Engine Efficiency Research Articles

Investigation of Engine Lubrication Oil Quality Using a Support Vector Machine and Electronic Nose

Monitoring the quality of engine oil improves engine efficiency and reduces engine maintenance costs. Several methods have been proposed for this purpose; however, most of them take too long to test oil quality. This paper introduces a fast, simple, and accurate method to determine oil quality using an electronic nose and artificial intelligence. The TU5 engine and 10-40W “Behran Super Pishtaz” engine oil were used in the experiments. Tests were conducted at six different quality levels. Oil properties such as viscosity, density, flash point, and freezing point were measured at each level. Additionally, oil smell signals were recorded using an olfactory machine at these quality levels. The fraction method was employed to adjust the sensors’ responses. Five statistical features were extracted from each signal, and these features were used to train and test a support vector machine (SVM) for classifying oil quality using the five-fold cross-validation method. The results indicated a statistically significant change in viscosity and density with variations in oil quality. The density increased as the quality decreased. Viscosity, however, initially decreased and then increased at later stages. An analysis of the sensory outputs revealed that changes in oil quality also affected these outputs, with the most pronounced sensitivity observed in the MQ135 and MQ138 sensors. The final accuracies of the SVM in classifying oil quality were 68.22%, 85.86%, and 95.44% for linear, radial basis function (RBF), and polynomial kernels, respectively. The SVM sensitivities for oil qualities A, B, C, D, E, and F were 97.99%, 97.37%, 95.51%, 92.67%, 94.48%, and 94.59%, respectively.

Comparative Evaluation of the Effect of Exhaust Gas Recirculation Usage on the Performance Characteristics and Emissions of a Natural Gas/Diesel Compression-Ignition Engine Operating at Part-Load Conditions

The use of natural gas as an alternative fuel in dual-fuel compression-ignition engines can lead to a substantial reduction in the majority of pollutant emissions compared to fossil fuels, while the thermal efficiency of the engine can be maintained at adequate levels. Its usage has increased widely in recent years, and significant efforts have been made to investigate the inherent physical and chemical processes that take place during this engine’s combustion, as well as the parameters that affect the operation of the engine and use natural gas as energy source. The scope of this study is to investigate the effect of EGR temperature (cold and hot) and rate (10% and 20%) on the performance characteristics and emissions of a dual-fuel compression-ignition engine operating at a specific engine operating point under dual-fuel (diesel–natural gas) conditions. For this reason, a phenomenological two-zone combustion model was developed. The results of the model were validated against the experimental data obtained from a single-cylinder direct-injection, turbocharged compression-ignition dual-fuel research engine operated under part-load conditions (IMEP = 0.52 Mpa and engine speed = 1500 rpm) and at various replacement percentages of diesel using methane (which was treated as a natural gas surrogate). The model results were in good agreement with the experimental results, revealing the ability of the model to be used in the aforementioned EGR analysis. The results of the study revealed that engine operation with 10% cold EGR does not significantly affect the engine performance characteristics, and combined with the addition of 80% gaseous fuel energy, can lead to a substantial reduction in NO and soot emissions, with a moderate increase in CO emissions. On the other hand, a significant finding of the present work is that engine operation with hot EGR under the investigated operating conditions, even though it had a beneficial effect on NO-specific emissions, led to a reduction in engine efficiency and may raise issues regarding the mechanical strength of the engine.

Towards a more powerful hydrogen engine

This research examines the operational efficiency of a single-cylinder hydrogen engine through simulations conducted in OpenModelica. The research focuses on analyzing the combustion characteristics, heat release, temperature, and pressure at varying engine speeds of 1700, 2000, and 2500 rpm. The combustion process is modeled Applying the Wiebe function to represent the proportion of fuel mass that is burned, while the heat released by combustion is calculated using a formula incorporating the fuel mass, lower heating value, and combustion efficiency. The study presents detailed simulations of in-cylinder temperature, heat release rate, and pressure profiles, and examines how these parameters vary with engine speed. Results reveal that peak temperatures and heat release rates increase with engine speed, indicating more intensive combustion at higher speeds. The pressure curves and power output also show significant changes with speed, with higher engine speeds leading to increased power output and improved engine efficiency. This study provides insights into the hydrogen engine's behavior under different operating conditions, highlighting the impact of engine speed on performance metrics.

