Challenges of CRISPR-based Gene Editing in Primary T Cells

Adaptive T cell immunotherapy holds great promise for the successful treatment of leukemia as well as other types of cancers. More recently, it was also shown to be an effective treatment option for chronic virus infections in immunosuppressed patients. Autologous or allogeneic T cells used for immunotherapy are usually genetically modified to express novel T cell or chimeric antigen receptors. The production of such cells was significantly simplified with the CRISPR/Cas system allowing deletion or insertion of novel genes at specific locations within the genome. In this review, we describe recent methodological breakthroughs important for the conduction of these genetic modifications, summarize crucial points to be considered when conducting such experiments, and highlight the potential pitfalls of these approaches.

Climate Change on Fertility and Reproductive Processes of Female Livestock

The effects of climate change continues to be a growing modern-day challenge. Climate change-induced heat stress disrupts reproductive and fertility systems in livestock. In males, it modifies the physiology of the spermatogenic cycle resulting to poor quality semen and high prevalence of secondary sperm defects. In female livestock, heat stress decreases the production of gonadotrophins, results to hormonal imbalance, decreases the quality of oocytes, and lengthens the oestrous period leading to infertility. These effects can be reversed through genetic modifications, nutritive supplementation, physical cooling mechanisms, and hormonal therapies. The successful implementation of the ameliorative strategies is pegged on improved research and their combined administration. Ultimately, climate change mitigation and adaptation are indispensable to overcome fertility problems in livestock among other environmental effects of the climate variations.

REGULATION OF REPRODUCTIVE CLONING AND INHERITABLE GENETIC MODIFICATIONS ON HUMANS - PERILS AND DEFICIENCIES

Harms arising from reproductive cloning or inheritable genetic modifications, for the time being, seem significant. This is supported by the simple fact that the first cloned monkeys were short-lived or by the fact that inheritable genetic modifications still carry a high chance of getting “off-target” results, which could result in serious health problems. Inheritable genetic modifications, in particular, have a high therapeutic potential, and it is suggested that this technology’s comprehension is shifting from an absolute ban, to concerns over safety issues. International law can prove to be facilitative when it comes to deciding which new technology should be prohibited, restricted or allowed, having in mind possible consequences and the so-called phenomenon of reproductive tourism. Legally binding regulation of both technologies has proven challenging at the universal level. However, there has been some progress in Europe on that matter. Harms arising from inheritable genetic modifications seem even higher than in the case of reproductive cloning, since they have the potential to affect the whole of humanity, including future generations. The Criminal Code of Serbia and the Constitution of the Republic of Serbia prohibit reproductive cloning. However, the prohibition of inheritable genetic modifications on humans is not regulated explicitly in the Criminal Code of Serbia, making this technology seem more acceptable or less harmful.

Recent Molecular Tools for the Genetic Manipulation of Highly Industrially Important Mucoromycota Fungi

Mucorales is the largest and most well-studied order of the phylum Mucormycota and is known for its rapid growth rate and various industrial applications. The Mucorales fungi are a fascinating group of filamentous organisms with many uses in research and the industrial and medical fields. They are widely used biotechnological producers of various secondary metabolites and other value-added products. Certain members of Mucorales are extensively used as model organisms for genetic and molecular investigation and have extended our understanding of the metabolisms of other members of this order as well. Compared with other fungal species, our understanding of Mucoralean fungi is still in its infancy, which could be linked to their lack of effective genetic tools. However, recent advancements in molecular tools and approaches, such as the construction of recyclable markers, silencing vectors, and the CRISPR-Cas9-based gene-editing system, have helped us to modify the genomes of these model organisms. Multiple genetic modifications have been shown to generate valuable products on a large scale and helped us to understand the morphogenesis, basic biology, pathogenesis, and host–pathogen interactions of Mucoralean fungi. In this review, we discuss various conventional and modern genetic tools and approaches used for efficient gene modification in industrially important members of Mucorales.

