Expanding the CRISPR toolbox in Culicine mosquitoes: in vitro validation of Pol III promoters

CRISPR–Cas9-based “gene drive” technologies have been proposed as a novel and effective means of controlling human diseases vectored by mosquitoes. However, more complex designs than those demonstrated to date—and an expanded molecular toolbox with which to build them—will be required to overcome the issues of resistance formation/evolution and drive spatial/temporal limitation. Foreseeing this need, we assessed the sgRNA transcriptional activities of 33 phylogenetically diverse insect Polymerase III promoters using three disease-relevant Culicine mosquito cell lines (Aedes aegypti, Aedes albopictus, and Culex quinquefasciatus). We show that U6 promoters work across species with a range of transcriptional activity levels and find 7SK promoters to be especially promising because of their broad phylogenetic activity. We further show that U6 promoters can be substantially truncated without affecting transcriptional levels. These results will be of great utility to researchers involved in developing the next generation of gene drives

Developing a cell-based fluorescent assay for screening Dicer-activating compounds

MicroRNAs are ~22 nucleotide long RNA strands which regulate gene expression by binding to the 3’UTRs of messenger RNAs. MicroRNAs are predicted to regulate about a half of all protein-coding genes in the human genome thus affecting many cellular processes. One crucial part of microRNA biogenesis is the cleaving of pre-miRNA strands into mature microRNAs by the type III RNase enzyme, Dicer. Dicer has been shown to be downregulated due to aging and in many disease states. Particularly central nervous system disorders are linked to dysregulated microRNA processing. According to the latest studies, Dicer is crucial to the survival of dopaminergic neurons and conditional Dicer knockout mice show severe nigrostriatal dopaminergic cell loss, which is a hallmark of Parkinson’s disease. By activating Dicer with a small-molecule drug, enoxacin, the survival of dopaminergic cells exposed to stress is significantly improved. However, enoxacin, which is a fluoroquinolone antibiotic, activates Dicer only at high concentrations (10-100 μM) and is polypharmacological, which may cause detrimental side effects. Therefore, enoxacin is not a suitable drug candidate for Dicer deficiencies and better Dicer-activating drug candidates are needed. The aim of this work was to develop a cell-based fluorescent assay to screen for Dicer-activating compounds. Assays which measure Dicer activity have already been developed, but they have some pitfalls which don’t make them optimal to use for high-throughput screening of Dicer-activating compounds. Some are cell-free enzyme-based assays and thus neglect Dicer in its native context. The RNA to be processed by Dicer does not represent a common mammalian RNA type. Most assays do not have internal normalizing factors, such as a second reporter protein to account for e.g. cell death, or the analysis method is not feasible for high-throughput screening data. Considering these disadvantages, the study started by designing a reporter plasmid in silico. The plasmid expresses two fluorescent proteins, mCherry (red) and EGFP (green), and a mCherry transcripttargeting siRNA implemented into a pre-miR155 backbone which is processed by Dicer. Thus, measuring the ratios of red and green fluorescence intensities will give an indication on Dicer activity. The plasmid also has additional regulatory elements for stabilizing expression levels. The plasmid was then produced by molecular cloning methods and its functionality was tested with Dicer-modulating compounds. The assay was optimised by testing it in different cell lines and varying assay parameters, and stable cell lines were created to make large-scale screening more convenient. Finally, a small-scale screen was done with ten pharmacologically active compounds. Transiently transfected, in Chinese hamster ovarian cells, mCherry silencing was too efficient for reliable detection of improvement in silencing efficiency due to floor effect. With an inducible, Tet-On, system in FLP-IN 293 T-Rex cells, the expression could be controlled by administering doxycycline and the improvement in silencing was quantifiable. The assay seemed to be functional after 72 hours and 120 hours of incubation using enoxacin (100 μM) as a positive control. However, the screening found no compounds to significantly reduce mCherry/EGFP fluorescence ratio and, additionally, the effect of enoxacin was abolished. Therefore, a more thorough analysis on the effects of enoxacin was done and, although statistically significant, enoxacin was only marginally effective in reducing mCherry/EGFP fluorescence ratio after 72 hours of treatment. It should be noted from the small-scale screening that metformin and BDNF, compounds previously shown to elevate