Effect of high compression ratio on thermal efficiency and unburned ammonia emissions of a dual-fuel high-pressure direct injection marine ammonia engine

Effect of high compression ratio on thermal efficiency and unburned ammonia emissions of a dual-fuel high-pressure direct injection marine ammonia engine

Dendrite neural network scheme for estimating output power and efficiency for a class of solar free-piston Stirling engine

ABSTRACT This paper presents the dendrite neural network (DNN) scheme for predicting two challenging parameters, namely the output power and efficiency of the solar free-piston Stirling engine (FPSE). This paper utilizes vital characteristics to predict the output power and efficiency accurately. MSE and regression analysis are employed to investigate the reliability of the proposed DNN structure. As a result, not only was the MSE close to zero, but the regression outcomes were also very close to 1, which confirmed the reliability of the DNN. In the next step, several experimental case studies are used to study the effectiveness of the proposed DNN more thoroughly. Therefore, a maximum error of 5% was observed, confirming good agreement between the empirical data and the predictions made by the DNN. Lastly, through the simulation and experimental investigations, it can be concluded that the method is reliable and can be used for other types of FPSEs.

Advancing the C[formula omitted] low-temperature oxidation chemistry through species measurements in a rapid compression machine. Part B: n-Butane

Studying the oxidation of n-butane, a major component of LNG, is critical to improve the efficiency of transportation engines. Furthermore, its negative temperature coefficient (NTC) behavior provides insights into the oxidation of larger hydrocarbons. Several studies have investigated n-butane oxidation at engine-operating pressures using various methods, including ignition delay time (IDT) measurements in rapid compression machines (RCMs) and shock tubes, flame velocities, and species concentrations in flow reactors. While these species measurements provide deeper insights into oxidation networks than IDTs, they are limited to either low-pressure or highly diluted conditions. To address this gap, this study measures species concentrations during n-butane oxidation at 30 bar in the NTC region (742 K and 855 K, respectively), at stoichiometric and moderate dilution levels in an RCM. A novel two-valve setup allowed gas sample extraction for off-line gas chromatography-mass spectrometry analysis. Complementary IDT data were obtained in the temperature range of 680−910 K, at pressures of 15 and 30 bar, and equivalence ratios of 0.5, 1.0, and 2.0. The results suggest that while current n-butane models reasonably predict its autoignition characteristics, they fall short in predicting the formation of key oxidation intermediates at engine-relevant conditions. In this context, the n-butane submechanism within the NUIGMech1.3 framework was updated. Modifications involve recently computed thermochemical data for critical intermediates and adjustments to rate constants, using analogies with structurally similar molecules such as n-propane and n-pentane. The present model reproduces reasonably well both the measured IDT and species concentrations documented herein and data from the literature. Nevertheless, the model slightly underestimates the reactivity within the NTC domain and the formation of some intermediates at the NTC peak. This study highlights the importance of integrating species concentration and IDT measurements at application-relevant conditions to refine kinetic mechanisms and significantly advances the understanding of C4 hydrocarbon oxidation chemistry.Novelty and Significance StatementThe novelty of this research lies in the measurement of species concentrations during the ignition delay of n-butane mixtures in an RCM at high pressures near the NTC minimum and maximum using a novel two-valve gas sampling setup. This, in combination with new thermochemical data and rate rules based on analogies with propane and n-pentane, allowed the refinement of the n-butane sub-mechanism within the NUIGMech1.3 framework.By combining species concentration measurements with ignition delay times in the RCM, this study examines the oxidation of n-butane, a major component of LPG, under conditions that closely mimic engine environments, overcoming the limitations of previous studies limited to highly dilute conditions. This research is part of a broader investigation of C4 oxidation chemistry, along with our companion work on 1-butene. The resulting kinetic model is capable of reproducing most of the available n-butane and 1-butene validation targets.

