Efficient deletion of microRNAs using CRISPR/Cas9 with dual guide RNAs

MicroRNAs (miRNAs) are short non-coding RNAs that play crucial roles in gene regulation, exerting post-transcriptional silencing, thereby influencing cellular function, development, and disease. Traditional loss-of-function methods for studying miRNA functions, such as miRNA inhibitors and sponges, present limitations in terms of specificity, transient effects, and off-target effects. Similarly, CRISPR/Cas9-based editing of miRNAs using single guide RNAs (sgRNAs) also has limitations in terms of design space for generating effective gRNAs. In this study, we introduce a novel approach that utilizes CRISPR/Cas9 with dual guide RNAs (dgRNAs) for the rapid and efficient generation of short deletions within miRNA genomic regions. Through the expression of dgRNAs through single-copy lentiviral integration, this approach achieves over a 90% downregulation of targeted miRNAs within a week. We conducted a comprehensive analysis of various parameters influencing efficient deletion formation. In addition, we employed doxycycline (Dox)-inducible expression of Cas9 from the AAVS1 locus, enabling homogeneous, temporal, and stage-specific editing during cellular differentiation. Compared to miRNA inhibitory methods, the dgRNA-based approach offers higher specificity, allowing for the deletion of individual miRNAs with similar seed sequences, without affecting other miRNAs. Due to the increased design space, the dgRNA-based approach provides greater flexibility in gRNA design compared to the sgRNA-based approach. We successfully applied this approach in two human cell lines, demonstrating its applicability for studying the mechanisms of human erythropoiesis and pluripotent stem cell (iPSC) biology and differentiation. Efficient deletion of miR-451 and miR-144 resulted in blockage of erythroid differentiation, and the deletion of miR-23a and miR-27a significantly affected iPSC survival. We have validated the highly efficient deletion of genomic regions by editing protein-coding genes, resulting in a significant impact on protein expression. This protocol has the potential to be extended to delete multiple miRNAs within miRNA clusters, allowing for future investigations into the cooperative effects of the cluster members on cellular functions. The protocol utilizing dgRNAs for miRNA deletion can be employed to generate efficient pooled libraries for high-throughput comprehensive analysis of miRNAs involved in different biological processes

Design to Control a Thermal Runaway in Battery Electric Vehicles (BEVs)

The rapid electrification of vehicles has neccessitated advancements in battery technology, impacting various components within the car battery pack. This project focuses on the development of thermal barriers, which serve as crucial components that prevent potential fires from thermal runaway incidents in the battery pack from reaching the vehicle's interior compartment. Currently, thermal barriers are predominantly composed of mica. The objective of this project is to identify a more sustainable alternative material to mica that possesses similar properties while considering economic, social, and environmental factors.  Following the product development process, a comprehensive background study and benchmark analysis were conducted. Subsequently, the specific requirements for the new material were defined, and potential suppliers providing alternative materials were identified. Torch tests and dielectric strength tests were performed to assess thermal conductivity, fire resistance, and dielectric properties, which were deemed critical attributes. The test results unveiled a novel material comprised of a combination of two distinct materials from different suppliers. One material was a composite consisting of glass fiber and carbon, exhibiting exceptional thermal properties. The other material was an electrical-grade paper boasting excellent electrical properties. When combined, these materials formed a composite that paralleled the properties of mica. The composite material had a total thickness of 2 mm, slightly thicker than the current implementation of mica. To explore the feasibility of a thinner alternative, an estimation was conducted, suggesting that a thinner sheet of the composite material could be possible. In conclusion, this study demonstrates the feasibility of replacing mica with an alternative material. Further testing and investigation are required to determine the optimal implementation and minimum thickness of the new material. However, based on the successful test results and similarity in properties, it can be affirmed that the new material has the potential to replace mica as a sustainable thermal barrier in the battery pack.Denna avhandling fokuserar på utvecklingen av hållbara termiska barriärer som ersättning för mica i batteripacken i elektriska bilar, vilket har blivit nödvändigt på grund av den snabba elektrifieringen av fordon. Syftet med studien är att identifiera ett mer hållbart alternativ till mica och samtidigt ta hänsyn till ekonomiska, sociala och miljömässiga faktorer. Genom att följa produktutvecklingsprocessen har en omfattande bakgrundsstudie och benchmark-analys genomförts. Därefter definierades specifika krav för det nya materialet, och potentiella leverantörer av alternativa material identifierades. Väsentliga attribut som termisk ledningsförmåga, brandsäkerhet och elektriska egenskaper utvärderades genom att utföra flam-tester och tester för den elektriska ledningsförmågan. Resultaten av testerna resulterade i ett innovativt material bestående av en kombination av två olika material från olika leverantörer. En komposit av glasfiber och kol visade exceptionella termiska egenskaper, medan ett elektriskt kvalitetspapper uppvisade utmärkta elektriska egenskaper. När dessa material kombinerades bildade de en komposit som liknade micas egenskaper. Det komposita materialet hade en total tjocklek på 2 mm, något tjockare än mica. Genom en uppskattning undersöktes möjligheten att använda en tunnare variant av det komposita materialet. Sammanfattningsvis visar denna studie att det är genomförbart att ersätta mica med det föreslagna alternativa materialet. Vidare tester och undersökningar krävs för att optimera implementeringen och fastställa den minsta möjliga tjockleken på det nya materialet. Baserat på de positiva testresultaten och likheten i egenskaper kan det konstateras att det föreslagna materialet har potential att vara en hållbar termisk barriär i batteripaket

