Cancer cells depend on nucleotides for proliferation. Inhibition of nucleotide metabolism by antimetabolites is a well-established anticancer therapy. However, resistance and toxicity to antimetabolite treatments reduce their effectiveness. Here, we focus on the pyrimidine de novo synthesis pathway, which is crucial for cancer cell proliferation, yet its pharmacological targeting in cancer has been without much clinical success so far. Hence, it is important to understand how cancer cells cope with the insufficiency of this pathway. Here, we describe a procedure to prepare subcutaneous tumor model deficient in de novo pyrimidine synthesis. For examination of metabolic responses to de novo synthesis blockade in tumors, we propose application of MALDI imaging that allows spatially resolved examination of metabolic responses to de novo synthesis blockade in tumors.
RNA interference (RNAi) designates sequence-specific mRNA degradation mediated by small RNAs generated from long double-stranded RNA (dsRNA) by RNase III Dicer. RNAi appears inactive in mammalian cells except for mouse oocytes, where high RNAi activity exists because of an N-terminally truncated Dicer isoform, denoted DicerO. DicerO processes dsRNA into small RNAs more efficiently than the full-length Dicer expressed in somatic cells. DicerO is expressed from an oocyte-specific promoter of retrotransposon origin, which is silenced in other cell types. In this work, we evaluated CRISPR-based strategies for epigenetic targeting of the endogenous Dicer gene to restore DicerO expression and, consequently, RNAi. We show that reactivation of DicerO expression can be achieved in mouse embryonic stem cells, but it is not sufficient to establish a robust canonical RNAi response.
- MeSH
- buňky 3T3 MeSH
- DEAD-box RNA-helikasy antagonisté a inhibitory genetika MeSH
- embryonální kmenové buňky cytologie metabolismus MeSH
- malá interferující RNA genetika MeSH
- myši MeSH
- promotorové oblasti (genetika) * MeSH
- ribonukleasa III antagonisté a inhibitory genetika MeSH
- RNA interference MeSH
- sekvence CRISPR * MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
According to Darwin's theory, endless evolution leads to a revolution. One such example is the Clustered Regularly Interspaced Palindromic Repeats (CRISPR)-Cas system, an adaptive immunity system in most archaea and many bacteria. Gene editing technology possesses a crucial potential to dramatically impact miscellaneous areas of life, and CRISPR-Cas represents the most suitable strategy. The system has ignited a revolution in the field of genetic engineering. The ease, precision, affordability of this system is akin to a Midas touch for researchers editing genomes. Undoubtedly, the applications of this system are endless. The CRISPR-Cas system is extensively employed in the treatment of infectious and genetic diseases, in metabolic disorders, in curing cancer, in developing sustainable methods for fuel production and chemicals, in improving the quality and quantity of food crops, and thus in catering to global food demands. Future applications of CRISPR-Cas will provide benefits for everyone and will save countless lives. The technology is evolving rapidly; therefore, an overview of continuous improvement is important. In this review, we aim to elucidate the current state of the CRISPR-Cas revolution in a tailor-made format from its discovery to exciting breakthroughs at the application level and further upcoming trends related to opportunities and challenges including ethical concerns.
- MeSH
- Archaea metabolismus MeSH
- Bacteria metabolismus MeSH
- CRISPR-Cas systémy * MeSH
- dějiny 20. století MeSH
- dějiny 21. století MeSH
- dobytek MeSH
- editace genu metody MeSH
- genetické inženýrství dějiny metody MeSH
- genom MeSH
- lidé MeSH
- sekvence CRISPR MeSH
- zemědělské plodiny genetika MeSH
- zvířata MeSH
- Check Tag
- dějiny 20. století MeSH
- dějiny 21. století MeSH
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- historické články MeSH
- přehledy MeSH
Triticeae cereals are among the most important crop plants grown worldwide and being used for animal feed, food and beverages. Although breeding efforts evolved over the last ten thousand years our today's crop plants, biotechnological methods would help to speed up the process and incorporate traits impossible by conventional breeding. The main research topics were related to cover the future demand on our agricultural practices to supply sufficient food for a growing world population. Target traits are resistances against viral and fungal diseases, improvement of water and nitrogen use efficiency, to tackle plant architecture, both below and aboveground and to develop varieties that could grow on dry or salty locations. Other applications are considering accumulation of useful compounds or decreasing allergenicity. This review will summarize methods to generate the material including a section how genome engineering using gRNA/Cas (CRISPR/Cas) technology could further improve the methodology and will give an overview about recent and future applications.
- MeSH
- mezinárodní spolupráce MeSH
- Nobelova cena MeSH
- sekvence CRISPR MeSH
- výzkumní pracovníci MeSH
- Publikační typ
- novinové články MeSH
With recent advances in novel gene-editing tools such as RNAi, ZFNs, TALENs, and CRISPR-Cas9, the possibility of altering microalgae toward designed properties for various application is becoming a reality. Alteration of microalgae genomes can modify metabolic pathways to give elevated yields in lipids, biomass, and other components. The potential of such genetically optimized microalgae can give a "domino effect" in further providing optimization leverages down the supply chain, in aspects such as cultivation, processing, system design, process integration, and revolutionary products. However, the current level of understanding the functional information of various microalgae gene sequences is still primitive and insufficient as microalgae genome sequences are long and complex. From this perspective, this work proposes to link up this knowledge gap between microalgae genetic information and optimized bioproducts using Artificial Intelligence (AI). With the recent acceleration of AI research, large and complex data from microalgae research can be properly analyzed by combining the cutting-edge of both fields. In this work, the most suitable class of AI algorithms (such as active learning, semi-supervised learning, and meta-learning) are discussed for different cases of microalgae applications. This work concisely reviews the current state of the research milestones and highlight some of the state-of-art that has been carried out, providing insightful future pathways. The utilization of AI algorithms in microalgae cultivation, system optimization, and other aspects of the supply chain is also discussed. This work opens the pathway to a digitalized future for microalgae research and applications.