MoCloro: an extension of the Chlamydomonas reinhardtii modular cloning toolkit for microalgal chloroplast engineering.

Photosynthetic microalgae are promising green cell factories for the sustainable production of high-value chemicals and biopharmaceuticals. The chloroplast organelle is being developed as a chassis for synthetic biology as it contains its own genome (the plastome) and some interesting advantages, such as high recombinant protein titers and a diverse and dynamic metabolism. However, chloroplast engineering is currently hampered by the lack of standardized cloning tools and Design-Build-Test-Learn workflows to ease genomic and metabolic engineering. The MoClo (Modular Cloning) toolkit based on Golden Gate assembly was recently developed in the model eukaryotic green microalgae Chlamydomonas reinhardtii to facilitate nuclear transformation and engineering. Here, we present MoCloro as an extension of the MoClo that allows chloroplast genome engineering. Briefly, a Golden Gate-compatible chloroplast transformation vector (pWF.K.4) was constructed, which contains homologous arms for integration at the petA site in the plastome. A collection of standardized parts (promoters, terminators, reporter and selection marker genes) was created following the MoClo syntax to enable easy combinatorial assembly of multi-cassettes in the destination pWF.K.4 vector. The functionality of the biobricks was assayed by constructing and assessing the expression of several multigenic constructs. Finally, a generic vector pK4 was constructed for easy Golden Gate cloning of 5' and 3' homologous arms, allowing targeting at alternative plastome integration sites. This work represents a significant advancement in technology aimed at facilitating more efficient and rapid chloroplast transformation and engineering of green microalgae.

Engineering efficiency through CI/CD pipeline optimization

Engineering efficiency through CI/CD pipeline optimization

Dual-Stage Energy Recovery from Internal Combustion Engines

Waste heat recovery is one of the most investigated solutions for increasing the efficiency of powertrains in the transportation sector. A major portion of thermal energy is wasted via exhaust gases. Almost one third of fuel energy is lost, and its recovery as propulsion energy is a promising goal. Moreover, this enables the increased electrification or hybridization of powertrains, assuming the energy recovered is converted into electrical form and used to fulfill different vehicles’ needs. The present study focuses on a dual-stage energy recovery system designed to enhance the efficiency of internal combustion engines (ICEs) in heavy-duty vehicles (HDVs). The system combines a turbocompound unit for direct heat recovery (DHR) and an organic Rankine cycle (ORC) for indirect heat recovery (IHR). These technologies aim to exploit waste heat from exhaust gases, converting it into electrical energy. In this regard, electrical energy can be stored in a battery for it to be available for the energy needs of powertrains that use hybrid propulsion and for driving pumps and compressors on board, following recent technologies of auxiliaries on demand. The proposed setup was modeled and analyzed under off-design conditions to evaluate energy recovery potential and engine performance impacts. From this point of view, in fact, any device that operates on exhaust gas introduces a pressure loss, increasing engine backpressure, whose effect is an increase in specific fuel consumption. An estimate of this negative effect is presented in this paper based on experimental data measured in a F1C IVECO™ engine. An average net recovery of 5–6% of engine power has been demonstrated, with an important prevalence of the turbocompound with respect to the ORC section. The results demonstrate the viability of integrating DHR and IHR stages, with implications for advancing sustainable transportation technologies.