Infection of wild-type mice by SARS-CoV-2 B.1.351 variant indicates a possible novel cross-species transmission route

COVID-19 is identified as a zoonotic disease caused by SARS-CoV-2, which also can cross-transmit to many animals but not mice. Genetic modifications of SARS-CoV-2 or mice enable the mice susceptible to viral infection. Although neither is the natural situation, they are currently utilized to establish mouse infection models. Here we report a direct contact transmission of SARS-CoV-2 variant B.1.351 in wild-type mice. The SARS-CoV-2 (B.1.351) replicated efficiently and induced significant pathological changes in lungs and tracheas, accompanied by elevated proinflammatory cytokines in the lungs and sera. Mechanistically, the receptor-binding domain (RBD) of SARS-CoV-2 (B.1.351) spike protein turned to a high binding affinity to mouse angiotensin-converting enzyme 2 (mACE2), allowing the mice highly susceptible to SARS-CoV-2 (B.1.351) infection. Our work suggests that SARS-CoV-2 (B.1.351) expands the host range and therefore increases its transmission route without adapted mutation. As the wild house mice live with human populations quite closely, this possible transmission route could be potentially risky. In addition, because SARS-CoV-2 (B.1.351) is one of the major epidemic strains and the mACE2 in laboratory-used mice is naturally expressed and regulated, the SARS-CoV-2 (B.1.351)/mice could be a much convenient animal model system to study COVID-19 pathogenesis and evaluate antiviral inhibitors and vaccines.

A cotransformation system of the unicellular red alga Cyanidioschyzon merolae with blasticidin S deaminase and chloramphenicol acetyltransferase selectable markers

Background The unicellular red alga Cyanidioschyzon merolae exhibits a very simple cellular and genomic architecture. In addition, procedures for genetic modifications, such as gene targeting by homologous recombination and inducible/repressible gene expression, have been developed. However, only two markers for selecting transformants, uracil synthase (URA) and chloramphenicol acetyltransferase (CAT), are available in this alga. Therefore, manipulation of two or more different chromosomal loci in the same strain in C. merolae is limited. Results This study developed a nuclear targeting and transformant selection system using an antibiotics blasticidin S (BS) and the BS deaminase (BSD) selectable marker by homologous recombination in C. merolae. In addition, this study has succeeded in simultaneously modifying two different chromosomal loci by a single-step cotransformation based on the combination of BSD and CAT selectable markers. A C. merolae strain that expresses mitochondrion-targeted mSCARLET (with the BSD marker) and mVENUS (with the CAT marker) from different chromosomal loci was generated with this procedure. Conclusions The newly developed BSD selectable marker enables an additional genetic modification to the already generated C. merolae transformants based on the URA or CAT system. Furthermore, the cotransformation system facilitates multiple genetic modifications. These methods and the simple nature of the C. merolae cellular and genomic architecture will facilitate studies on several phenomena common to photosynthetic eukaryotes.

Extracellular Vesicles and Their Interplay with Biological Membranes

Most cells secrete vesicles into the extracellular environment to interact with other cells. These extracellular vesicles (EVs), have undergone a paradigm shift upon the discovery that they also transport important material including proteins, lipids and nucleic acids. As natural cargo carriers, EVs are not recognised by the immune system as foreign substances, and consequently evade removal by immune cells. These intrinsic biological properties of EVs have led to further research on utilising EVs as potential diagnostic biomarkers and drug delivery systems (DDSs). However, the internalisation of EVs by target cells is still not fully understood. Moreover, it is unclear whether EVs can cross certain biological membranes like the blood-brain barrier (BBB) naturally, or require genetic modifications to do so. Hence, this review aims to evaluate the relationship between the composition of EVs and their association with different biological membranes they encounter before successfully releasing their cargo into target cells. This review identifies specific biomarkers detected in various EVs and important biological barriers present in the gastrointestinal, placental, immunological, neurological, lymphatic, pulmonary, renal and intracellular environments, and provides a recommendation on how to engineer EVs as potential drug carriers based on key proteins and lipids involved in crossing these barriers.

Genetic factors and cancer: diagnosis, prognosis and future perspectives

Genetics is specifically responsible for several pathologies or, at the least, it is associated with a wide range of them, either as a primary causal agent (congenital genetic diseases) or secondary, being a factor within several possible for a given disease. One of the most critical genetic concepts is developed from the phenotype, equivalent to the genotype associated with the environment. In other words, for a condition to manifest itself, cancer, for example, we need a genetic alteration within the environment, which somehow influences carcinogenesis from stochastic or induced interactions. Cancer cases are approximately 80% and 90% associated with external causes, and environmental changes are mainly motivated by human actions, habits, and behavior, leading to an increased risk of different types of cancer. These changes lead to the formation of a cycle since man promotes environmental changes, leading to genetic modifications responsible for 10-20% of cancer formation. Although the percentage seems not to be significant, we have, in fact, several genetic mechanisms that will lead to the emergence of the most diverse types of cancer, including polymorphisms, mutations, oxidative stress, oncogenes, and genes that regulate the cell cycle, including apoptosis.