Double-stranded DNA Targets Research Articles

Structure Reveals a Mechanism of CRISPR-RNA-Guided Nuclease Recruitment and Anti-CRISPR Viral Mimicry

Bacteria and archaea have evolved sophisticated adaptive immune systems that rely on CRISPR RNA (crRNA)-guided detection and nuclease-mediated elimination of invading nucleic acids. Here, we present the cryo-electron microscopy (cryo-EM) structure of the type I-F crRNA-guided surveillance complex(Csy complex) from Pseudomonas aeruginosa bound to a double-stranded DNA target. Comparison of this structure to previously determined structures of this complex reveals a ∼180-degree rotation of the C-terminal helical bundle on the "large" Cas8f subunit. We show that the double-stranded DNA (dsDNA)-induced conformational change in Cas8f exposes a Cas2/3 "nuclease recruitment helix" that is structurally homologous to a virally encoded anti-CRISPR protein (AcrIF3). Structural homology between Cas8f and AcrIF3 suggests that AcrIF3 is a mimic of the Cas8f nuclease recruitment helix.

Target-dependent nickase activities of the CRISPR-Cas nucleases Cpf1 and Cas9.

Clustered regularly interspaced short palindromic repeat (CRISPR) machineries are prokaryotic immune systems that have been adapted as versatile gene editing and manipulation tools. We found that CRISPR-Cas nucleases Cpf1 (also known as Cas12a) and Cas9 exhibit differential guide RNA sequence requirements for cleavage of the two strands of target DNA in vitro. As a consequence of the differential guide RNA requirements, both Cas9 and Cpf1 enzymes can exhibit potent nickase activities on an extensive class of mismatched dsDNA targets. These properties allow the production of efficient nickases for a chosen dsDNA target sequence, without modification of the nuclease protein, using guide RNAs with a variety of patterns of mismatch to the intended DNA target. In parallel to the nicking activities observed with purified Cas9 in vitro, we observed sequence-dependent nicking for both perfectly matched and partially mismatched target sequences in a Sacchromyces cerevisae system. Our findings have implications for CRISPR spacer acquisition, off-target potential of CRISPR gene editing/manipulation, and tool development using homology directed nicking.

Polymerase Chain Reaction-Dynamic Light Scattering Sensor for DNA and Protein by Using Both Replication and Cleavage Properties of Taq Polymerase.

The incorporation of AuNPs into polymerase chain reaction (PCR) has become a promising strategy to develop sensitive sensing platforms, due to desirable optical properties of AuNPs and the exponential amplification power of PCR. However, the combination of AuNPs to PCR usually fails to reach expected sensitivity along with additional steps. Here we report a one-step and universal PCR-based biosensor for size-dependent detection of nucleic acids and proteins by using the dynamic light scattering (DLS) technique. This sensing system employs a DNA modified AuNPs (AuNPs-DNA) probe to serve as the TaqMan like signal probe, in which DNA on the surface of AuNPs can be cleaved by Taq polymerase during the extension process of PCR, causing the aggregation of AuNPs to be detected by DLS. This strategy enables sequence-specific recognition of target double-stranded DNA (dsDNA), overcoming the background signal change that triggered by the increase of the PCR cycle. It further demonstrates its applicability in detecting a foodborne pathogen Listeria monocytogenes with a detection limit of 1.2 fg μL-1, which is more sensitive than colorimetric methods. Moreover, by utilizing a proximity assay strategy, this biosensor shows the capability to the homogeneous detection of protein, showing a detection limit of 1.0 pM for a model thrombin. Together, the work demonstrates a reliable strategy to incorporate AuNPs into PCR amplification process as a signal probe, instead of the post-PCR process.

