The limited number of well-characterised model bacteria cannot address all the challenges in a circular bioeconomy. Therefore, there is a growing demand for new production strains with enhanced resistance to extreme conditions, versatile metabolic capabilities and the ability to utilise cost-effective renewable resources while efficiently generating attractive biobased products. Particular thermophilic microorganisms fulfil these requirements. Non-virulent Gram-negative Caldimonas thermodepolymerans DSM15344 is one such attractive thermophile that efficiently converts a spectrum of plant biomass sugars into high quantities of polyhydroxyalkanoates (PHA)-a fully biodegradable substitutes for synthetic plastics. However, to enhance its biotechnological potential, the bacterium needs to be 'domesticated'. In this study, we established effective homologous recombination and transposon-based genome editing systems for C. thermodepolymerans. By optimising the electroporation protocol and refining counterselection methods, we achieved significant improvements in genetic manipulation and constructed the AI01 chassis strain with improved transformation efficiency and a ΔphaC mutant that will be used to study the importance of PHA synthesis in Caldimonas. The advances described herein highlight the need for tailored approaches when working with thermophilic bacteria and provide a springboard for further genetic and metabolic engineering of C. thermodepolymerans, which can be considered the first model of thermophilic PHA producer.

• Klíčová slova
• Caldimonas thermodepolymerans, gene deletion, genetic engineering, polyhydroxyalkanoates, thermophiles,
• MeSH
• editace genu * metody   MeSH
• elektroporace MeSH
• genom bakteriální MeSH
• homologní rekombinace MeSH
• metabolické inženýrství metody   MeSH
• polyhydroxyalkanoáty * metabolismus   biosyntéza   MeSH
• transpozibilní elementy DNA genetika   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• polyhydroxyalkanoáty * MeSH
• transpozibilní elementy DNA MeSH

Postponing the adoption of genome editing (GE) is costly, with lengthy regulatory processes contributing to postponement. Accelerating agricultural research and development (R&D) transfer is important for stimulating sustainable agricultural transitions and enhancing global food security. Using the MAGNET model, we incorporate dynamic R&D accumulation and compare economic projections in scenarios with accelerated R&D transfer. We calculate the cost of delay (COD) from postponing GE adoption. The results show that accelerating R&D transfer in high-income countries impacts economic performance, welfare, and food affordability globally; the annuity of COD ranges from losses of -$1.1 billion (Brazil) to gains of $18.5 billion (Europe). A 3-year acceleration of R&D transfer in all countries benefits middle and low-income countries the most (e.g. China, India, other Asian countries, and Sub-Saharan African countries), with the annuity of COD ranging from -$4.8 billion (Brazil) to $83.9 billion (China). Therefore, streamlining the GE regulatory framework is essential for enhancing food security and global welfare.

• Klíčová slova
• Agricultural R&D transfer, computable general equilibrium model, cost of delay, genome editing, global food security,
• MeSH
• editace genu ekonomika   MeSH
• lidé MeSH
• výzkum ekonomika   trendy   MeSH
• zajištění potravin * ekonomika   MeSH
• zásobování potravinami ekonomika   MeSH
• zemědělské plodiny genetika   růst a vývoj   ekonomika   MeSH
• zemědělství * ekonomika   metody   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

Micronutrient deficiency conditions, such as anemia, are the most prevalent global health problem due to inadequate iron and folate in dietary sources. Biofortification advancements can propel the rapid amelioration of nutritionally beneficial components in crops that are required to combat the adverse effects of micronutrient deficiencies on human health. To date, several strategies have been proposed to increase micronutrients in plants to improve food quality, but very few approaches have intrigued `clustered regularly interspaced short palindromic repeats' (CRISPR) modules for the enhancement of iron and folate concentration in the edible parts of plants. In this review, we discuss two important approaches to simultaneously enhance the bioavailability of iron and folate concentrations in rice endosperms by utilizing advanced CRISPR-Cas9-based technology. This includes the 'tuning of cis-elements' and 'enhancer re-shuffling' in the regulatory components of genes that play a vital role in iron and folate biosynthesis/transportation pathways. In particular, base-editing and enhancer re-installation in native promoters of selected genes can lead to enhanced accumulation of iron and folate levels in the rice endosperm. The re-distribution of micronutrients in specific plant organs can be made possible using the above-mentioned contemporary approaches. Overall, the present review discusses the possible approaches for synchronized iron and folate biofortification through modification in regulatory gene circuits employing CRISPR-Cas9 technology.