Design to Control a Thermal Runaway in Battery Electric Vehicles (BEVs)

The rapid electrification of vehicles has neccessitated advancements in battery technology, impacting various components within the car battery pack. This project focuses on the development of thermal barriers, which serve as crucial components that prevent potential fires from thermal runaway incidents in the battery pack from reaching the vehicle's interior compartment. Currently, thermal barriers are predominantly composed of mica. The objective of this project is to identify a more sustainable alternative material to mica that possesses similar properties while considering economic, social, and environmental factors.  Following the product development process, a comprehensive background study and benchmark analysis were conducted. Subsequently, the specific requirements for the new material were defined, and potential suppliers providing alternative materials were identified. Torch tests and dielectric strength tests were performed to assess thermal conductivity, fire resistance, and dielectric properties, which were deemed critical attributes. The test results unveiled a novel material comprised of a combination of two distinct materials from different suppliers. One material was a composite consisting of glass fiber and carbon, exhibiting exceptional thermal properties. The other material was an electrical-grade paper boasting excellent electrical properties. When combined, these materials formed a composite that paralleled the properties of mica. The composite material had a total thickness of 2 mm, slightly thicker than the current implementation of mica. To explore the feasibility of a thinner alternative, an estimation was conducted, suggesting that a thinner sheet of the composite material could be possible. In conclusion, this study demonstrates the feasibility of replacing mica with an alternative material. Further testing and investigation are required to determine the optimal implementation and minimum thickness of the new material. However, based on the successful test results and similarity in properties, it can be affirmed that the new material has the potential to replace mica as a sustainable thermal barrier in the battery pack.Denna avhandling fokuserar på utvecklingen av hållbara termiska barriärer som ersättning för mica i batteripacken i elektriska bilar, vilket har blivit nödvändigt på grund av den snabba elektrifieringen av fordon. Syftet med studien är att identifiera ett mer hållbart alternativ till mica och samtidigt ta hänsyn till ekonomiska, sociala och miljömässiga faktorer. Genom att följa produktutvecklingsprocessen har en omfattande bakgrundsstudie och benchmark-analys genomförts. Därefter definierades specifika krav för det nya materialet, och potentiella leverantörer av alternativa material identifierades. Väsentliga attribut som termisk ledningsförmåga, brandsäkerhet och elektriska egenskaper utvärderades genom att utföra flam-tester och tester för den elektriska ledningsförmågan. Resultaten av testerna resulterade i ett innovativt material bestående av en kombination av två olika material från olika leverantörer. En komposit av glasfiber och kol visade exceptionella termiska egenskaper, medan ett elektriskt kvalitetspapper uppvisade utmärkta elektriska egenskaper. När dessa material kombinerades bildade de en komposit som liknade micas egenskaper. Det komposita materialet hade en total tjocklek på 2 mm, något tjockare än mica. Genom en uppskattning undersöktes möjligheten att använda en tunnare variant av det komposita materialet. Sammanfattningsvis visar denna studie att det är genomförbart att ersätta mica med det föreslagna alternativa materialet. Vidare tester och undersökningar krävs för att optimera implementeringen och fastställa den minsta möjliga tjockleken på det nya materialet. Baserat på de positiva testresultaten och likheten i egenskaper kan det konstateras att det föreslagna materialet har potential att vara en hållbar termisk barriär i batteripaket

