Synthetic biology: at the crossroads of genetic engineering and human therapeutics-a Keystone Symposia report.

Synthetic biology has the potential to transform cell- and gene-based therapies for a variety of diseases. Sophisticated tools are now available for both eukaryotic and prokaryotic cells to engineer cells to selectively achieve therapeutic effects in response to one or more disease-related signals, thus sparing healthy tissue from potentially cytotoxic effects. This report summarizes the Keystone eSymposium "Synthetic Biology: At the Crossroads of Genetic Engineering and Human Therapeutics," which took place on May 3 and 4, 2021. Given that several therapies engineered using synthetic biology have entered clinical trials, there was a clear need for a synthetic biology symposium that emphasizes the therapeutic applications of synthetic biology as opposed to the technical aspects. Presenters discussed the use of synthetic biology to improve T cell, gene, and viral therapies, to engineer probiotics, and to expand upon existing modalities and functions of cell-based therapies.

Plant genome engineering from lab to field-a Keystone Symposia report.

Facing the challenges of the world's food sources posed by a growing global population and a warming climate will require improvements in plant breeding and technology. Enhancing crop resiliency and yield via genome engineering will undoubtedly be a key part of the solution. The advent of new tools, such as CRIPSR/Cas, has ushered in significant advances in plant genome engineering. However, several serious challenges remain in achieving this goal. Among them are efficient transformation and plant regeneration for most crop species, low frequency of some editing applications, and high attrition rates. On March 8 and 9, 2021, experts in plant genome engineering and breeding from academia and industry met virtually for the Keystone eSymposium "Plant Genome Engineering: From Lab to Field" to discuss advances in genome editing tools, plant transformation, plant breeding, and crop trait development, all vital for transferring the benefits of novel technologies to the field.

CRISPR: History and perspectives to the future.

This review summarizes the information about the history and future of the CRISPR/Cas9 method. Genome editing can be perceived as a group of technologies that allow scientists to change the DNA of an organism. These technologies involve the deletion, insertion, or modification of the genome at a specific site in a DNA sequence. Gene therapy in humans has a perspective to be used to eliminate the gene responsible for a particular genetic disorder. The review focuses on the key elements of this promising method and the possibility of its application in the treatment of cancer and genetic diseases.

The advancements, challenges, and future implications of the CRISPR/Cas9 system in swine research.

Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (CRISPR/Cas9) genome editing technology has dramatically influenced swine research by enabling the production of high-quality disease-resistant pig breeds, thus improving yields. In addition, CRISPR/Cas9 has been used extensively in pigs as one of the tools in biomedical research. In this review, we present the advancements of the CRISPR/Cas9 system in swine research, such as animal breeding, vaccine development, xenotransplantation, and disease modeling. We also highlight the current challenges and some potential applications of the CRISPR/Cas9 technologies.

Rodent Models to Analyze the Glioma Microenvironment.

Animal models are still indispensable for understanding the basic principles of glioma development and invasion. Preclinical approaches aim to analyze the treatment efficacy of new drugs before translation into clinical trials is possible. Various animal disease models are available, but not every approach is useful for addressing specific questions. In recent years, it has become increasingly evident that the tumor microenvironment plays a key role in the nature of glioma. In addition to providing an overview, this review evaluates available rodent models in terms of usability for research on the glioma microenvironment.

Therapeutic and diagnostic relevance of Crispr technology.

CRISPR is a family of DNA repeats providing immunity against viral and plasmid invading DNA in bacteria and archaea. The system consist of an endonuclease Cas, guided by a RNA sequence, able to cleave the DNA double strand at a specific site. The discovery of Crispr function in 2007 has revolutionized genetic engineering by giving to the world the most powerful and precise tool for targeted genome editing. The aim of this review is to synthesize the current knowledge on Crispr/cas system and its application in biomedical field. In particular, we focus on the relevance of this new tool in progressing our comprehension for biological mechanisms and improving our ability to treat and prevent genetic diseases, to control microbial virulence and to generate animal models for basic and clinical research. We discuss also the ethical issues that may prevent the application of Crispr technology in living beings.

Genetic engineering of T cells for immunotherapy.

Genetically engineered T cell immunotherapies have provided remarkable clinical success to treat B cell acute lymphoblastic leukaemia by harnessing a patient's own T cells to kill cancer, and these approaches have the potential to provide therapeutic benefit for numerous other cancers, infectious diseases and autoimmunity. By introduction of either a transgenic T cell receptor or a chimeric antigen receptor, T cells can be programmed to target cancer cells. However, initial studies have made it clear that the field will need to implement more complex levels of genetic regulation of engineered T cells to ensure both safety and efficacy. Here, we review the principles by which our knowledge of genetics and genome engineering will drive the next generation of adoptive T cell therapies.