Kinematic and vibration analysis of a single-cylinder engine: Effects of connecting rod length, fuel type, and engine speed

AbstractThis paper emphasizes the role of fuel substitution in mitigating vibrations and enhancing the performance and reliability of internal combustion engines (ICEs). Using MATLAB for modal analysis, the study investigates natural frequencies and identifies potential resonance issues within critical engine components. The primary focus is on analyzing a four-stroke single-cylinder engine under various speeds and measuring the effects of fuel type on engine performance and efficiency. A comparative assessment evaluated vibration levels between natural gas and petrol by measuring engine vibrations across a range of speeds and analyzing the impact of varying dimensions of internal moving components on performance parameters. Results indicate minimal differences between the engines at lower speeds (1,500–2,000 rpm), while significant increases in vibrations were observed at higher speeds (2,500–3,000 rpm) for the gas engine, highlighting potential mechanical instability. At higher speeds, vibrations peak at the top of the engine, reaching 200 m s−2 with petrol and 240 m s−2 with natural gas. The experimental setup, including accelerometer modules and control systems, enabled real-time vibration monitoring. Numerical results showed that the longest connecting rod (197 mm) yielded the highest displacement, velocity, and acceleration metrics. These findings underscore the advantages of using natural gas as a fuel, including its availability, cost-effectiveness, and environmentally friendly nature, as it produces significantly lower harmful emissions compared to petrol. Additionally, the study offers insights into vibration-damping mechanisms and the influence of modifications to internal moving parts, such as crankshaft diameter and connecting rod length, on engine performance and stability.

Effect of coatings on the tribological performance of engine valve train: A review

Coatings play a crucial role in engine valve train components, reducing friction and wear to enhance engine efficiency and longevity. Common coatings in internal combustion engines include Diamond-Like Carbon (DLC) and Hard coatings, all of which improve tribological performance. These coatings are vital for valve trains, which face high stress and temperature variations. Deposition techniques like Physical Vapor Deposition (PVD), Chemical Vapor Deposition (CVD), and Plasma-Assisted Chemical Vapor Deposition (PACVD) influence coating performance. DLC coatings, often metalloid-doped, are noted for their low friction and high hardness. Hard coatings excel in wear resistance and friction reduction. This review brings together experimental findings on tribological coatings for engine valve train components, focusing on materials, deposition methods, testing conditions, and performance metrics. It highlights the best coatings for reducing wear and friction, ultimately enhancing the performance of internal combustion engines. Among the coatings evaluated, Chromium Nitride with Tungsten Carbide/Carbon (CrN + WC/C) stood out. This coating significantly reduced friction torque at low to moderate rotational speeds (with reductions of 20%–30% at 800 rpm and 1400 rpm) and maintained this reduction at high speeds (30% reduction at 2000 rpm). Additionally, under high load conditions, the CrN + WC/C coating achieved a remarkable 75% reduction in mean friction torque (reducing it from 4 to 1 Nm at both 800 and 2000 rpm). These tests were conducted using SAE 10W-30 oil at 80°C. The consistent performance of CrN + WC/C across varied conditions makes it the superior choice. CrN + WC/C coatings optimize the efficiency and longevity of valve train components by reducing friction, wear, and maintenance needs, significantly enhancing engine system reliability.

Designer Organs: Ethical Genetic Modifications in the Era of Machine Perfusion.

Gene therapy is a rapidly developing field, finally yielding clinical benefits. Genetic engineering of organs for transplantation may soon be an option, thanks to convergence with another breakthrough technology, ex vivo machine perfusion (EVMP). EVMP allows access to the functioning organ for genetic manipulation prior to transplant. EVMP has the potential to enhance genetic engineering efficiency, improve graft survival, and reduce posttransplant complications. This will enable genetic modifications with a vast variety of applications, while raising questions on the ethics and regulation of this emerging technology. This review provides an in-depth discussion of current methodologies for delivering genetic vectors to transplantable organs, particularly focusing on the enabling role of EVMP. Organ-by-organ analysis and key characteristics of various vector and treatment options are assessed. We offer a road map for research and clinical translation, arguing that achieving scientific benchmarks while creating anticipatory governance is necessary to secure societal benefit from this technology.