Structural insights into a high fidelity variant of SpCas9

The RNA-guided endonucleases of the CRISPR-Cas9 system, including the most widely used Cas9 from Streptococcus pyogenes (SpCas9), are becoming a robust genome editing tool in model organisms and hold immense promise for therapeutic applications. Many strategies have been employed to overcome the limitations caused by SpCas9’s off-target effects and its stringent requirement for the protospacer adjacent motif (PAM) sequence. However, the structural mechanisms underlying these strategies remain undefined. Here, we present crystal structure of a SpCas9 variant, xCas9 3.7 that has broad PAM compatibility and high DNA targeting specificity, in complex with a single-guide RNA and its double-stranded DNA targets. Structural comparison revealed that salt bridge-stabilized R1335 is critical for the stringent selection of PAM sequence by SpCas9. Unrestricted rotamerization of this residue by the E1219V mutation in xCas9 3.7 lessens the stringency for PAM recognition and allows SpCas9 to recognize multiple PAM sequences as further supported by biochemical data. Compared to those in wild-type (WT) SpCas9, REC2 and REC3 domains in xCas9 3.7 undergo striking conformational changes, leading to reduced contact with DNA substrate. SpCas9 mutants engineered to display less interaction with DNA and have conformationally more flexible REC2 and REC3 domains display enhanced specificity for DNA substrates in both biochemical and cellular assays. Taken together, our findings reveal the structural mechanisms underlying the broadened PAM compatibility and high DNA fidelity of xCas9 3.7, which can assist rational engineering of more efficient SpCas9 variants and probably other Cas9 orthologs.

Mechanistic Insights into the cis- and trans-Acting DNase Activities of Cas12a

CRISPR-Cas12a (Cpf1) is an RNA-guided DNA-cutting nuclease that has been repurposed for genome editing. Upon target DNA binding, Cas12a cleaves both the target DNA in cis and non-target single-stranded DNAs (ssDNAs) in trans. To elucidate the molecular basis for both DNase cleavage modes, we performed structural and biochemical studies on Francisella novicida Cas12a. We show that guide RNA-target strand DNA hybridization conformationally activates Cas12a, triggering its trans-acting, non-specific, single-stranded DNase activity. In turn, cis cleavage of double-stranded DNA targets is a result of protospacer adjacent motif (PAM)-dependent DNA duplex unwinding, electrostatic stabilization of the displaced non-target DNA strand, and ordered sequential cleavage of the non-target and target DNA strands. Cas12a releases the PAM-distal DNA cleavage product and remains bound to the PAM-proximal DNA cleavage product in a catalytically competent, trans-active state. Together, these results provide a revised model for the molecular mechanisms of both the cis- and the trans-acting DNase activities of Cas12a enzymes, enabling their further exploitation as genome editing tools.

Assessing Oligonucleotide Binding to Double-Stranded DNA.

Sequence-specific targeting of double-stranded DNA (dsDNA) using synthetic oligonucleotides (ONs) has been under investigation in different therapeutic approaches. Several methods can be used to evaluate ONs effect and binding capacity to their target sequence. Here we describe some of the methods, which have been frequently used for assessing ONs binding to dsDNA.

Phage AcrIIA2 DNA Mimicry: Structural Basis of the CRISPR and Anti-CRISPR Arms Race

CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR-associated proteins) systems provide prokaryotic cells with adaptive immunity against invading bacteriophages. Bacteriophages counteract bacterial responses by encoding anti-CRISPR inhibitor proteins (Acr). However, the structural basis for their inhibitory actions remains largely unknown. Here, we report the crystal structure of the AcrIIA2-SpyCas9-sgRNA (single-guide RNA) complex at 3.3Å resolution. We show thatAcrIIA2 binds SpyCas9 at a position similar to the target DNA binding region. More specifically, AcrIIA2 interacts with the protospacer adjacent motif (PAM) recognition residues of Cas9, preventing target double-stranded DNA (dsDNA) detection. Thus, phage-encoded AcrIIA2 appears to act as a DNA mimic that blocks subsequent dsDNA binding by virtue of its highly acidic residues, disabling bacterial Cas9 by competing with target dsDNA binding with a binding motif distinct from AcrIIA4. Our study provides a more detailed mechanistic understanding of AcrIIA2-mediated inhibition of SpyCas9, the most widely used genome-editing tool, opening new avenues for improved regulatory precision during genome editing.

