular processes that involve DNA compaction and extraction. As previously reported, the mechanical tension on the DNA reduces or inhibits the CRISPR/Cas12a cleavage reaction. Yet, it remains unknown that if Cas12a cleavage inhibition by tension is reversible. Here I show that the inhibited DNA cleavage can be rescued when the tension is relieved. I employed a high-throughput single-molecule technique to monitor the effect of stretching on hundreds of the target DNA in the same microchamber. The target DNA molecules were tethered to the surface and labeled with magnetic beads. Cas12a/crRNA complexes were introduced when the target DNA was stretched. Later, the target DNA was relaxed by removing the magnetic field. Shortly after this step, I observed the cleavage process of the relaxed DNA tethers. This observation has many implications for the CRISPR/Cas12a gene-editing technique. As it can explain the heterogenous targeting efficiencies reported among the cell cultures with the same guide RNA and targeting strategy.

Employing template-directed CRISPR-based editing of the OsALS gene to create herbicide tolerance in Basmati rice.

Rice (Oryza sativa) is one of the primary food crops which contributes major portion of daily calorie intake. It is used as model crop for various genome editing studies. Basmati rice was also explored for establishing non-homologous end joining-based genome editing. But it was not clear whether homology-directed repair (HDR)-based genome editing can be done in Basmati rice. The current study was designed to establish HDR-based genome editing in Basmati rice to develop herbicide tolerance. There is severe weed spread when rice is grown via direct planted rice method in various countries to save labour and water resources. Therefore, the use of herbicides is necessary to control weeds. These herbicides can also affect cultivated rice which creates the need to develop herbicide-tolerant rice. In current study, we introduced a point mutation in Acetolactate Synthase gene to convert tryptophan to leucine at position 548. For this purpose, different constructs for HDR were tested with different RNA scaffold and orientation of repair templates. Out of four different architectures, the one having repair template identical to the target DNA strand precisely edited the target site. We successfully established template-directed CRISPR-Cas9 system in Super Basmati rice by detecting desired substitutions at the target site in Acetolactate Synthase locus. Moreover, this editing of Acetolactate Synthase gene resulted in the production of herbicide tolerance in Super Basmati rice. This study suggests that such type of HDR system can be used to precisely edit other genes for crop improvement.

Twisting and swiveling domain motions in Cas9 to recognize target DNA duplexes, make double-strand breaks, and release cleaved duplexes.

The CRISPR-associated protein 9 (Cas9) has been engineered as a precise gene editing tool to make double-strand breaks. CRISPR-associated protein 9 binds the folded guide RNA (gRNA) that serves as a binding scaffold to guide it to the target DNA duplex via a RecA-like strand-displacement mechanism but without ATP binding or hydrolysis. The target search begins with the protospacer adjacent motif or PAM-interacting domain, recognizing it at the major groove of the duplex and melting its downstream duplex where an RNA-DNA heteroduplex is formed at nanomolar affinity. The rate-limiting step is the formation of an R-loop structure where the HNH domain inserts between the target heteroduplex and the displaced non-target DNA strand. Once the R-loop structure is formed, the non-target strand is rapidly cleaved by RuvC and ejected from the active site. This event is immediately followed by cleavage of the target DNA strand by the HNH domain and product release. Within CRISPR-associated protein 9, the HNH domain is inserted into the RuvC domain near the RuvC active site via two linker loops that provide allosteric communication between the two active sites. Due to the high flexibility of these loops and active sites, biophysical techniques have been instrumental in characterizing the dynamics and mechanism of the CRISPR-associated protein 9 nucleases, aiding structural studies in the visualization of the complete active sites and relevant linker structures. Here, we review biochemical, structural, and biophysical studies on the underlying mechanism with emphasis on how CRISPR-associated protein 9 selects the target DNA duplex and rejects non-target sequences.

Full-Length Model of SaCas9-sgRNA-DNA Complex in Cleavage State.

Staphylococcus aureus Cas9 (SaCas9) is a widely used genome editing tool. Understanding its molecular mechanisms of DNA cleavage could effectively guide the engineering optimization of this system. Here, we determined the first cryo-electron microscopy structure of the SaCas9-sgRNA-DNA ternary complex. This structure reveals that the HNH nuclease domain is tightly bound to the cleavage site of the target DNA strand, and is in close contact with the WED and REC domains. Moreover, it captures the complete structure of the sgRNA, including the previously unresolved stem-loop 2. Based on this structure, we build a full-length model for the ternary complex in cleavage state. This model enables identification of the residues for the interactions between the HNH domain and the WED and REC domains. Moreover, we found that the stem-loop 2 of the sgRNA tightly binds to the PI and RuvC domains and may also regulate the position shift of the RuvC domain. Further mutagenesis and molecular dynamics simulations supported the idea that the interactions of the HNH domain with the WED and REC domains play an important role in the DNA cleavage. Thus, this study provides new mechanistic insights into the DNA cleavage of SaCas9 and is also useful for guiding the future engineering of SaCas9-mediated gene editing systems.

Structural insights into target DNA recognition and cleavage by the CRISPR-Cas12c1 system.