Understanding the CMAS corrosion behavior of high‐entropy (La0.2Sm0.2Er0.2Y0.2Yb0.2)2Ce2O7

AbstractCeramic thermal barrier coating (TBC) materials are used to protect the superalloys from the damage of harmful high‐temperature airflow and improve the efficiency of jet and gas turbine engines. However, the long‐term application of TBC materials and the robustness of these materials can be destroyed by aggressive calcium‐magnesium‐alumina‐silicate (CMAS) melt during high‐temperature service. Increasing the configuration entropy of material by doping multiple principal components has become a research hotspot in the design of corrosion‐resistant thermal barrier coating material and has opened an infinite space of chemical composition, structure, and material properties. In this study, high‐entropy (La0.2Sm0.2Er0.2Y0.2Yb0.2)2Ce2O7 was synthesized and its CMAS corrosion behavior was investigated by experimental investigation and first‐principles calculation. The effects of the increase of configurational entropy and the subsequent potential effects on the CMAS corrosion behavior of Ce‐based fluorite oxides have been sufficiently investigated. By compared with control samples, the high‐entropy (La0.2Sm0.2Er0.2Y0.2Yb0.2)2Ce2O7 possesses the minimum infiltration depth of CMAS melts and the denser corrosion reaction layer, indicating the best corrosion resistance. The corrosion resistance mechanism of high‐entropy (La0.2Sm0.2Er0.2Y0.2Yb0.2)2Ce2O7 was studied by first‐principles calculation. The greater stability and resistance to segregation of CMAS melt in the CMAS/(La0.2Sm0.2Er0.2Y0.2Yb0.2)2Ce2O7 system, the poor adsorption capacity for CMAS melt which leads to a weak infiltration ability of CMAS melt; the weakest interfacial chemical reaction at the interface indicated by the smallest value of Griffith rupture work and the least species migration of high‐entropy fluorite oxide can be responsible to the enhanced corrosion resistance. Our work reveals that increasing the configuration entropy can be an effective strategy for TBC material to enhance corrosion resistance.

Influence of Biodiesel from Used Cooking Oil and Sunflower Oil on Engine Efficiency and Emission Profiles

This study evaluates the performance and emissions characteristics of a compression ignition engine fueled with biodiesel blends derived from used cooking oil (UO) and sunflower oil (SF) at concentrations of 5%, 10%, 20%, and 50%. Tests were conducted under different load conditions (20%, 50%, and 100%) across engine speeds ranging from 1500 to 3600 rpm, focusing on effective power, torque, brake specific fuel consumption (BSFC), and emissions of NOx, CO, HC, particulate matter (PM), smoke, and CO2. Consistent engine operating conditions were maintained for all fuel blends. The results indicated that increasing the biodiesel concentration led to a decrease in brake power and torque—up to 3.18% reduction for SF50 compared to diesel—due to the lower calorific value of biodiesel. For SF biodiesel, the BSFC increased with higher biodiesel content, while for UO biodiesel the results varied across concentrations. Emissions analysis revealed lower CO and HC at 2500 rpm for all biodiesel blends relative to diesel, while NOx emissions varied depending on fuel type and concentration. In terms of particles, both PM and smoke were measured, and while PM showed different results across blends, smoke was lower for all blends compared to diesel. Our overall analysis shows that biodiesel blends up to 20% can be effectively used in diesel engines without substantial modifications, offering a balance between performance and reduced emissions.