CRISPR: No Sign of Slowing Down

CRISPR: No Sign of Slowing Down

Polymer-mineral scaffold augments in vivo equine multipotent stromal cell osteogenesis

BackgroundUse of bioscaffolds to direct osteogenic differentiation of adult multipotent stromal cells (MSCs) without exogenous proteins is a contemporary approach to bone regeneration. Identification of in vivo osteogenic contributions of exogenous MSCs on bioscaffolds after long-term implantation is vital to understanding cell persistence and effect duration.MethodsThis study was designed to quantify in vivo equine MSC osteogenesis on synthetic polymer scaffolds with distinct mineral combinations 9 weeks after implantation in a murine model. Cryopreserved, passage (P)1, equine bone marrow-derived MSCs (BMSC) and adipose tissue-derived MSCs (ASC) were culture expanded to P3 and immunophenotyped with flow cytometry. They were then loaded by spinner flask on to scaffolds composed of tricalcium phosphate (TCP)/hydroxyapatite (HA) (40:60; HT), polyethylene glycol (PEG)/poly-l-lactic acid (PLLA) (60:40; GA), or PEG/PLLA/TCP/HA (36:24:24:16; GT). Scaffolds with and without cells were maintained in static culture for up to 21 days or implanted subcutaneously in athymic mice that were radiographed every 3 weeks up to 9 weeks. In vitro cell viability and proliferation were determined. Explant composition (double-stranded (ds)DNA, collagen, sulfated glycosaminoglycan (sGAG), protein), equine and murine osteogenic target gene expression, microcomputed tomography (μCT) mineralization, and light microscopic structure were assessed.ResultsThe ASC and BMSC number increased significantly in HT constructs between 7 and 21 days of culture, and BMSCs increased similarly in GT constructs. Radiographic opacity increased with time in GT-BMSC constructs. Extracellular matrix (ECM) components and dsDNA increased significantly in GT compared to HT constructs. Equine and murine osteogenic gene expression was highest in BMSC constructs with mineral-containing scaffolds. The HT constructs with either cell type had the highest mineral deposition based on μCT. Regardless of composition, scaffolds with cells had more ECM than those without, and osteoid was apparent in all BMSC constructs.ConclusionsIn this study, both exogenous and host MSCs appear to contribute to in vivo osteogenesis. Addition of mineral to polymer scaffolds enhances equine MSC osteogenesis over polymer alone, but pure mineral scaffold provides superior osteogenic support. These results emphasize the need for bioscaffolds that provide customized osteogenic direction of both exo- and endogenous MSCs for the best regenerative potential.

Programmable RNA Cleavage and Recognition by a Natural CRISPR-Cas9 System from Neisseria meningitidis

The microbial CRISPR systems enable adaptive defense against mobile elements and also provide formidable tools for genome engineering. The Cas9 proteins are type II CRISPR-associated, RNA-guided DNA endonucleases that identify double-stranded DNA targets by sequence complementarity and protospacer adjacent motif (PAM) recognition. Here we report that the type II-C CRISPR-Cas9 from Neisseria meningitidis (Nme) is capable of programmable, RNA-guided, site-specific cleavage and recognition of single-stranded RNA targets and that this ribonuclease activity is independent of the PAM sequence. We define the mechanistic feature and specificity constraint for RNA cleavage by NmeCas9 and also show that nuclease null dNmeCas9 binds to RNA target complementary to CRISPR RNA. Finally, we demonstrate that NmeCas9-catalyzed RNA cleavagecan be blocked by three families of type II-C anti-CRISPR proteins. These results fundamentally expand the targeting capacities of CRISPR-Cas9 and highlight the potential utility of NmeCas9 as a single platform to target both RNA and DNA.

How Well Can DNA Rupture DNA? Shearing and Unzipping Forces inside DNA Nanostructures.

A purely DNA nanomachine must support internal stresses across short DNA segments with finite rigidity, producing effects that can be qualitatively very different from experimental observations of isolated DNA in fixed-force ensembles. In this article, computational simulations are used to study how well the rigidity of a driving DNA duplex can rupture a double-stranded DNA target into single-stranded segments and how well this stress can discriminate between unzipping or shearing geometries. This discrimination is found to be maximized at an optimal length but deteriorates as the driving duplex is either lengthened or shortened. This differs markedly from a fixed-force ensemble and has implications for the design parameters and limitations of dynamic DNA nanomachines.