Genetic engineering in organoids.

Three-dimensional organoids have been widely used for developmental and disease modeling. Organoids are derived from both adult and pluripotent stem cells. Various types are available for mimicking almost all major organs and tissues in the mouse and human. While culture protocols for stepwise differentiation and long-term expansion are well established, methods for genetic manipulation in organoids still need further standardization. In this review, we summarized different methods for organoid genetics and provide the pros and cons of each method for designing an optimal strategy.

CRISPR/Cas9 Technology as a Modern Genetic Manipulation Tool for Recapitulating of Neurodegenerative Disorders in Large Animal Models.

BACKGROUND: Neurodegenerative diseases are often the consequence of alterations in structures and functions of the Central Nervous System (CNS) in patients. Despite obtaining massive genomic information concerning the molecular basis of these diseases and since the neurological disorders are multifactorial, causal connections between pathological pathways at the molecular level and CNS disorders development have remained obscure and need to be elucidated to a great extent. OBJECTIVE: Animal models serve as accessible and valuable tools for understanding and discovering the roles of causative factors in the development of neurodegenerative disorders and finding appropriate treatments. Contrary to rodents and other small animals, large animals, especially non-human primates (NHPs), are remarkably similar to humans; hence, they establish suitable models for recapitulating the main human's neuropathological manifestations that may not be seen in rodent models. In addition, they serve as useful models to discover effective therapeutic targets for neurodegenerative disorders due to their similarity to humans in terms of physiology, evolutionary distance, anatomy, and behavior. METHODS: In this review, we recommend different strategies based on the CRISPR-Cas9 system for generating animal models of human neurodegenerative disorders and explaining in vivo CRISPR-Cas9 delivery procedures that are applied to disease models for therapeutic purposes. RESULTS: With the emergence of CRISPR/Cas9 as a modern specific gene-editing technology in the field of genetic engineering, genetic modification procedures such as gene knock-in and knock-out have become increasingly easier compared to traditional gene targeting techniques. Unlike the old techniques, this versatile technology can efficiently generate transgenic large animal models without the need to complicate lab instruments. Hence, these animals can accurately replicate the signs of neurodegenerative disorders. CONCLUSION: Preclinical applications of CRISPR/Cas9 gene-editing technology supply a unique opportunity to establish animal models of neurodegenerative disorders with high accuracy and facilitate perspectives for breakthroughs in the research on the nervous system disease therapy and drug discovery. Furthermore, the useful outcomes of CRISPR applications in various clinical phases are hopeful for their translation to the clinic in a short time.

Genetic engineering of crops for insect resistance: An overview.

Phytophagous insect incidence is a serious threat for reduction of crop productivity globally. There is an estimation of one fourth of crop is being destroyed by insects annually. Indeed, the development of insect-resistant crops is a great milestone in agriculture to increase crop yield and reduce pesticide dependency. Genetic engineering facilitates development of insect resistant crops by expressing bacterial δ-endotoxins and vegetative insecticidal proteins and other plant genes like lectins, protease inhibitors, etc. In addition, RNA interference and genome editing through CRISPR Cas9 also provides new solutions for the development of insect-resistant crops. The resultant genetically modified crops showed resistance against lepidopteran, dipteran, homopteran and coleopteran insects. The insect-resistant crops have made a significant economic impact worldwide in terms of higher yield and low pesticide usage. In this review, we focus on different strategies for developing transgenics against insect pest control by expressing different insecticidal proteins in crops.

An update and perspectives on the use of promoters in plant genetic engineering.

Genetically engineered plants have varied applications in agriculture for enhancing the values of food and feed. Genetic engineering aims to introduce selected genetic regions with desirable traits into target plants for both spatial and temporal expressions. Promoters are the key elements responsible for regulating gene expressions by modulating the transcription factors (TFs) through recognition of RNA polymerases. Based on their recognition and expression, RNA polymerases were categorized into RNA pol II and pol III promoters. Promoter activity and specificity are the two prime parameters in regulating the transgene expression. Since the use of constitutive promoters like Cauliflower mosaic virus (CaMV) 35S may lead to adverse effects on nontarget organisms or ecosystem, inducible/tissue specific promoters and/or the RNA pol III promoters provide myriad opportunities for gene expressions with controlled regulation and with minimum adverse effects. Besides their role in transgene expression, their influence in synthetic biology and genome editing are also discussed. This review provides an update on the importance, current prospects, and insight into the advantages and disadvantages of promoters reported thus far would help to utilize them in the endeavour to develop nutritionally and agronomically improved transgenic crops for commercialization.