Lubricant Viscosity Impact in Fuel Economy: Experimental Uncertainties Compensation

Climate constraints impose greenhouse gas emissions mitigation, and passenger cars have considerable contributions to contribute to this. To improve the engine efficiency of vehicles equipped with conventional powertrains, many technologies are available but with limited individual contribution. The experimental assessment of some technology regarding fuel economy measurement results is sometimes lower than test uncertainties. This study proposes a methodology to compensate the fuel economy for two test uncertainties: vehicle speed variations and battery recharging. The proposed method can be applied when investigating the effects of different vehicle design changes, including engine power cell design. In this work, the proposed method is demonstrated on the test of two oils: one 5W40, the other 5W20, both without FM. Applying the proposed methodology to experimental results, the expected higher influence of oil viscosity on urban conditions could be observed, and the experimental results presented a much better correlation with the vehicle numerical simulation. Applying the proposed compensation, fuel savings of using the 5W20 in comparison to the 5W40 oil was 3.5% under urban conditions and 2.0% on highways.

Entangled quantum Stirling heat engine based on two particles Heisenberg model with Dzyaloshinskii-Moriya interaction

Quantum heat engines have attracted significant attention in recent years due to their potential to surpass classical thermodynamic limits by leveraging quantum effects such as entanglement and coherence. In this study, we analyze a quantum Stirling heat engine characterized by a working substance composed of a two-particle Heisenberg model with Dzyaloshinskii–Moriya (DM) interaction under an external magnetic field. We investigate the impact of the antisymmetric interaction on the engine’s efficiency across varying coupling parameters. Our findings demonstrate that the utilization of a two-qubit Heisenberg model in an entangled quantum Stirling heat engine can significantly enhance efficiency and performance. By optimizing the antisymmetric exchange parameters, we achieve substantial enhancements in engine efficiency, with results demonstrating that the efficiency attains remarkably high values compared to other cycles utilizing the same working substance. These enhancements are primarily influenced by the DM interaction and the entangled states of the working substance, leading to superior performance.

Heat Transfer Modeling of Hydrogen-Fueled Spark Ignition Engine

Currently, green hydrogen, generated through renewable energy sources, stands out as one of the best substitutes for fossil fuels in mitigating pollutant emissions and consequent global warming. Particularly, the utilization of hydrogen in spark ignition engines has undergone extensive study in recent years. Many aspects have been analyzed: the conversion of gasoline engines to hydrogen operation, the combustion duration, the heat transfer, and, in general, the engine thermal efficiency. Hydrogen combustion is characterized by a smaller quenching distance compared to traditional hydrocarbon fuels such as gasoline or natural gas and this produces a smaller thermal boundary layer and consequently higher heat transfer. This paper presents findings from experimental trials and numerical simulations conducted on a hydrogen-powered CFR (cooperative fuel research) engine, focusing specifically on heat transfer with combustion chamber walls. The engine has also been fueled with methane and isooctane (two reference fuels); both the engine compression ratio and the air/fuel ratio have been changed in a wide range in order to compare the three fuels in terms of heat transfer, combustion duration, and engine thermal efficiency in different operating conditions. A numerical model has been calibrated with experimental data in order to predict the amount of heat transfer under the best thermal efficiency operating conditions. The results show that, when operated with hydrogen, the best engine efficiency is obtained with a compression ratio of 11.9 and an excess air ratio (λ) of 2.

Key Technologies to 50% Brake Thermal Efficiency for Gasoline Engine of Passenger Car

Review Key Technologies to 50% Brake Thermal Efficiency for Gasoline Engine of Passenger Car Xinke Miao, Bingxin Xu, Jun Deng, and Liguang Li * School of Automotive Studies, Tongji University, Shanghai 201804, China * Correspondence: liguang@tongji.edu.cn Received: 13 August 2024; Revised: 6 December 2024; Accepted: 17 December 2024; Published: 20 January 2025 Abstract: As fuel consumption and emissions regulations become increasingly stringent, various advanced strategies have been proposed to achieve higher efficiency in internal combustion engines. This paper reviews the advancements in thermal efficiency of gasoline engines and analyzes the key technological methods to achieve over 50% brake thermal efficiency (BTE). The technological routes proposed for high-efficiency gasoline engine are primarily focused on high compression ratios and lean combustion combined with novel combustion technologies. Supporting technologies mainly include Atkinson/Miller cycles, intake boosting, exhaust gas re-circulation (EGR), water injection, thermal barrier coatings, friction reduction, structural optimization, and combustion diagnostics and control.