Cryo-EM Structures Reveal Mechanism and Inhibition of DNA Targeting by a CRISPR-Cas Surveillance Complex

Prokaryotic cells possess CRISPR-mediated adaptive immune systems that protect them from foreign genetic elements, such as invading viruses. A central element of this immune system is an RNA-guided surveillance complex capable of targeting non-self DNA or RNA for degradation in a sequence- and site-specific manner analogous to RNA interference. Although the complexes display considerable diversity in their composition and architecture, many basic mechanisms underlying target recognition and cleavage are highly conserved. Using cryoelectron microscopy (cryo-EM), we show that the binding of target double-stranded DNA (dsDNA) to a type I-F CRISPR system yersinia (Csy) surveillance complex leads to large quaternary and tertiary structural changes in the complex that are likely necessary in the pathway leading to target dsDNA degradation by a trans-acting helicase-nuclease. Comparison of the structure of the surveillance complex before and after dsDNA binding, or in complex with three virally encoded anti-CRISPR suppressors that inhibit dsDNA binding, reveals mechanistic details underlying target recognition and inhibition.

Diagnosis of Tuberculosis Using Colorimetric Gold Nanoparticles on a Paper-Based Analytical Device.

We have developed a colorimetric sensing strategy employing gold nanoparticles and a paper-based analytical platform for the diagnosis of tuberculosis (TB). By utilizing the surface plasmon resonance effect, we were able to monitor changes in the color of a gold nanoparticle colloid based on the effects of single-stranded DNA probe molecules hybridizing with targeted double-stranded TB DNA. The hybridization event changes the surface charge density of the nanoparticles, causing them to aggregate to various degrees, which modifies the color of the solution in a manner that can be readily measured to determine the concentration of the targeted DNA analyte. In order to adapt this TB diagnosis method to resource-limited settings, we extended this label-free oligonucleotide and unmodified gold nanoparticle solution-based technique to a paper-based system that can be measured using a smartphone to obtain rapid parallel colorimetric results with low reagent consumption and without the need for sophisticated analytical equipment. In this study, we investigated various assay conditions, including the denaturing temperature and time, different oligonucleotide probe sequences, as well as the ratio of single stranded probe and double stranded target DNA. After optimizing these variables, we were able to achieve a detection limit of 1.95 × 10-2 ng/mL for TB DNA. Furthermore, multiple tests could be performed simultaneously with a 60 min turnaround time.

Karmoisin and cardinal ortholog genes participate in the ommochrome synthesis of Nilaparvata lugens (Hemiptera: Delphacidae).

Ommochrome is the major source for eye coloration of all insect species so far examined. Phenoxazinone synthetase (PHS) has always been regarded as the terminal step enzyme for ommochrome formation, which is encoded by cardinal or karmoisin genes. Our previous study indicated that the karmoisin ortholog gene (Nl-karmoisin) product in the brown planthopper (BPH) was a monocarboxylate transporter, while not a PHS. Here, based on full-length complementary DNA, the cardinal ortholog gene in BPH (Nl-cardinal) product was predicted to be a haem peroxidase rather than a PHS. We suggest for the first time that neither karmoisin nor cardinal encodes the PHS, but whether PHS participates in BPH eye pigmentation needs further research. Nymphal RNA interference (RNAi) experiments showed that knockdown Nl-cardinal transcript led the BPH ocelli and compound eye to color change from brown to red, while knockdown Nl-karmoisin only made the ocelli present the red phenotype. Notably, not only the Nl-cardinal transcript, dscd injection (Nl-cardinal targeting double-stranded DNA (dsRNA)) also significantly reduced the Nl-karmoisin transcript by 33.7%, while dska (Nl-karmoisin targeting dsRNA) injection did not significantly change the Nl-cardinal transcript. Considering the above RNAi and quantitative real-time polymerase chain reaction results, we propose that Nl-cardinal plays a more important role in ommochrome synthesis than Nl-karmoisin, and it may be an upstream gene of Nl-karmoisin. The present study suggested that both karmoisin and cardinal ortholog genes play a role in ommochrome synthesis in a hemimetabolous insect.

