Comparative Analysis of Cas9 Activators Across Multiple Species

Several groups have generated programmable transcription factors based on the versatile Cas9 protein, yet their relative potency and effectiveness across various cell types and species remain unexplored. Here, we compare Cas9 activator systems and examine their ability to induce robust gene expression in several human, mouse, and fly cell lines. We also explore the potential for improved activation through the combination of the most potent activator systems and assess the role of cooperativity in maximizing gene expression

UK–Russia Researcher Links Workshop: extracellular vesicles – mechanisms of biogenesis and roles in disease pathogenesis, M.V. Lomonosov Moscow State University, Moscow, Russia, 1–5 March 2015

The UK–Russia extracellular vesicles (EVs) workshop was held at the Medical Center of the M.V. Lomonosov Moscow State University, Moscow, Russia, with 56 attendees from UK and Russian universities and research institutes. The program consisted of 6 research sessions and was focused on studies of EVs isolated from in vitro model systems or biological fluids, including blood and urine. The multidisciplinary program included presentations on mechanisms of EV biogenesis, the role of EVs in disease pathogenesis, the diagnostic value of EVs, including their quantitation and cargo load, as well as the clinical use of EVs in regenerative medicine. Methodological challenges imposed by the nanoscale size of EVs as well as targeted delivery approaches for therapeutics were considered in a separate session on technologies. The main aim of the workshop was to overview challenges confronting EV researchers and to facilitate knowledge exchange between researchers with different backgrounds and skills. Given the lack of definitive EV nomenclature, specific terms (exosomes or microvesicles) were only applied in the meeting report to studies that carried out full EV characterization, including differential ultracentrifugation isolation approaches, comprehensive protein marker characterization, and single vesicle analysis (electron microscopy and nanoparticle analysis), to ascertain EV size and morphology following the International Society for Extracellular Vesicles standardization recommendations (1,2). In studies where characterization was not conclusive, the term EV is used

Cas9 gRNA engineering for genome editing, activation and repression

We demonstrate that by altering the length of Cas9-associated guide RNA(gRNA) we were able to control Cas9 nuclease activity and simultaneously perform genome editing and transcriptional regulation with a single Cas9 protein. We exploited these principles to engineer mammalian synthetic circuits with combined transcriptional regulation and kill functions governed by a single multifunctional Cas9 protein.National Human Genome Research Institute (U.S.) (P50 HG005550)United States. Department of Energy (DE-FG02-02ER63445)Wyss Institute for Biologically Inspired EngineeringUnited States. Army Research Office (DARPA W911NF-11-2-0054)National Science Foundation (U.S.)United States. National Institutes of Health (5R01CA155320-04)United States. National Institutes of Health (P50 GM098792)National Cancer Institute (U.S.) (5T32CA009216-34)Massachusetts Institute of Technology. Department of Biological EngineeringHarvard Medical School. Department of GeneticsDefense Threat Reduction Agency (DTRA) (HDTRA1-14-1-0006

Highly-efficient Cas9-mediated transcriptional programming

The RNA-guided nuclease Cas9 can be reengineered as a programmable transcription factor. However, modest levels of gene activation have limited potential applications. We describe an improved transcriptional regulator obtained through the rational design of a tripartite activator, VP64-p65-Rta (VPR), fused to nuclease-null Cas9. We demonstrate its utility in activating endogenous coding and noncoding genes, targeting several genes simultaneously and stimulating neuronal differentiation of human induced pluripotent stem cells (iPSCs).National Human Genome Research Institute (U.S.) (Grant P50 HG005550)United States. Dept. of Energy (Grant DE-FG02-02ER63445)Wyss Institute for Biologically Inspired EngineeringNational Science Foundation (U.S.). Graduate Research FellowshipMassachusetts Institute of Technology. Department of Biological EngineeringHarvard Medical School. Department of Genetic

Minimal information for studies of extracellular vesicles (MISEV2023): From basic to advanced approaches