Jet-Induced Compression Ignition (JICI)—Application of Spark-Assisted Compression Ignition (SACI) in a Combustion System with Active Pre-Chamber

<div>The application of short burn durations at lean engine operation has the potential to increase the efficiency of spark-ignition engines. To achieve short burn durations, spark-assisted compression ignition (SACI) as well as active pre-chamber (PC) combustion systems are suitable technologies. Since a combination of these two combustion concepts has the potential to achieve shorter burn durations than the application of only one of these concepts, the concept of jet-induced compression ignition (JICI) was investigated in this study. With the JICI, the fuel is ignited in the PC, and the combustion products igniting the charge in the main combustion chamber (MC) triggered the autoignition of the MC charge. A conventional gasoline fuel (RON 95 E10) and a Porsche synthetic fuel (POSYN) were investigated to assess the fuel influence on the JICI. Variations of the relative air/fuel ratio in the exhaust gas (λ<sub>ex</sub>) were performed to evaluate both the occurrence of the JICI and the dilution capability. To assess the sensitivity of the JICI, variations of the engine speed and the engine load were performed. When using RON 95 E10, a shift from a conventional PC combustion to the JICI was observed between λ<sub>ex</sub> = 2.3 and λ<sub>ex</sub> = 2.5. The variations of the engine speed and the engine load revealed an increased JICI intensity when the engine speed decreased and when the engine load increased. When using POSYN, no JICI was observed. The occurrence of the JICI was correlated to the knock resistances of the fuels, i.e., the lower knock resistance of RON 95 E10 yielded the JICI, whereas the higher one of POSYN did not. At λ<sub>ex</sub> = 2.8, applying POSYN resulted in an increase of the burn duration of 5.5°CA, which was a relative increase of 41%, compared to the use of RON 95 E10 due to the absence of the JICI in case of POSYN. However, the application of POSYN resulted in the highest net indicated efficiency (η<sub>i,net</sub>). In particular, the application of RON 95 E10 yielded a maximum of η<sub>i,net</sub> = 41.5% at λ<sub>ex</sub> = 2.6, whereas using POSYN resulted in a maximum of η<sub>i,net</sub> = 42.6% at λ<sub>ex</sub> = 2.2 due to the higher knock resistance of POSYN.</div>

Optimizing approaches for targeted integration of transgenic cassettes by integrase-mediated cassette exchange in mouse and human stem cells.

To enable robust expression of transgenes in stem cells, recombinase-mediated cassette exchange at safe harbor loci is frequently adopted. The choice of recombinase enzyme is a critical parameter to ensure maximum efficiency and accuracy of the integration event. We have explored the serine recombinase family of site-specific integrases and have directly compared the efficiency of PhiC31, W-beta, and Bxb1 integrase for targeted transgene integration at the Gt(ROSA)26Sor locus in mouse embryonic stem cells. All 3 integrases were found to be suitable for efficient engineering and long-term expression of each integrase was compatible with pluripotency, as evidenced by germline transmission. Bxb1 integrase was found to be 2-3 times more efficient than PhiC31 and W-beta. The Bxb1 system was adapted for cassette exchange at the AAVS1 locus in human induced pluripotent stem (iPS) cells, and the 2 commonly used ubiquitous promoters, CAG and Ef1α (EIF1A), were tested for their suitability in driving expression of the integrated transgenic cargo. AAVS1-integrated Ef1α promoter led to a very mosaic pattern of expression in targeted hiPS cells, whereas the AAVS1-integrated CAG promoter drove consistent and stable expression. To validate the system for the integration of functional machinery, the Bxb1 integrase system was used to integrate CAG-driven CRISPR-activation and CRISPR-inhibition machinery in human iPS cells and robust sgRNA-induced up- and downregulation of target genes was demonstrated.