Target Complementarity Research Articles

Efficient genome editing in grapevine using CRISPR/LbCas12a system

Clustered regularly interspaced short palindromic repeats (CRISPR) /Cas12a system, also known as CRISPR/Cpf1, has been successfully harnessed for genome engineering in many plants, but not in grapevine yet. Here we developed and demonstrated the efficacy of CRISPR/Cas12a from Lachnospiraceae bacterium ND2006 (LbCas12a) in inducing targeted mutagenesis by targeting the tonoplastic monosaccharide transporter1 (TMT1) and dihydroflavonol-4-reductase 1 (DFR1) genes in 41B cells. Knockout of DFR1 gene altered flavonoid accumulation in dfr1 mutant cells. Heat treatment (34℃) improved the editing efficiencies of CRISPR/LbCas12a system, and the editing efficiencies of TMT1-crRNA1 and TMT1-crRNA2 increased from 35.3% to 44.6% and 29.9% to 37.3% after heat treatment, respectively. Moreover, the sequences of crRNAs were found to be predominant factor affecting editing efficiencies irrespective of the positions within the crRNA array designed for multiplex genome editing. In addition, genome editing with truncated crRNAs (trucrRNAs) showed that trucrRNAs with 20 nt guide sequences were as effective as original crRNAs with 24 nt guides in generating targeted mutagenesis, whereas trucrRNAs with shorter regions of target complementarity ≤ 18 nt in length may not induce detectable mutations in 41B cells. All these results provide evidence for further applications of CRISPR/LbCas12a system in grapevine as a powerful tool for genome engineering.Graphical

Isospin-violating dark matter at liquid noble detectors: new constraints, future projections, and an exploration of target complementarity

There is no known reason that dark matter interactions with the Standard Model should couple to neutrons and protons in the same way. This isospin violation can have large consequences, modifying the sensitivity of existing and future direct detection experimental constraints by orders of magnitude. Previous works in the literature have focused on the zero-momentum limit which has its limitations when extending the analysis to the Non-Relativistic Effective Field Theory basis (NREFT). In this paper, we study isospin violation in a detailed manner, paying specific attention to the experimental setups of liquid noble detectors. We analyse two effective Standard Model gauge invariant models as interesting case studies as well as the more model-independent NREFT operators. This work demonstrates the high degree of complementarity between the target nuclei xenon and argon. Most notably, we show that the Standard Model gauge-invariant formulation of the standard spin-dependent interaction often generates a sizeable response from argon, a target nuclei with zero spin. This work is meant as an update and a useful reference to model builders and experimentalists.

Detection of Pancreatic Cancer miRNA with Biocompatible Nitrogen-Doped Graphene Quantum Dots.

Early-stage pancreatic cancer remains challenging to detect, leading to a poor five-year patient survival rate. This obstacle necessitates the development of early detection approaches based on novel technologies and materials. In this work, the presence of a specific pancreatic cancer-derived miRNA (pre-miR-132) is detected using the fluorescence properties of biocompatible nitrogen-doped graphene quantum dots (NGQDs) synthesized using a bottom-up approach from a single glucosamine precursor. The sensor platform is comprised of slightly positively charged (1.14 0.36 mV) NGQDs bound via stacking and/or electrostatic interactions to the negatively charged (−22.4 6.00 mV) bait ssDNA; together, they form a complex with a 20 nm average size. The NGQDs’ fluorescence distinguishes specific single-stranded DNA sequences due to bait–target complementarity, discriminating them from random control sequences with sensitivity in the micromolar range. Furthermore, this targetability can also detect the stem and loop portions of pre-miR-132, adding to the practicality of the biosensor. This non-invasive approach allows cancer-specific miRNA detection to facilitate early diagnosis of various forms of cancer.

Remission of Persistent Negative Symptoms and Psychosocial Consequences by Combined Clozapine and Cariprazine Treatment in a Patient With Long-Standing Treatment-Resistant Schizoaffective Disorder.

This patient case report describes a 45-year old white unmarried man with disability pension due to schizoaffective disorder, diagnosed at the age of 24. He lives in an apartment and has housing support. Retrospectively, the patient displayed prodromal markers of a disorder within the schizophrenia spectrum many years before the onset of frank psychosis, indeed since childhood. Over the years several symptoms and signs across schizophrenia domains have been manifest: positive, negative, cognitive, and affective, among which the negative and affective symptoms and signs were the earliest to appear. While the positive, disorganized, and catatonic symptoms responded to treatment – when duly tested and complied with – the negative and affective symptoms have been notoriously difficult to handle. We now report on the successful introduction of cariprazine (CAR) to his ongoing clozapine (CLZ) medication, the result of which has been a near-complete remission of his persistent negative and psychosocial issues. We interpret this remarkable alleviation of the patient's disease – and concomitant improvement of his quality of life – in terms of neuroreceptor target complementarity between CLZ and CAR, with particular emphasis on the contributions from the D3 and D2 receptor partial agonist components of the latter agent.