© 2024 The Authors. Journal of Extracellular Vesicles, published by Wiley Periodicals, LLC on behalf of the International Society for Extracellular Vesicles. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY), https://creativecommons.org/licenses/by/4.0/Extracellular vesicles (EVs), through their complex cargo, can reflect the state of their cell of origin and change the functions and phenotypes of other cells. These features indicate strong biomarker and therapeutic potential and have generated broad interest, as evidenced by the steady year-on-year increase in the numbers of scientific publications about EVs. Important advances have been made in EV metrology and in understanding and applying EV biology. However, hurdles remain to realising the potential of EVs in domains ranging from basic biology to clinical applications due to challenges in EV nomenclature, separation from non-vesicular extracellular particles, characterisation and functional studies. To address the challenges and opportunities in this rapidly evolving field, the International Society for Extracellular Vesicles (ISEV) updates its 'Minimal Information for Studies of Extracellular Vesicles', which was first published in 2014 and then in 2018 as MISEV2014 and MISEV2018, respectively. The goal of the current document, MISEV2023, is to provide researchers with an updated snapshot of available approaches and their advantages and limitations for production, separation and characterisation of EVs from multiple sources, including cell culture, body fluids and solid tissues. In addition to presenting the latest state of the art in basic principles of EV research, this document also covers advanced techniques and approaches that are currently expanding the boundaries of the field. MISEV2023 also includes new sections on EV release and uptake and a brief discussion of in vivo approaches to study EVs. Compiling feedback from ISEV expert task forces and more than 1000 researchers, this document conveys the current state of EV research to facilitate robust scientific discoveries and move the field forward even more rapidly.Peer reviewe

Minimal information for studies of extracellular vesicles (MISEV2023): From basic to advanced approaches

Extracellular vesicles (EVs), through their complex cargo, can reflect the state of their cell of origin and change the functions and phenotypes of other cells. These features indicate strong biomarker and therapeutic potential and have generated broad interest, as evidenced by the steady year-on-year increase in the numbers of scientific publications about EVs. Important advances have been made in EV metrology and in understanding and applying EV biology. However, hurdles remain to realising the potential of EVs in domains ranging from basic biology to clinical applications due to challenges in EV nomenclature, separation from non-vesicular extracellular particles, characterisation and functional studies. To address the challenges and opportunities in this rapidly evolving field, the International Society for Extracellular Vesicles (ISEV) updates its ‘Minimal Information for Studies of Extracellular Vesicles’, which was first published in 2014 and then in 2018 as MISEV2014 and MISEV2018, respectively. The goal of the current document, MISEV2023, is to provide researchers with an updated snapshot of available approaches and their advantages and limitations for production, separation and characterisation of EVs from multiple sources, including cell culture, body fluids and solid tissues. In addition to presenting the latest state of the art in basic principles of EV research, this document also covers advanced techniques and approaches that are currently expanding the boundaries of the field. MISEV2023 also includes new sections on EV release and uptake and a brief discussion of in vivo approaches to study EVs. Compiling feedback from ISEV expert task forces and more than 1000 researchers, this document conveys the current state of EV research to facilitate robust scientific discoveries and move the field forward even more rapidly

PROLinkSingle Step Circularization and Purification Procedure for the Generation of an Improved Variant of Human Growth Hormone

Human growth hormone (hGH) plays an important role during human development and is also an approved therapeutic for the treatment of several diseases. However, one major drawback of hGH is its short circulating half-life requiring frequent administration, which is inconvenient and painful for the patients. Recent publications indicate that circularization greatly increases the stability of proteins due to their protection from exoproteolytic attack and a higher thermal stability of the circular form. Using sortase A, a transpeptidase isolated from Staphylococcus aureus, we developed a single step solid-phase circularization and purification procedure resulting in a circular version of hGH with improved properties. We could show that circular hGH binds to the recombinant hGH receptor with binding kinetics similar to those of linear hGH and that circularization does not alter the biological activity of hGH in vitro. Besides, circular hGH showed almost complete resistance toward exoproteolytic attack and slightly increased thermal stability which could possibly translate into an extended plasma half-life. The single step solid-phase circularization and purification procedure is in principle a generic process, which could also be applied for other proteins that meet the requirements for circularization