Plant abiotic stress tolerance: Insights into resilience build-up.

Climate change and the consequential unpredictable environmental stress conditions negatively impact crop productivity. It has thus become a challenge to develop solutions for food security and sustainable agriculture in the backdrop of increasing population pressure and dwindling land and water resources. This further necessitates that focus of international research should be on curtailing yield losses through improved crop breeding practices and genetic manipulation for the development of resistant crop varieties. Plants being sessile, have developed a complex regulatory network of genetic machinery which includes transcription factors, small RNAs, signalling pathways, stress sensors and defense pathways. Needless to say, research efforts have exploited this genetic reservoir for manipulating crop plants for tolerance or resistance against different stresses. In the past few decades, significant achievement has been made for developing transgenic plants for a wide variety of single or multiple stress tolerance associated traits. Several regulatory mechanisms have been identified to fine tune and tailor the tolerance response in target sensitive crops. The advent of metabolic engineering has added new dimensions to manipulate stress tolerance pathways. Novel strategies are needed to develop stable, superior performing lines under challenging field environment without yield penalty and significant success has to be achieved to translate the research outcome from lab-to-land to reach farmer's fields.

Application of different types of CRISPR/Cas-based systems in bacteria.

As important genome editing tools, CRISPR/Cas systems, especially those based on type II Cas9 and type V Cas12a, are widely used in genetic and metabolic engineering of bacteria. However, the intrinsic toxicity of Cas9 and Cas12a-mediated CRISPR/Cas tools can lead to cell death in some strains, which led to the development of endogenous type I and III CRISPR/Cas systems. However, these systems are hindered by complicated development and limited applications. Thus, further development and optimization of CRISPR/Cas systems is needed. Here, we briefly summarize the mechanisms of different types of CRISPR/Cas systems as genetic manipulation tools and compare their features to provide a reference for selecting different CRISPR/Cas tools. Then, we show the use of CRISPR/Cas technology for bacterial strain evolution and metabolic engineering, including genome editing, gene expression regulation and the base editor tool. Finally, we offer a view of future directions for bacterial CRISPR/Cas technology.

Current achievements and future prospects of genetic engineering in Indian mustard (Brassica juncea L. Czern & Coss.).

MAIN CONCLUSION: Transgenic technology in Indian mustard has expedited crop improvement programs. Further, there is a need to optimize gene editing protocols and find out the suitable target genes to harvest the benefits of gene editing technology in this important edible oilseed crop. Brassica juncea is an economically and industrially important oilseed crop being grown mainly in India and in some parts of Canada, Russia, China and Australia. Besides being consumed as edible oil, it also has numerous applications in food and paint industry. However, its overall production and productivity are being hampered by a number of biotic and abiotic stress factors. Further, its oil and seedmeal quality needs to be improved for increasing food as well as feed value. However, the lack of resistant crossable germplasm or varieties necessitated the use of genetic engineering interventions in Indian mustard crop improvement. A number of genes conferring resistance to biotic stresses including lectins for aphids' control, chitinase, glucanase and osmotin for disease control and for abiotic stresses, CODA, LEA and ion antiporter genes have been transferred to Indian mustard. Both antisense and RNAi technologies have been employed for improving oil and seedmeal quality. Efforts have been made to improve the phytoremediation potential of this crop through genetic engineering approach. The deployment of barnase/barstar gene system for developing male sterile and restorer lines has really expedited hybrid development programs in Indian mustard. Further, there is a need to optimize gene editing protocols and to find out suitable target genes for gene editing in this crop. In this review paper, authors have attempted to review various genetic transformation efforts carried out in Indian mustard for its improvement to combat biotic and abiotic stress challenges, quality improvement and hybrid development.

[Progress in engineering application of human adenovirus].

Human adenoviruses are widespread causative agent that induces respiratory diseases, epidemic keratoconjunctivitis and other related diseases. Adenoviruses are commonly used in experimental and clinical areas. It is one of the most commonly used virus vectors in gene therapy, and it has attracted a lot of attention and has a high research potential in tumor gene therapy and virus oncolytic. Here, we summarize the biological characteristics, epidemiology and current application of adenovirus, in order to provide reference for engineering application of adenovirus.