Cas12c is the recently characterized dual RNA-guided DNase effector of type V-C CRISPR-Cas (clustered regularly interspaced short palindromic repeats and CRISPR-associated protein) systems. Due to minimal requirements for a protospacer adjacent motif (PAM), Cas12c is an attractive candidate for genome editing. Here we report the crystal structure of Cas12c1 in complex with single guide RNA (sgRNA) and target double-stranded DNA (dsDNA) containing the 5'-TG-3' PAM. Supported by biochemical and mutation assays, this study reveals distinct structural features of Cas12c1 and the associated sgRNA, as well as the molecular basis for PAM recognition, target dsDNA unwinding, heteroduplex formation and recognition, and cleavage of non-target and target DNA strands. Cas12c1 recognizes the PAM through a mechanism that is interdependent on sequence identity and Cas12c1-induced conformational distortion of the PAM region. Another special feature of Cas12c1 is the cleavage of both non-target and target DNA strands at a single, uniform site with indistinguishable cleavage capacity and order. Location of the sgRNA seed region and minimal length of target DNA required for triggering Cas12c1 DNase activity were also determined. Our findings provide valuable information for developing the CRISPR-Cas12c1 system into an efficient, high-fidelity genome editing tool.

Enhancement of CRISPR/Cas12a trans-cleavage activity using hairpin DNA reporters.

The RNA programmed non-specific (trans) nuclease activity of CRISPR-Cas Type V and VI systems has opened a new era in the field of nucleic acid-based detection. Here, we report on the enhancement of trans-cleavage activity of Cas12a enzymes using hairpin DNA sequences as FRET-based reporters. We discover faster rate of trans-cleavage activity of Cas12a due to its improved affinity (Km) for hairpin DNA structures, and provide mechanistic insights of our findings through Molecular Dynamics simulations. Using hairpin DNA probes we significantly enhance FRET-based signal transduction compared to the widely used linear single stranded DNA reporters. Our signal transduction enables faster detection of clinically relevant double stranded DNA targets with improved sensitivity and specificity either in the presence or in the absence of an upstream pre-amplification step.

An ingenious electrochemical aptasensor for detection of CYFRA 21–1 based on dual-output toehold mediated strand displacement reaction

Herein, a “signal-on” electrochemical aptasensor based on dual-output toehold mediated strand displacement reaction (TMSDR) was developed to detect cytokeratin fragment antigen 21–1 (CYFRA 21–1). First, the methylene blue (MB) labeled signal probe (SP) was combined with a long template strand (TS) to form the three-strands duplex, which was immobilized on magnetic beads. The sequence of TS was innovatively designed with two toehold zones. Thus, with the presence of target DNA and Fuel strand (F), double SP and target were released through TMSDR. Besides, the released target could participate in the next cycle, achieving the further amplification. The released SP was selected by magnetic separation, then added to the gold electrode (GE) to hybridize with the hairpin DNA. Ultimately, the target can be detected by recording the current response of the MB. Under optimized conditions, the response peak current was linear with the concentration of CYFRA 21–1 in range from 10 pM to 1 μM with detection limit of 8.079 pM. The proposed signal amplification strategy was enzyme-free in homogenous solution and the using of magnetic beads improved the recognition efficiency and simplified the experimental steps, which endowed the sensor with super-sensitivity.

Protein and DNA Yield Current Enhancements, Slow Translocations, and an Enhanced Signal-to-Noise Ratio under a Salt Imbalance.

Nanopores are a promising single-molecule sensing device class that captures molecular-level information through resistive or conductive pulse sensing (RPS and CPS). The latter has not been routinely utilized in the nanopore field despite the benefits it could provide, specifically in detecting subpopulations of a molecule. A systematic study was conducted here to study the CPS-based molecular discrimination and its voltage-dependent characteristics. CPS was observed when the cation movement along both electrical and chemical gradients was favored, which led to an ∼3× improvement in SNR (i.e., signal-to-noise ratio) and an ∼8× increase in translocation time. Interestingly, a reversal of the salt gradient reinstates the more conventional resistive pulses and may help elucidate RPS-CPS transitions. The asymmetric salt conditions greatly enhanced the discrimination of DNA configurations including linear, partially folded, and completely folded DNA states, which could help detect subpopulations in other molecular systems. These findings were then utilized for the detection of a Cas9 mutant, Cas9d10a─a protein with broad utilities in genetic engineering and immunology─bound to DNA target strands and the unbound Cas9d10a + sgRNA complexes, also showing significantly longer event durations (>1 ms) than typically observed for proteins.

A Mutated Nme1Cas9 Is a Functional Alternative RNase to Both LwaCas13a and RfxCas13d in the Yeast S. cerevisiae.

CRISPR–Cas systems provide powerful biological tools for genetic manipulation and gene expression regulation. Class 2 systems, comprising type II, type V, and type VI, have the significant advantage to require a single effector Cas protein (Cas9, Cas12, and Cas13 respectively) to cleave nucleic acids upon binding the crRNA. Both Cas9 and Cas12 recognize DNA and induce a double-strand break in it. In contrast, Cas13 bind and cleave RNA exclusively. However, some Cas9 homologs have shown RNase activity as well. Here, we harnessed Nme1Cas9, LwaCas13a, and RfxCas13d to carry out gene downregulation in Saccharomyces cerevisiae by triggering mRNA degradation. To avoid potential DNA damage, we mutated Nme1Cas9 into d16ANme1Cas9 that lost the nuclease activity of the RuvC domain but retained the active HNH domain, able to act on the target DNA strand and, therefore, on the corresponding transcript. Our results showed that d16ANme1Cas9 is a functional RNase in vivo, although with moderate activity since it provoked a fluorescence reduction from 21% to 32%. Interestingly, d16ANme1Cas9 works in a PAM-independent way nor demands helper PAMmer molecules. LwaCas13a and RfxCas13d appeared substantially unfunctional in S. cerevisiae, though they were shown to perform well in mammalian cells. To the best of our knowledge, this is the first report about the working in vivo of a variant of Nme1Cas9 as an RNase and the issues connected with the usage of Cas13 proteins in S. cerevisiae.