Structural Basis for the Canonical and Non-canonical PAM Recognition by CRISPR-Cpf1

The RNA-guided Cpf1 (also known as Cas12a) nuclease associates with a CRISPR RNA (crRNA) and cleaves the double-stranded DNA target complementary to the crRNA guide. The two Cpf1 orthologs from Acidaminococcus sp. (AsCpf1) and Lachnospiraceae bacterium (LbCpf1) have been harnessed for eukaryotic genome editing. Cpf1 requires a specific nucleotide sequence, called a protospacer adjacent motif (PAM), for target recognition. Besides the canonical TTTV PAM, Cpf1 recognizes suboptimal C-containing PAMs. Here, we report four crystal structures of LbCpf1 in complex with the crRNA and its target DNA containing either TTTA, TCTA, TCCA, or CCCA as the PAM. These structures revealed that, depending on the PAM sequences, LbCpf1 undergoes conformational changes to form altered interactions with the PAM-containing DNA duplexes, thereby achieving the relaxed PAM recognition. Collectively, the present structures advance our mechanistic understanding of the PAM-dependent, crRNA-guided DNA cleavage by the Cpf1 family nucleases.

Structural Basis for the Altered PAM Recognition by Engineered CRISPR-Cpf1

The RNA-guided Cpf1 nuclease cleaves double-stranded DNA targets complementary to the CRISPR RNA (crRNA), and it has been harnessed for genome editing technologies. Recently, Acidaminococcus sp. BV3L6 (AsCpf1) was engineered to recognize altered DNA sequences as the protospacer adjacent motif (PAM), thereby expanding the target range ofCpf1-mediated genome editing. Whereas wild-type AsCpf1 recognizes the TTTV PAM, the RVR (S542R/K548V/N552R) and RR (S542R/K607R) variants can efficiently recognize the TATV and TYCV PAMs, respectively. However, their PAM recognition mechanisms remained unknown. Here we present the 2.0Å resolution crystal structures of the RVR and RR variants bound to a crRNA and its target DNA. The structures revealed that the RVR and RR variants primarily recognize the PAM-complementary nucleotides via the substituted residues. Our high-resolution structures delineated the altered PAM recognition mechanisms of the AsCpf1 variants, providing a basis for the further engineering of CRISPR-Cpf1.

Pipette tip biosensors for bacterial double-stranded DNA using bioluminescence induced by zinc finger luciferase

The authors describe a pipette type of biosensor for detecting target genes and using a zinc finger protein fused to luciferase (ZF luciferase). The ZF protein binds to a specific DNA sequence, and the target double-stranded (ds) DNA can be detected by monitoring the enzymatic activity of ZF luciferase. A small avidin-immobilized reaction plate is placed on a plastic pipette tip (referred to as Biologi tip). The dsDNA detection procedures are carried out by using a programmable dispensing robot equipped with a photodetector. These procedures include (a) the aspiration of an analyte to capture the biotinylated target dsDNA (a product of a polymerase chain reaction) on the small reaction plate inside the pipette tip, (b) the introduction of ZF luciferase and luciferin into the pipette tip, and (c) migration of the pipette tip to the detection port to measure bioluminescence on the small reaction plate. The emission originating from luciferase activity is observed on the reaction plate containing immobilized biotin-tagged target dsDNA, whereas plates containing non-target or biotinylated single-stranded DNA only do not yield a signal. The intensity of emission increases proportionally to the concentration of dsDNA, and the detection limit of the target dsDNA is as low as 62 pM. An actual genomic DNA sample from Escherichia coli O157 was successfully detected by this automatic analyzer using the Biologi tip equipped with a reaction plate. This indicates that this system has a large potential for practical applications, including in particular point-of-care analyses in hygiene control, food safety testing, and clinical diagnosis.