Australia-India Indo-Pacific Oceans initiative: regional collaborative arrangements in marine ecology in the Indo-Pacific baseline study

This Report collects together a series of baseline studies on Regional Collaborative Arrangements in Marine Ecology in the Indo Pacific. The findings from these studies are the basis of a set of recommendations for the Australian Government to promote greater Indo-Pacific cooperation in marine ecology.Published versio

Sex Differences in Adult Incarceration After Pediatric Traumatic Brain Injury

Pediatric traumatic brain injury (pTBI) is a major risk factor associated with adulthood incarceration. Most research into the link between pTBI and adulthood incarceration has focused on incarcerated males, who comprise the vast majority of incarcerated adults, particularly in industrialized nations. In this review, we sought to identify sex-related differences in the incidence and pathophysiology of pTBI and subsequent risk of adulthood incarceration. A scoping review was undertaken using PubMed, Scopus, Ovid, and the Cochrane Library. Articles analyzing sex-related differences in pTBI and adult incarceration rates, studies conducted on an incarcerated population, and cohort studies, cross-sectional studies, clinical trials, systematic reviews, or meta-analyses were included in this review. Of the 85 unique results, 25 articles met our inclusion criteria. Male children are 1.5 times more likely to suffer a TBI than females; however, the prevalence of incarcerated adults with a history of pTBI is ?35?45% for both sexes. Neurophysiologically, female sex hormones are implicated in neuroprotective roles, mitigating central nervous system (CNS) damage post-TBI, although this role may be more complex, given that injury severity and sequelae have been correlated with male sex whereas increased mortality has been correlated with female sex. Further investigation into the relationship between estrogen and subsequent clinical measurements of CNS function is needed to develop interventions that may alleviate the pathophysiological consequences of pTBI

DataSheet1_Efficient deletion of microRNAs using CRISPR/Cas9 with dual guide RNAs.PDF

MicroRNAs (miRNAs) are short non-coding RNAs that play crucial roles in gene regulation, exerting post-transcriptional silencing, thereby influencing cellular function, development, and disease. Traditional loss-of-function methods for studying miRNA functions, such as miRNA inhibitors and sponges, present limitations in terms of specificity, transient effects, and off-target effects. Similarly, CRISPR/Cas9-based editing of miRNAs using single guide RNAs (sgRNAs) also has limitations in terms of design space for generating effective gRNAs. In this study, we introduce a novel approach that utilizes CRISPR/Cas9 with dual guide RNAs (dgRNAs) for the rapid and efficient generation of short deletions within miRNA genomic regions. Through the expression of dgRNAs through single-copy lentiviral integration, this approach achieves over a 90% downregulation of targeted miRNAs within a week. We conducted a comprehensive analysis of various parameters influencing efficient deletion formation. In addition, we employed doxycycline (Dox)-inducible expression of Cas9 from the AAVS1 locus, enabling homogeneous, temporal, and stage-specific editing during cellular differentiation. Compared to miRNA inhibitory methods, the dgRNA-based approach offers higher specificity, allowing for the deletion of individual miRNAs with similar seed sequences, without affecting other miRNAs. Due to the increased design space, the dgRNA-based approach provides greater flexibility in gRNA design compared to the sgRNA-based approach. We successfully applied this approach in two human cell lines, demonstrating its applicability for studying the mechanisms of human erythropoiesis and pluripotent stem cell (iPSC) biology and differentiation. Efficient deletion of miR-451 and miR-144 resulted in blockage of erythroid differentiation, and the deletion of miR-23a and miR-27a significantly affected iPSC survival. We have validated the highly efficient deletion of genomic regions by editing protein-coding genes, resulting in a significant impact on protein expression. This protocol has the potential to be extended to delete multiple miRNAs within miRNA clusters, allowing for future investigations into the cooperative effects of the cluster members on cellular functions. The protocol utilizing dgRNAs for miRNA deletion can be employed to generate efficient pooled libraries for high-throughput comprehensive analysis of miRNAs involved in different biological processes.</p