Dicer levels, showed similar effects to enoxacin. The quality of the assay in terms of high-throughput screening was determined by calculating Zfactors and coefficients of variations for the experiments, which showed that the variability of the assay was acceptable, but the differences between controls was not large enough for reliable screening. In conclusion, the effects of metformin and BDNF should be further studied and regarding the assay, more optimisation is needed for large-scale, high-throughput, screening to be done with minimal resources.MikroRNA:t ovat noin 22 nukleotidiä pitkiä RNA-juosteita, jotka estävät geenien ilmentymistä sitoutumalla lähetti-RNA:n 3’UTR-alueille. MikroRNA:t osallistuvat laajalti moneen soluprosessiin säätelemällä noin puolta kaikista proteiineja koodaavista geeneistä. MikroRNA:n ilmentymisessä, eräs tärkeä vaihe on III tyypin RNaasin, Dicerin, suorittama pre-miRNA:n prosessointi valmiiksi mikroRNA-juosteeksi. Dicerin toiminnan ja ilmentymisen on mitattu heikentyvän ikääntymisen johdosta, sekä useissa eri taudeissa. Erityisesti keskushermostotautien ja mikroRNA prosessointiin liittyvien ongelmien välillä on löydetty yhteys. Tuoreimpien tutkimusten mukaan Dicerilla on tärkeä rooli myös dopaminergisten hermosolujen selviytymisen kannalta ja lisäksi Dicer muuntogeenisillä hiirillä mustatumakkeen dopamiinihermosolut kuolevat, joka on Parkinsonin taudin keskeisin patofysiologinen ilmiö. Dicerin aktiivisuuden tehostamisella, käyttäen enoksasiinia, on suojaava vaikutus dopamiinihermosoluille. Enoksasiini, joka on fluorokinoloneihin kuuluva antimikrobinen yhdiste, tehostaa Diceria vain suurilla pitoisuuksilla (10-100 μM). Lisäksi se on polyfarmakologinen voiden aiheuttaa paljon vakavia haittavaikutuksia, joten se ei ole optimaalinen lääkeaine Dicer-puutoksiin liitettyjen tautien hoitamiseen. Tämän erikoistyön tavoitteena oli kehittää solupohjainen, fluoresenssiin perustuva menetelmä, jolla voisi seuloa parempia Diceria aktivoivia yhdisteitä. Dicerin aktiivisuutta mittaavia menetelmiä on jo kehitetty aiemmin muutamia, mutta ne eivät ole optimaalisia Diceria-aktivoivien yhdisteiden seulomiseksi. Osa kokeista on entsyymipohjaisia eivätkä ne ota huomioon solunsisäistä endogeenistä Diceria. Kokeissa käytettävä RNA, jonka Dicer prosessoi, ei edusta yleisiä nisäkässolujen RNA-tyyppejä. Tietyissä kokeissa ei ole sisäistä suhteuttavaa tekijää (esimerkiksi toista fluoresoivaa proteiinia) tai niillä ei ole mahdollista suorittaa laajoja seulontoja. Työssä suunniteltiin ensiksi edellä mainitut puutteet huomioon ottaen reportteriplasmidi in silico. Plasmidi ilmentää kahta fluoresoivaa proteiinia, mCherry:ä (punainen) ja EGFP:tä,(vihreä) sekä mCherry:n ilmentymistä estävää siRNA-juostetta pre-miR155:n runkoon liitettynä, jonka Dicer prosessoi. Näin ollen, mittaamalla punaisen ja vihreän fluoresenssi-intensiteettien suhdetta, voidaan tutkia Dicerin aktiivisuutta. Plasmidissa on myös useita säätelyelementtejä ilmentymisen tasaamiseksi. Plasmidi valmistettiin molekyylikloonausmenetelmin ja sen toiminnollisuutta testattiin Dicerin aktiivisuuteen vaikuttavilla yhdisteillä. Seulontakoetta optimoitiin eri solulinjoilla ja olosuhdemuutoksilla, ja lisäksi valmistettiin stabiileja solulinjoja laajamittaisen seulonnan helpottamiseksi. Lopuksi suoritettiin pienen mittakaavan seulonta kymmenelle farmakologisesti aktiiviselle yhdisteelle. Ohimenevästi transfektoituna, Kiinanhamsterin munasarjasoluissa, mCherryn hiljentäminen oli niin tehokasta, että hiljentämisen tehostamista ei voitu luotettavasti mitata. Hallitsemalla ilmentymistä doksisykliinin avulla, Tet-On-systeemillä FLP-IN 293 T-Rex soluilla, saatiin ilmentymistä kontrolloitua ja mittaukset luotettaviksi. Seulontakoe saatiin toimimaan 72 tunnin ja 120 tunnin aikapisteillä käyttäen enoksasiinia positiivisena kontrollina. Seulonnasta ei löydetty mCherry/EGFP fluoresenssien suhdetta merkitsevästi vähentäviä yhdisteitä ja lisäksi enoksasiinin vaikutus ei ollut enää tilastollisesti merkitsevä. Tämän perusteella suoritettiin laajempi analyysi enoksasiinin vaikutuksista, missä havaittiin, että sen vaikutus mCherry/EGFP fluoresenssien suhteen vähentämisessä oli, vaikkakin tilastollisesti merkitsevä, hyvin vähäinen 72 tunnin kokeessa. Huomioitavaa pienen mittakaavan seulonnasta on, että metformiinin ja BDNF:n, joiden on aiemmin osoitettu lisäävän Dicerin ilmentymistä, vaikutukset olivat vastaavia enoksasiinin vaikutukseen. Seulontakokeen laatu laajamittaisen seulontakokeen suhteen määritettiin laskemalla kokeille Z-tekijän sekä hajonnan koeffisienttien arvot. Nämä osoittivat, että kokeen hajonta oli hyväksyttävä, mutta ero kontrollien välillä oli liian pieni, jotta koetta voisi käyttää luotettavasti seulomiseen. Tärkeimpinä johtopäätöksinä, metformiinin ja BDNF:n vaikutuksia Diceriin tulisi tutkia tarkemmin, ja koetta on optimoitava lisää, jotta laajamittaisia seulontoja voidaan suorittaa mahdollisimman vähillä resursseilla