Does Cas9-Catalyzed DNA Cleavage Generate Blunt Ends or Staggered Ends? Insight from Molecular Dynamic Simulations

The CRISPR-associated endonuclease Cas9 from Streptococcus pyogenes (spCas9) along with a single guide RNA (sgRNA) has merged as a versatile toolbox for genome editing. Despite recent advances in the mechanistic studies on spCas9-sgRNA-mediated target double-stranded DNA (dsDNA) recognition and cleavage, it is still unclear how the catalytic Mg2+ ions induce the conformation changes toward the catalytic active state. It also remains controversial whether Cas9 generates blunt-ended or staggered-ended breaks with overhangs in the target DNA. To investigate these issues, here we performed the first all-atom molecular dynamics simulations of the spCas9-sgRNA-dsDNA system with and without Mg2+ bound. The simulation results show that binding of two Mg2+ ions at the RuvC domain active site could lead to structurally and energetically favorable coordination ready for the non-target DNA strand cleavage. Importantly, we argue that Cas9-catalyzed target DNA cleavage produces 1-bp staggered ends rather than generally assumed blunt ends.

Guiding the Self-Assembly of RecA Proteinfilaments on DNA Scaffolds to Create Rationally Designed Nanostructures

Over the past three decades DNA self-assembly has been used to create increasingly complex nanoscale structures. DNA nanotubes with tunable persistence length have provided an attractive model system for protein filaments and structures with rationally defined nanoscale shapes, made using the “DNA origami” technique are promising for applications such as drug delivery and nano-plasmonics. However, the scale-up necessary for many DNA origami applications remains prohibited by cost and less than perfect yields. We seek to combine the ability of rational design afforded by DNA origami with the cost-efficient, high yield assembly of protein filaments to enable the construction of nanostructures with new functionalities. For a protein to be used in combination with DNA nanostructures, it must interact with DNA in a controllable way. RecA protein is an ideal choice because of its ability to form rigid filaments on DNA. Here we demonstrate the construction of a nanostructures including tetrahedra, rectangular shapes as well as micrometer scale filaments from DNA origami and RecA protein. We first fold specific sections of M13 single-stranded DNA, using the DNA origami technique, to create a mechanically flexible skeleton. Unfolded sections of the M13 DNA sequence are made double-stranded using a DNA polymerase gap-filling reaction. This skeleton is then rigidified by the addition of RecA, which self-assembles into filaments of defined length at specific double-stranded DNA target sites. Our method greatly improves upon conventional DNA origami in several ways. By reducing the number of distinct input components, we not only reduce cost but also the number of possible mis-folding events, allowing for higher yield nanostructures. Furthermore, the direct incorporation of proteins with origami adds a new level of functionality, thus broadening the scope of potential origami applications.

Inhibition of CRISPR/Cas9-Mediated Genome Engineering by a Type I Interferon-Induced Reduction in Guide RNA Expression.

Clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9-mediated genome engineering technology is a powerful tool for generation of cells and animals with engineered mutations in their genomes. In order to introduce the CRISPR/Cas9 system into target cells, nonviral and viral vectors are often used; however, such vectors trigger innate immune responses associated with production of type I interferons (IFNs). We have recently demonstrated that type I IFNs inhibit short-hairpin RNA-mediated gene silencing, which led us to hypothesize that type I IFNs may also inhibit CRISPR/Cas9-mediated genome mutagenesis. Here we investigated this hypothesis. A single-strand annealing assay using a reporter plasmid demonstrated that CRISPR/Cas9-mediated cleavage efficiencies of the target double-stranded DNA were significantly reduced by IFNα. A mismatch recognition nuclease-dependent genotyping assay also demonstrated that IFNα reduced insertion or deletion (indel) mutation levels by approximately half. Treatment with IFNα did not alter Cas9 protein expression levels, whereas the copy numbers of guide RNA (gRNA) were significantly reduced by IFNα stimulation. These results indicate that type I IFNs significantly reduce gRNA expression levels following introduction of the CRISPR/Cas9 system in the cells, leading to a reduction in the efficiencies of CRISPR/Cas9-mediated genome mutagenesis. Our findings provide important clues for the achievement of efficient genome engineering using the CRISPR/Cas9 system.