• Klíčová slova
• Biofortification, CRISPR, enhancer re-shuffling, folic acid, iron,
• MeSH
• biofortifikace * MeSH
• CRISPR-Cas systémy * MeSH
• editace genu metody   MeSH
• geneticky modifikované rostliny * metabolismus   genetika   MeSH
• kyselina listová * metabolismus   MeSH
• lidé MeSH
• rýže (rod) metabolismus   genetika   MeSH
• železo * metabolismus   MeSH
• zemědělské plodiny * metabolismus   genetika   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH
• Názvy látek
• kyselina listová * MeSH
• železo * MeSH

The International Mouse Phenotyping Consortium (IMPC) systematically produces and phenotypes mouse lines with presumptive null mutations to provide insight into gene function. The IMPC now uses the programmable RNA-guided nuclease Cas9 for its increased capacity and flexibility to efficiently generate null alleles in the C57BL/6N strain. In addition to being a valuable novel and accessible research resource, the production of 3313 knockout mouse lines using comparable protocols provides a rich dataset to analyze experimental and biological variables affecting in vivo gene engineering with Cas9. Mouse line production has two critical steps - generation of founders with the desired allele and germline transmission (GLT) of that allele from founders to offspring. A systematic evaluation of the variables impacting success rates identified gene essentiality as the primary factor influencing successful production of null alleles. Collectively, our findings provide best practice recommendations for using Cas9 to generate alleles in mouse essential genes, many of which are orthologs of genes linked to human disease.

• Klíčová slova
• Cas9, Genome editing, Knockout, Mouse,
• MeSH
• alely MeSH
• CRISPR-Cas systémy MeSH
• editace genu * metody   MeSH
• esenciální geny * MeSH
• fenotyp MeSH
• genetické inženýrství metody   MeSH
• myši inbrední C57BL MeSH
• myši knockoutované * MeSH
• myši MeSH
• zvířata MeSH
• Check Tag
• mužské pohlaví MeSH
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

A tool for precise, target-specific, efficient, and affordable genome editing is a dream for many researchers, from those who conduct basic research to those who use it for applied research. Since 2012, we have tool that almost fulfils such requirements; it is based on clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) systems. However, even CRISPR/Cas has limitations and obstacles that might surprise its users. In this review, we focus on the most frequently used variant, CRISPR/Cas9 from Streptococcus pyogenes, and highlight key factors affecting its mutagenesis outcomes: (i) factors affecting the CRISPR/Cas9 activity, such as the effect of the target sequence, chromatin state, or Cas9 variant, and how long it remains in place after cleavage; and (ii) factors affecting the follow-up DNA repair mechanisms including mostly the cell type and cell cycle phase, but also, for example, the type of DNA ends produced by Cas9 cleavage (blunt/staggered). Moreover, we note some differences between using CRISPR/Cas9 in plants, yeasts, and animals, as knowledge from individual kingdoms is not fully transferable. Awareness of these factors can increase the likelihood of achieving the expected results of plant genome editing, for which we provide detailed guidelines.

• Klíčová slova
• CRISPR/Cas, Cell cycle, DNA repair, cleavage, editing, mutagenesis, plants, post-cleavage trimming, staggered ends,
• MeSH
• CRISPR-Cas systémy * MeSH
• editace genu * MeSH
• oprava DNA * MeSH
• rostliny * genetika   MeSH
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH

Genetic editing of the germline using CRISPR/Cas9 technology has made it possible to alter livestock traits, including the creation of resistance to viral diseases. However, virus adaptability could present a major obstacle in this effort. Recently, chickens resistant to avian leukosis virus subgroup J (ALV-J) were developed by deleting a single amino acid, W38, within the ALV-J receptor NHE1 using CRISPR/Cas9 genome editing. This resistance was confirmed both in vitro and in vivo. In vitro resistance of W38-/- chicken embryonic fibroblasts to all tested ALV-J strains was shown. To investigate the capacity of ALV-J for further adaptation, we used a retrovirus reporter-based assay to select adapted ALV-J variants. We assumed that adaptive mutations overcoming the cellular resistance would occur within the envelope protein. In accordance with this assumption, we isolated and sequenced numerous adapted virus variants and found within their envelope genes eight independent single nucleotide substitutions. To confirm the adaptive capacity of these substitutions, we introduced them into the original retrovirus reporter. All eight variants replicated effectively in W38-/- chicken embryonic fibroblasts in vitro while in vivo, W38-/- chickens were sensitive to tumor induction by two of the variants. Importantly, receptor alleles with more extensive modifications have remained resistant to the virus. These results demonstrate an important strategy in livestock genome engineering towards antivirus resistance and illustrate that cellular resistance induced by minor receptor modifications can be overcome by adapted virus variants. We conclude that more complex editing will be necessary to attain robust resistance.

• MeSH
• CRISPR-Cas systémy MeSH
• editace genu MeSH
• fibroblasty virologie   metabolismus   MeSH
• kur domácí * virologie   MeSH
• kuřecí embryo MeSH
• molekulární evoluce MeSH
• nemoci drůbeže virologie   genetika   MeSH
• odolnost vůči nemocem genetika   MeSH
• proteiny virového obalu genetika   metabolismus   MeSH
• ptačí leukóza * virologie   genetika   MeSH
• virus ptačí leukózy * genetika   fyziologie   MeSH
• zvířata MeSH
• Check Tag
• kuřecí embryo MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• proteiny virového obalu MeSH

Protein phosphorylation, the most common and essential post-translational modification, belongs to crucial regulatory mechanisms in plants, affecting their metabolism, intracellular transport, cytoarchitecture, cell division, growth, development, and interactions with the environment. Protein kinases and phosphatases, two important families of enzymes optimally regulating phosphorylation, have now become important targets for gene editing in crops. We review progress on gene-edited protein kinases and phosphatases in crops using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9). We also provide guidance for computational prediction of alterations and/or changes in function, activity, and binding of protein kinases and phosphatases as consequences of CRISPR/Cas9-based gene editing with its possible application in modern crop molecular breeding towards sustainable agriculture.