Multiple Sequence Alignments Enhance Boundary Definition of RNA Structures

Self-contained structured domains of RNA sequences have often distinct molecular functions. Determining the boundaries of structured domains of a non-coding RNA (ncRNA) is needed for many ncRNA gene finder programs that predict RNA secondary structures in aligned genomes because these methods do not necessarily provide precise information about the boundaries or the location of the RNA structure inside the predicted ncRNA. Even without having a structure prediction, it is of interest to search for structured domains, such as for finding common RNA motifs in RNA-protein binding assays. The precise definition of the boundaries are essential for downstream analyses such as RNA structure modelling, e.g., through covariance models, and RNA structure clustering for the search of common motifs. Such efforts have so far been focused on single sequences, thus here we present a comparison for boundary definition between single sequence and multiple sequence alignments. We also present a novel approach, named RNAbound, for finding the boundaries that are based on probabilities of evolutionarily conserved base pairings. We tested the performance of two different methods on a limited number of Rfam families using the annotated structured RNA regions in the human genome and their multiple sequence alignments created from 14 species. The results show that multiple sequence alignments improve the boundary prediction for branched structures compared to single sequences independent of the chosen method. The actual performance of the two methods differs on single hairpin structures and branched structures. For the RNA families with branched structures, including transfer RNA (tRNA) and small nucleolar RNAs (snoRNAs), RNAbound improves the boundary predictions using multiple sequence alignments to median differences of −6 and −11.5 nucleotides (nts) for left and right boundary, respectively (window size of 200 nts)

Paternal effects in a wild-type zebrafish implicate a role of sperm-derived small RNAs

While the importance of maternal effects has long been appreciated, a growing body of evidence now points to the paternal environment having an important influence on offspring phenotype. Indeed, research on rodent models suggests that paternal stress leaves an imprint on the behaviour and physiology of offspring via nonge netic information carried in the spermatozoa; however, fish have been understudied with regard to these sperm-mediated effects. Here, we investigated whether the ze brafish was subjected to heritable influences of paternal stress by exposing males to stressors (conspecific-derived alarm cue, chasing and bright light) before mating and assessing the behavioural and endocrine responses of their offspring, including their behavioural response to conspecific-derived alarm cue. We found that after males are exposed to stress, their larval offspring show weakened responses to stressors. Small RNA sequencing subsequently revealed that the levels of several small noncoding RNAs, including microRNAs, PIWI-interacting RNAs and tRNA-derived small RNAs, were altered in the spermatozoa of stressed fathers, suggesting that stress-induced alterations to the spermatozoal RNA landscape may contribute to shaping offspring phenotype. The work demonstrates that paternal stress should not be overlooked as a source of phenotypic variation and that spermatozoal small RNAs may be important intergenerational messengers in fish