Tertiary motifs as building blocks for the design of protein-binding peptides.

Despite advances in protein engineering, the de novo design of small proteins or peptides that bind to a desired target remains a difficult task. Most computational methods search for binder structures in a library of candidate scaffolds, which can lead to designs with poor target complementarity and low success rates. Instead of choosing from pre‐defined scaffolds, we propose that custom peptide structures can be constructed to complement a target surface. Our method mines tertiary motifs (TERMs) from known structures to identify surface‐complementing fragments or “seeds.” We combine seeds that satisfy geometric overlap criteria to generate peptide backbones and score the backbones to identify the most likely binding structures. We found that TERM‐based seeds can describe known binding structures with high resolution: the vast majority of peptide binders from 486 peptide‐protein complexes can be covered by seeds generated from single‐chain structures. Furthermore, we demonstrate that known peptide structures can be reconstructed with high accuracy from peptide‐covering seeds. As a proof of concept, we used our method to design 100 peptide binders of TRAF6, seven of which were predicted by Rosetta to form higher‐quality interfaces than a native binder. The designed peptides interact with distinct sites on TRAF6, including the native peptide‐binding site. These results demonstrate that known peptide‐binding structures can be constructed from TERMs in single‐chain structures and suggest that TERM information can be applied to efficiently design novel target‐complementing binders.

The impact of operator interference and target complementarity in dark matter direct detection experiments

We present a method to determine the limits on the DM-nucleon interaction strengths for one experiment in the non-relativistic effective theory by taking the interference of operators into account. Further, we extend the method to combine several experiments. To apply the developed methods, we use data from the XENON1T and PICO6O collaborations. The relaxation caused by the interference among operators can be up to four orders of magnitude. The strengthening of the limits by combining the analysis of both experiments can be up to four orders of magnitude.

Improvement criteria for different advanced technologies towards bio-stabilized leachate based on molecular subcategories of DOM

Considering dissolved organic matter (DOM) molecules, the present study is an attempt to unravel the individual removal targets of nine advanced treatment technologies for bio-stabilized landfill leachate. For the eight DOM molecular subcategories, preferable technologies and removal rates were as follows: lipids ‒ powdered activated carbon (PAC) adsorption (97%) and Fenton (97%); proteins ‒ extended electrolysis (92%) and Fenton (92%); and lignins/carboxylic rich alicyclic molecules (CRAM)-like organics ‒ Fenton (90%) and extended electrolysis (75%). As to individual technologies, Fenton, PAC adsorption, extended electrolysis, and reverse osmosis (RO) had the highest removal rates based on the intensity and abundance of DOM. As to the improved technology combinations, “Fenton with PAC adsorption” and “PAC adsorption with reverse osmosis” were then recommended according to the target complementarity in compound intensity and abundance. The study suggested that the treatment strategy of an unknown recalcitrantly biodegraded wastewater could be designed in a tailored way based on the subcategorized DOM characteristics of the refractory wastewater.

Structural and functional analyses reveal the contributions of the C- and N-lobes of Argonaute protein to selectivity of RNA target cleavage

Some gene transcripts have cellular functions as regulatory noncoding RNAs. For example, ∼23-nucleotide (nt)-long siRNAs are loaded into Argonaute proteins. The resultant ribonucleoprotein assembly, the RNA-induced silencing complex (RISC), cleaves RNAs that are extensively base-paired with the loaded siRNA. To date, base complementarity is recognized as the major determinant of specific target cleavage (or slicing), but little is known about how Argonaute inspects base pairing before cleavage. A hallmark of Argonaute proteins is their bilobal structure, but despite the significance of this structure for curtailing slicing activity against mismatched targets, the molecular mechanism remains elusive. Here, our structural and functional studies of a bilobed yeast Argonaute protein and its isolated catalytic C-terminal lobe (C-lobe) revealed that the C-lobe alone retains almost all properties of bilobed Argonaute: siRNA-duplex loading, passenger cleavage/ejection, and siRNA-dependent RNA cleavage. A 2.1 Å-resolution crystal structure revealed that the catalytic C-lobe mirrors the bilobed Argonaute in terms of guide-RNA recognition and that all requirements for transitioning to the catalytically active conformation reside in the C-lobe. Nevertheless, we found that in the absence of the N-terminal lobe (N-lobe), target RNAs are scanned for complementarity only at positions 5-14 on a 23-nt guide RNA before endonucleolytic cleavage, thereby allowing for some off-target cleavage. Of note, acquisition of an N-lobe expanded the range of the guide RNA strand used for inspecting target complementarity to positions 2-23. These findings offer clues to the evolution of the bilobal structure of catalytically active Argonaute proteins.

Enhanced Proofreading Governs CRISPR-Cas9 Targeting Accuracy

The RNA-guided CRISPR-Cas9 nuclease from Streptococcus pyogenes (SpCas9) has been widely repurposed for genome editing. DNA cleavage of Cas9 is controlled by the conformational state of the HNH nuclease domain, but the mechanism that governs HNH activation at on-target DNA while reducing cleavage activity at off-target sites remains poorly understood. Using single-molecule Förster resonance energy transfer (smFRET), we identified an intermediate state of Streptococcus pyogenes Cas9, representing a conformational checkpoint between DNA binding and cleavage. Upon DNA binding, the HNH domain transitions between multiple conformations before docking into its active state. We showed that the high-fidelity (SpCas9-HF1) and enhanced specificity (eSpCas9(1.1)) Cas9 variants are trapped in an inactive state when bound to mismatched targets. We find that a non-catalytic domain within Cas9, REC3, recognizes target complementarity and governs the HNH nuclease to regulate overall catalytic competence. Exploiting this observation, we designed a new hyper-accurate Cas9 variant (HypaCas9) that demonstrates high genome-wide specificity without compromising on-target activity in human cells. These results offer a more comprehensive model to rationalize and modify the balance between target recognition and nuclease activation for precision genome editing.

Enhanced proofreading governs CRISPR-Cas9 targeting accuracy.

The RNA-guided CRISPR-Cas9 nuclease from Streptococcus pyogenes (SpCas9) has been widely repurposed for genome editing1–4. High-fidelity (SpCas9-HF1) and enhanced specificity (eSpCas9(1.1)) variants exhibit substantially reduced off-target cleavage in human cells, but the mechanism of target discrimination and the potential to further improve fidelity were unknown5–9. Using single-molecule Förster resonance energy transfer (smFRET) experiments, we show that both SpCas9-HF1 and eSpCas9(1.1) are trapped in an inactive state10 when bound to mismatched targets. We find that a non-catalytic domain within Cas9, REC3, recognizes target complementarity and governs the HNH nuclease to regulate overall catalytic competence. Exploiting this observation, we designed a new hyper-accurate Cas9 variant (HypaCas9) that demonstrates high genome-wide specificity without compromising on-target activity in human cells. These results offer a more comprehensive model to rationalize and modify the balance between target recognition and nuclease activation for precision genome editing.

Target RNA Secondary Structure Is a Major Determinant of miR159 Efficacy.

In plants, microRNA (miRNA)-target complementarity has long been considered the predominant factor determining the silencing outcome of the miRNA-target interaction, although the efficacy of such interactions have rarely been appraised in plants. Here, we perform in planta silencing efficacy assays on seven Arabidopsis MYB genes, all of which contain conserved miR159-binding sites of analogous complementarity. These genes were found to be differentially silenced by miR159; MYB81, MYB97, MYB101, MYB104, and DUO1 were all poorly silenced, whereas MYB33 and MYB65 were strongly silenced. Curiously, this is consistent with previous genetic analysis defining MYB33 and MYB65 as the major functional targets of miR159. Neither the free energy of miR159-target complementarity, nor miRNA binding site accessibility, as determined by flanking region AU content, could fully explain the discrepancy of miR159 silencing efficacy. Instead, we found that MYB33 and MYB65 were both predicted to contain a distinctive RNA secondary structure abutting the miR159 binding site. The structure is composed of two stem-loops (SLs) that are predicted to form in MYB33/65 homologs of species as evolutionary distant as gymnosperms. Functional analysis found that the RNA structure in MYB33 correlated with strong silencing efficacy; introducing mutations to disrupt either SL attenuated miR159 efficacy, while introducing complementary mutations to restore the SLs, but not the sequence, restored strong miR159-mediated silencing. Therefore, it appears that this RNA secondary structure demarcates MYB33/65 as sensitive targets of miR159, which underpins the narrow functional specificity of Arabidopsis miR159.

Reconfiguring the firm’s core technological portfolio through open innovation: focusing on technological M&A

PurposeThe purpose of this study is to investigate the effects of open innovation, especially focusing on technological M&A, on subsequent innovation and changes to the firm’s core technological portfolio.Design/methodology/approachThe study suggests three types of core technological areas, based on prior focus and experience in technological categories. These are 1) the existing core area, in which the acquirer firm retains its knowledge and expertise, 2) the enhanced core area, where knowledge and expertise in the acquirer firm’s insufficient areas are strengthened, and 3) the new core area, i.e. new knowledge fields in which the acquirer firm ventures into. The study then analyzes the effects of two key knowledge characteristics of the target firm, similarity and complementarity, on post-M&A innovation outcomes in each of the three core technological areas.FindingsThe results confirm that while none of the investigated knowledge characteristics of the target firm is advantageous for post-M&A innovation outcomes in existing core areas, similarity of the target firm does facilitate post-M&A innovation outcomes in enhanced core areas. Moreover, the results confirm that complementarity of the target firm is beneficial for post-M&A innovation outcomes in new core areas.Originality/valueThe study explains the reconfiguration mechanism of a firm’s core technological portfolio. It also suggests an extended framework to analyze innovation outcomes in more detail. Moreover, the study helps to explain why most M&As result in failure.

A Dynamic Search Process Underlies MicroRNA Targeting

Argonaute proteins play a central role in mediating post-transcriptional gene regulation by microRNAs (miRNAs). Argonautes use the nucleotide sequences in miRNAs as guides for identifying target messenger RNAs for repression. Here, we used single-molecule FRET to directly visualize how human Argonaute-2 (Ago2) searches for and identifies target sites in RNAs complementary to its miRNA guide. Our results suggest that Ago2 initially scans for target sites with complementarity to nucleotides 2-4 of the miRNA. This initial transient interaction propagates into a stable association when target complementarity extends to nucleotides 2-8. This stepwise recognition process is coupled to lateral diffusion of Ago2 along the target RNA, which promotes the target search by enhancing the retention of Ago2 on the RNA. The combined results reveal the mechanisms that Argonaute likely uses to efficiently identify miRNA target sites within the vast and dynamic agglomeration of RNA molecules in the living cell.

Complementarity of dark matter detectors in light of the neutrino background

Direct detection dark matter experiments looking for WIMP-nucleus elastic scattering will soon be sensitive to an irreducible background from neutrinos which will drastically affect their discovery potential. Here we explore how the neutrino background will affect future ton-scale experiments considering both spin-dependent and spin-independent interactions. We show that combining data from experiments using different targets can improve the dark matter discovery potential due to target complementarity. We find that in the context of spin-dependent interactions, combining results from several targets can greatly enhance the subtraction of the neutrino background for WIMP masses below 10 GeV/c$^2$ and therefore probe dark matter models to lower cross-sections. In the context of target complementarity, we also explore how one can tune the relative exposures of different target materials to optimize the WIMP discovery potential.

Determinants beyond Both Complementarity and Cleavage Govern MicroR159 Efficacy in Arabidopsis

Plant microRNAs (miRNAs) are critical regulators of gene expression, however little attention has been given to the principles governing miRNA silencing efficacy. Here, we utilize the highly conserved Arabidopsis miR159-MYB33/MYB65 regulatory module to explore these principles. Firstly, we show that perfect central complementarity is not required for strong silencing. Artificial miR159 variants with two cleavage site mismatches can potently silence MYB33/MYB65, fully complementing a loss-of-function mir159 mutant. Moreover, these miR159 variants can cleave MYB33/MYB65 mRNA, however cleavage appears attenuated, as the ratio of cleavage products to full length transcripts decreases with increasing central mismatches. Nevertheless, high levels of un-cleaved MYB33/MYB65 transcripts are strongly silenced by a non-cleavage mechanism. Contrary to MIR159a variants that strongly silenced endogenous MYB33/MYB65, artificial MYB33 variants with central mismatches to miR159 are not efficiently silenced. We demonstrate that differences in the miRNA:target mRNA stoichiometry underlie this paradox. Increasing miR159 abundance in the MYB33 variants results in a strong silencing outcome, whereas increasing MYB33 transcript levels in the MIR159a variants results in a poor silencing outcome. Finally, we identify highly conserved nucleotides that flank the miR159 binding site in MYB33, and demonstrate that they are critical for efficient silencing, as mutation of these flanking nucleotides attenuates silencing at a level similar to that of central mismatches. This implies that the context in which the miRNA binding site resides is a key determinant in controlling the degree of silencing and that a miRNA “target site” encompasses sequences that extend beyond the miRNA binding site. In conclusion, our findings dismiss the notion that miRNA:target complementarity, underpinned by central matches, is the sole dictator of the silencing outcome.

Improving CRISPR-Cas nuclease specificity using truncated guide RNAs.