- MeSH
- lidé MeSH
- Nobelova cena * MeSH
- sekvence CRISPR MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- úvodníky MeSH
CRISPR is a prokaryotic defence system that was adapted as a tool for genome editing and has become one of the most important discoveries of this century. CRISPR-associated endonucleases cleave DNA at precise sites, which are marked by complementary short-guided RNA. The recently developed versions of endonucleases are compatible with a broad range of PAM motifs, have a higher specificity and enable a specific nucleotide to be replaced.
- MeSH
- DNA genetika MeSH
- editace genu * MeSH
- endonukleasy genetika metabolismus MeSH
- guide RNA, Kinetoplastida genetika MeSH
- lidé MeSH
- sekvence CRISPR genetika MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
RNA-guided, engineered nucleases derived from the prokaryotic adaptive immune system CRISPR-Cas represent a powerful platform for gene deletion and editing. When used as a therapeutic approach, direct delivery of Cas9 protein and single-guide RNA (sgRNA) could circumvent the safety issues associated with plasmid delivery and therefore represents an attractive tool for precision genome engineering. Gene deletion or editing in adipose tissue to enhance its energy expenditure, fatty acid oxidation, and secretion of bioactive factors through a "browning" process presents a potential therapeutic strategy to alleviate metabolic disease. Here, we developed "CRISPR-delivery particles," denoted CriPs, composed of nano-size complexes of Cas9 protein and sgRNA that are coated with an amphipathic peptide called Endo-Porter that mediates entry into cells. Efficient CRISPR-Cas9-mediated gene deletion of ectopically expressed GFP by CriPs was achieved in multiple cell types, including a macrophage cell line, primary macrophages, and primary pre-adipocytes. Significant GFP loss was also observed in peritoneal exudate cells with minimum systemic toxicity in GFP-expressing mice following intraperitoneal injection of CriPs containing Gfp-targeting sgRNA. Furthermore, disruption of a nuclear co-repressor of catabolism, the Nrip1 gene, in white adipocytes by CriPs enhanced adipocyte browning with a marked increase of uncoupling protein 1 (UCP1) expression. Of note, the CriP-mediated Nrip1 deletion did not produce detectable off-target effects. We conclude that CriPs offer an effective Cas9 and sgRNA delivery system for ablating targeted gene products in cultured cells and in vivo, providing a potential therapeutic strategy for metabolic disease.
- MeSH
- bílá tuková tkáň cytologie metabolismus MeSH
- buněčné linie MeSH
- CRISPR-Cas systémy MeSH
- editace genu MeSH
- energetický metabolismus * MeSH
- genový targeting metody MeSH
- lidé MeSH
- myši inbrední C57BL MeSH
- nuclear receptor interacting protein 1 genetika metabolismus MeSH
- plazmidy genetika metabolismus MeSH
- reportérové geny MeSH
- sekvence CRISPR MeSH
- tukové buňky metabolismus MeSH
- uncoupling protein 1 genetika metabolismus MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- hodnotící studie MeSH
- práce podpořená grantem MeSH
- Research Support, N.I.H., Extramural MeSH
Fanconi anemia (FA) is an inherited condition characterized by impaired DNA repair, physical anomalies, bone marrow failure, and increased incidence of malignancy. Gene editing holds great potential to precisely correct the underlying genetic cause such that gene expression remains under the endogenous control mechanisms. This has been accomplished to date only in transformed cells or their reprogrammed induced pluripotent stem cell counterparts; however, it has not yet been reported in primary patient cells. Here we show the ability to correct a mutation in Fanconi anemia D1 (FANCD1) primary patient fibroblasts. The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system was employed to target and correct aFANCD1gene deletion. Homologous recombination using an oligonucleotide donor was achieved and a pure population of modified cells was obtained by using inhibitors of poly adenosine diphosphate-ribose polymerase (poly ADP-ribose polymerase).FANCD1function was restored and we did not observe any promiscuous cutting of the CRISPR/Cas9 at off target sites. This consideration is crucial in the context of the pre-malignant FA phenotype. Altogether we show the ability to correct a patient mutation in primaryFANCD1cells in a precise manner. These proof of principle studies support expanded application of gene editing for FA.
- MeSH
- buněčné linie MeSH
- CRISPR-Cas systémy * MeSH
- delece genu MeSH
- editace genu metody MeSH
- Fanconiho anemie genetika metabolismus terapie MeSH
- fibroblasty metabolismus MeSH
- genetická terapie metody MeSH
- kultivované buňky MeSH
- lidé MeSH
- protein BRCA2 genetika metabolismus MeSH
- sekvence CRISPR MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