In silico selection of an aptamer to estrogen receptor alpha using computational docking employing estrogen response elements as aptamer-alike molecules

Aptamers, the chemical-antibody substitute to conventional antibodies, are primarily discovered through SELEX technology involving multi-round selections and enrichment. Circumventing conventional methodology, here we report an in silico selection of aptamers to estrogen receptor alpha (ERα) using RNA analogs of human estrogen response elements (EREs). The inverted repeat nature of ERE and the ability to form stable hairpins were used as criteria to obtain aptamer-alike sequences. Near-native RNA analogs of selected single stranded EREs were modelled and their likelihood to emerge as ERα aptamer was examined using AutoDock Vina, HADDOCK and PatchDock docking. These in silico predictions were validated by measuring the thermodynamic parameters of ERα -RNA interactions using isothermal titration calorimetry. Based on the in silico and in vitro results, we selected a candidate RNA (ERaptR4; 5′-GGGGUCAAGGUGACCCC-3′) having a binding constant (Ka) of 1.02 ± 0.1 × 108 M−1 as an ERα-aptamer. Target-specificity of the selected ERaptR4 aptamer was confirmed through cytochemistry and solid-phase immunoassays. Furthermore, stability analyses identified ERaptR4 resistant to serum and RNase A degradation in presence of ERα. Taken together, an efficient ERα-RNA aptamer is identified using a non-SELEX procedure of aptamer selection. The high-affinity and specificity can be utilized in detection of ERα in breast cancer and related diseases

Paternal effects in a wild‐type zebrafish implicate a role of sperm‐derived small RNAs

While the importance of maternal effects has long been appreciated, a growing body of evidence now points to the paternal environment having an important influence on offspring phenotype. Indeed, research on rodent models suggests that paternal stress leaves an imprint on the behaviour and physiology of offspring via nongenetic information carried in the spermatozoa; however, fish have been understudied with regard to these sperm‐mediated effects. Here, we investigated whether the zebrafish was subjected to heritable influences of paternal stress by exposing males to stressors (conspecific‐derived alarm cue, chasing and bright light) before mating and assessing the behavioural and endocrine responses of their offspring, including their behavioural response to conspecific‐derived alarm cue. We found that after males are exposed to stress, their larval offspring show weakened responses to stressors. Small RNA sequencing subsequently revealed that the levels of several small noncoding RNAs, including microRNAs, PIWI‐interacting RNAs and tRNA‐derived small RNAs, were altered in the spermatozoa of stressed fathers, suggesting that stress‐induced alterations to the spermatozoal RNA landscape may contribute to shaping offspring phenotype. The work demonstrates that paternal stress should not be overlooked as a source of phenotypic variation and that spermatozoal small RNAs may be important intergenerational messengers in fish

The Application of CRISPR Technology to High Content Screening in Primary Neurons

Axon growth is coordinated by multiple interacting proteins that remain incompletely characterized. High content screening (HCS), in which manipulation of candidate genes is combined with rapid image analysis of phenotypic effects, has emerged as a powerful technique to identify key regulators of axon outgrowth. Here we explore the utility of a genome editingapproach referred to as CRISPR (Clustered Regularly Interspersed Palindromic Repeats) for knockout screening in primary neurons. In the CRISPR approach a DNA-cleaving Cas enzyme is guided to genomic target sequences by user-created guide RNA (sgRNA), where it initiates a double-stranded break that ultimately results in frameshift mutation and loss of protein production. Using electroporation of plasmid DNA that co-expresses Cas9enzyme and sgRNA, we first verified the ability of CRISPR targeting to achieve protein-level knockdown in cultured postnatal cortical neurons. Targeted proteins included NeuN (RbFox3) and PTEN, a well-studied regulator of axon growth. Effective knockdown lagged at least four days behind transfection, but targeted proteins were eventually undetectable by immunohistochemistry in \u3e 80% of transfected cells. Consistent with this, anti-PTEN sgRNA produced no changes in neurite outgrowth when assessed three days post-transfection. When week-long cultures were replated, however, PTEN knockdown consistently increased neurite lengths. These CRISPR-mediated PTEN effects were achieved using multi-well transfection and automated phenotypic analysis, indicating the suitability of PTEN as a positive control for future CRISPR-based screening efforts. Combined, these data establish an example of CRISPR-mediated protein knockdown in primary cortical neurons and its compatibility with HCS workflows