Clustered, regularly interspaced, short palindromic repeat (CRISPR) RNA-guided nucleases (RGNs) are highly efficient genome editing tools. CRISPR-associated 9 (Cas9) RGNs are directed to genomic loci by guide RNAs (gRNAs) containing 20 nucleotides that are complementary to a target DNA sequence. However, RGNs can induce mutations at sites that differ by as many as five nucleotides from the intended target. Here we report that truncated gRNAs, with shorter regions of target complementarity <20 nucleotides in length, can decrease undesired mutagenesis at some off-target sites by 5,000-fold or more without sacrificing on-target genome editing efficiencies. In addition, use of truncated gRNAs can further reduce off-target effects induced by pairs of Cas9 variants that nick DNA (paired nickases). Our results delineate a simple, effective strategy to improve the specificities of Cas9 nucleases or paired nickases.

AutomiG, a Biosensor to Detect Alterations in miRNA Biogenesis and in Small RNA Silencing Guided by Perfect Target Complementarity

Defects in miRNA biogenesis or activity are associated to development abnormalities and diseases. In Drosophila, miRNAs are predominantly loaded in Argonaute-1, which they guide for silencing of target RNAs. The miRNA pathway overlaps the RNAi pathway in this organism, as miRNAs may also associate with Argonaute-2, the mediator of RNAi. We set up a gene construct in which a single inducible promoter directs the expression of the GFP protein as well as two miRNAs perfectly matching the GFP sequences. We show that self-silencing of the resulting automiG gene requires Drosha, Pasha, Dicer-1, Dicer-2 and Argonaute-2 loaded with the anti-GFP miRNAs. In contrast, self-silencing of the automiG gene does not involve Argonaute-1. Thus, automiG reports in vivo for both miRNA biogenesis and Ago-2 mediated silencing, providing a powerful biosensor to identify situations where miRNA or siRNA pathways are impaired. As a proof of concept, we used automiG as a biosensor to screen a chemical library and identified 29 molecules that strongly inhibit miRNA silencing, out of which 5 also inhibit RNAi triggered by long double-stranded RNA. Finally, the automiG sensor is also self-silenced by the anti-GFP miRNAs in HeLa cells and might be easily used to identify factors involved in miRNA biogenesis and silencing guided by perfect target complementarity in mammals.

What can(not) be measured with ton-scale dark matter direct detection experiments

Direct searches for dark matter have prompted in recent years a great deal of excitement within the astroparticle physics community, but the compatibility between signal claims and null results of different experiments is far from being a settled issue. In this context, we study here the prospects for constraining the dark matter parameter space with the next generation of ton-scale detectors. Using realistic experimental capabilities for a wide range of targets (including fluorine, sodium, argon, germanium, iodine and xenon), the role of target complementarity is analysed in detail while including the impact of astrophysical uncertainties in a self-consistent manner. We show explicitly that a multi-target signal in future direct detection facilities can determine the sign of the ratio of scalar couplings fn/fp, but not its scale. This implies that the scalar-proton cross-section is left essentially unconstrained if the assumption fp ∼ fn is relaxed. Instead, we find that both the axial-proton cross-section and the ratio of axial couplings an/ap can be measured with fair accuracy if multi-ton instruments using sodium and iodine will eventually come online. Moreover, it turns out that future direct detection data can easily discriminate between elastic and inelastic scatterings. Finally, we argue that, with weak assumptions regarding the WIMP couplings and the astrophysics, only the dark matter mass and the inelastic parameter (i.e. mass splitting) may be inferred from the recoil spectra — specifically, we anticipate an accuracy of tens of GeV (tens of keV) in the measurement of the dark matter mass (inelastic parameter).

Individual mRNA expression profiles reveal the effects of specific microRNAs

The effect of a microRNA on the levels of its target mRNAs can be measured within a single gene expression profile

Selecting effective siRNAs based on guide RNA structure.

In RNA interference, guide RNAs direct RNA-induced silencing complexes to mRNA targets, mediating cleavage and ultimately leading to gene silencing. We have observed that unstructured guide strands, which either completely lack complementary bases or in which internal base pairing is thermodynamically unlikely, confer strongest silencing, whereas structures with base-paired ends are inactive. Thus, the structure of the guide strand represents a major determinant of small interfering RNA activity. Here we describe a detailed computational protocol for identification of unstructured guide strands for a given mRNA target sequence. Sequentially, all guide sequences with target complementarity are simulated, their corresponding structures are folded and unstructured guide strands are selected and rated according to thermodynamic parameters. Although this procedure is new and remains to be validated by the community, it allows reliable identification of highly active siRNAs that can be used for functional target validation or drug development.