• Klíčová slova
• CRISPR/Cas9, crops, gene editing, in silico prediction, protein kinases, protein phosphatases,
• MeSH
• CRISPR-Cas systémy * MeSH
• editace genu * MeSH
• fosfatasy genetika   metabolismus   MeSH
• proteinkinasy * genetika   metabolismus   MeSH
• rostlinné proteiny genetika   metabolismus   MeSH
• šlechtění rostlin * metody   MeSH
• zemědělské plodiny * genetika   MeSH
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH
• Názvy látek
• fosfatasy MeSH
• proteinkinasy * MeSH
• rostlinné proteiny MeSH

Diamond-Blackfan anemia (DBA) is a rare genetic disorder affecting the bone marrow's ability to produce red blood cells, leading to severe anemia and various physical abnormalities. Approximately 75% of DBA cases involve heterozygous mutations in ribosomal protein (RP) genes, classifying it as a ribosomopathy, with RPS19 being the most frequently mutated gene. Non-RP mutations, such as in GATA1, have also been identified. Current treatments include glucocorticosteroids, blood transfusions, and hematopoietic stem cell transplantation (HSCT), with HSCT being the only curative option, albeit with challenges like donor availability and immunological complications. Gene therapy, particularly using lentiviral vectors and CRISPR/Cas9 technology, emerges as a promising alternative. This review explores the potential of gene therapy, focusing on lentiviral vectors and CRISPR/Cas9 technology in combination with non-integrating lentiviral vectors, as a curative solution for DBA. It highlights the transformative advancements in the treatment landscape of DBA, offering hope for individuals affected by this condition.

• Klíčová slova
• CRISPR/Cas9, Diamond–Blackfan anemia, gene therapy, hematopoietic stem cell transplantation, lentiviral vector, non-integrating lentiviral vector, rare genetic disorder, ribosomal protein genes, ribosomopathy,
• MeSH
• CRISPR-Cas systémy genetika   MeSH
• Diamondova-Blackfanova anemie * genetika   terapie   MeSH
• editace genu metody   MeSH
• genetická terapie * metody   MeSH
• genetické vektory MeSH
• Lentivirus genetika   MeSH
• lidé MeSH
• mutace genetika   MeSH
• ribozomální proteiny genetika   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH
• Názvy látek
• ribozomální proteiny MeSH

CRISPR/Cas technology is a powerful tool for genome engineering in Aspergillus oryzae as an industrially important filamentous fungus. Previous study has reported the application of the CRISPR/Cpf1 system based on the Cpf1 (LbCpf1) from Lachnospiraceae bacterium in A. oryzae. However, multiplex gene editing have not been investigated using this system. Here, we presented a new CRISPR/Cpf1 multiplex gene editing system in A. oryzae, which contains the Cpf1 nuclease (FnCpf1) from Francisella tularensis subsp. novicida U112 and CRISPR-RNA expression cassette. The crRNA cassette consisted of direct repeats and guide sequences driven by the A. oryzae U6 promoter and U6 terminator. Using the constructed FnCpf1 gene editing system, the wA and pyrG genes were mutated successfully. Furthermore, simultaneous editing of wA and pyrG genes in A. oryzae was performed using two guide sequences targeting these gene loci in a single crRNA array. This promising CRISPR/Cpf1 genome-editing system provides a powerful tool for genetically engineering A. oryzae.

• Klíčová slova
• Aspergillus oryzae, CRISPR/Cpf1 system, Filamentous fungi, Multiplex gene editing,
• MeSH
• Aspergillus oryzae * genetika   MeSH
• editace genu MeSH
• Francisella * MeSH
• vodící RNA, systémy CRISPR-Cas MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• vodící RNA, systémy CRISPR-Cas MeSH

The incidence and the burden of cardiovascular disease (CVD), coronary heart disease (CHD), type 2 diabetes mellitus (T2DM), and the metabolic syndrome are greatly increasing in our societies. Together, they account for 31% of all deaths worldwide. This chapter focuses on the role of two revolutionary discoveries that are changing the future of medicine, induced pluripotent stem cells (iPSCs) and CRISPR/Cas9 technology, in the study, and the cure of cardiovascular and metabolic diseases.We summarize the state-of-the-art knowledge about the possibility of editing iPSC genome for therapeutic applications without hampering their pluripotency and differentiation, using CRISPR/Cas technology, in the field of cardiovascular and metabolic diseases.

• Klíčová slova
• Cardiovascular, Epigenetics, Gene editing, Induced pluripotent stem cells (iPSC), Metabolism,
• MeSH
• diabetes mellitus 2. typu * genetika   terapie   MeSH
• editace genu MeSH
• indukované pluripotentní kmenové buňky * MeSH
• kardiovaskulární systém * MeSH
• lidé MeSH
• metabolické nemoci * genetika   terapie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH