Genome editing offers revolutionized solutions for plant breeding to sustain food production to feed the world by 2050. Therefore, genome-edited products are increasingly recognized via more relaxed legislation and community adoption. The world population and food production are disproportionally growing in a manner that would have never matched each other under the current agricultural practices. The emerging crisis is more evident with the subtle changes in climate and the running-off of natural genetic resources that could be easily used in breeding in conventional ways. Under these circumstances, affordable CRISPR-Cas-based gene-editing technologies have brought hope and charged the old plant breeding machine with the most energetic and powerful fuel to address the challenges involved in feeding the world. What makes CRISPR-Cas the most powerful gene-editing technology? What are the differences between it and the other genetic engineering/breeding techniques? Would its products be labeled as "conventional" or "GMO"? There are so many questions to be answered, or that cannot be answered within the limitations of our current understanding. Therefore, we would like to discuss and answer some of the mentioned questions regarding recent progress in technology development. We hope this review will offer another view on the role of CRISPR-Cas technology in future of plant breeding for food production and beyond.

• Klíčová slova
• Biotechnology regulation, CRISPR-Cas, Crop breeding, Genome engineering, New plant breeding techniques,
• MeSH
• CRISPR-Cas systémy genetika   MeSH
• editace genu * metody   MeSH
• geneticky modifikované rostliny genetika   MeSH
• genom rostlinný genetika   MeSH
• šlechtění rostlin * metody   MeSH
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH

Agrobacterium-mediated transformation is one of the most commonly used genetic transformation method that involves transfer of foreign genes into target plants. Agroinfiltration, an Agrobacterium-based transient approach and the breakthrough discovery of CRISPR/Cas9 holds trending stature to perform targeted and efficient genome editing (GE). The predominant feature of agroinfiltration is the abolishment of Transfer-DNA (T-DNA) integration event to ensure fewer biosafety and regulatory issues besides showcasing the capability to perform transcription and translation efficiently, hence providing a large picture through pilot-scale experiment via transient approach. The direct delivery of recombinant agrobacteria through this approach carrying CRISPR/Cas cassette to knockout the expression of the target gene in the intercellular tissue spaces by physical or vacuum infiltration can simplify the targeted site modification. This review aims to provide information on Agrobacterium-mediated transformation and implementation of agroinfiltration with GE to widen the horizon of targeted genome editing before a stable genome editing approach. This will ease the screening of numerous functions of genes in different plant species with wider applicability in future.

• Klíčová slova
• Agrobacterium, CRISPR/Cas9, genome editing, targeted site modification, transfer-DNA, transgene-free,
• MeSH
• Agrobacterium genetika   MeSH
• CRISPR-Cas systémy * MeSH
• editace genu metody   MeSH
• genom rostlinný * MeSH
• mutageneze MeSH
• regulace genové exprese u rostlin MeSH
• rostlinné proteiny genetika   MeSH
• rostliny genetika   metabolismus   MeSH
• transformace genetická MeSH
• zemědělské plodiny genetika   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH
• Názvy látek
• rostlinné proteiny MeSH

Tick saliva is a rich source of antihemostatic, anti-inflammatory, and immunomodulatory molecules that actively help the tick to finish its blood meal. Moreover, these molecules facilitate the transmission of tick-borne pathogens. Here we present the functional and structural characterization of Iripin-8, a salivary serpin from the tick Ixodes ricinus, a European vector of tick-borne encephalitis and Lyme disease. Iripin-8 displayed blood-meal-induced mRNA expression that peaked in nymphs and the salivary glands of adult females. Iripin-8 inhibited multiple proteases involved in blood coagulation and blocked the intrinsic and common pathways of the coagulation cascade in vitro. Moreover, Iripin-8 inhibited erythrocyte lysis by complement, and Iripin-8 knockdown by RNA interference in tick nymphs delayed the feeding time. Finally, we resolved the crystal structure of Iripin-8 at 1.89 Å resolution to reveal an unusually long and rigid reactive center loop that is conserved in several tick species. The P1 Arg residue is held in place distant from the serpin body by a conserved poly-Pro element on the P' side. Several PEG molecules bind to Iripin-8, including one in a deep cavity, perhaps indicating the presence of a small-molecule binding site. This is the first crystal structure of a tick serpin in the native state, and Iripin-8 is a tick serpin with a conserved reactive center loop that possesses antihemostatic activity that may mediate interference with host innate immunity.

• Klíčová slova
• Ixodes ricinus, blood coagulation, crystal structure, parasite, saliva, serpin, tick,
• MeSH
• aktivace komplementu účinky léků   imunologie   fyziologie   MeSH
• erytrocyty metabolismus   MeSH
• exprese genu genetika   MeSH
• hemokoagulace účinky léků   fyziologie   MeSH
• klíště enzymologie   genetika   metabolismus   MeSH
• komplement metabolismus   MeSH
• lymeská nemoc MeSH
• nymfa MeSH
• proteiny členovců metabolismus   MeSH
• regulace genové exprese genetika   MeSH
• serpiny metabolismus   ultrastruktura   MeSH
• slinné žlázy metabolismus   MeSH
• sliny chemie   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• komplement MeSH
• proteiny členovců MeSH
• serpiny MeSH

Rapid developments in the field of plant genome editing using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) systems necessitate more detailed consideration of the delivery of the CRISPR system into plants. Successful and safe editing of plant genomes is partly based on efficient delivery of the CRISPR system. Along with the use of plasmids and viral vectors as cargo material for genome editing, non-viral vectors have also been considered for delivery purposes. These non-viral vectors can be made of a variety of materials, including inorganic nanoparticles, carbon nanotubes, liposomes, and protein- and peptide-based nanoparticles, as well as nanoscale polymeric materials. They have a decreased immune response, an advantage over viral vectors, and offer additional flexibility in their design, allowing them to be functionalized and targeted to specific sites in a biological system with low cytotoxicity. This review is dedicated to describing the delivery methods of CRISPR system into plants with emphasis on the use of non-viral vectors.

• Klíčová slova
• CRISPR, exosomes and liposomes, genome editing, nanoparticles,
• MeSH
• CRISPR-Cas systémy * MeSH
• editace genu * MeSH
• exozómy chemie   MeSH
• genetická terapie * MeSH
• genom rostlinný MeSH
• liposomy chemie   MeSH
• nanočástice chemie   MeSH
• rostliny genetika   MeSH
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH
• Názvy látek
• liposomy MeSH

Genome-editing (GE) is having a tremendous influence around the globe in the life science community. Among its versatile uses, the desired modifications of genes, and more importantly the transgene (DNA)-free approach to develop genetically modified organism (GMO), are of special interest. The recent and rapid developments in genome-editing technology have given rise to hopes to achieve global food security in a sustainable manner. We here discuss recent developments in CRISPR-based genome-editing tools for crop improvement concerning adaptation, opportunities, and challenges. Some of the notable advances highlighted here include the development of transgene (DNA)-free genome plants, the availability of compatible nucleases, and the development of safe and effective CRISPR delivery vehicles for plant genome editing, multi-gene targeting and complex genome editing, base editing and prime editing to achieve more complex genetic engineering. Additionally, new avenues that facilitate fine-tuning plant gene regulation have also been addressed. In spite of the tremendous potential of CRISPR and other gene editing tools, major challenges remain. Some of the challenges are related to the practical advances required for the efficient delivery of CRISPR reagents and for precision genome editing, while others come from government policies and public acceptance. This review will therefore be helpful to gain insights into technological advances, its applications, and future challenges for crop improvement.

• Klíčová slova
• CRISPR, DNA-free genome editing, base editing, crop improvement, genome editing, prime editing,
• MeSH
• CRISPR-Cas systémy * MeSH
• editace genu metody   MeSH
• genom rostlinný MeSH
• šlechtění rostlin metody   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH

Komagataella phaffii (syn. Pichia pastoris) is one of the most commonly used host systems for recombinant protein expression. Achieving targeted genetic modifications had been hindered by low frequencies of homologous recombination (HR). Recently, a CRISPR/Cas9 genome editing system has been implemented for P. pastoris enabling gene knockouts based on indels (insertion, deletions) via non-homologous end joining (NHEJ) at near 100% efficiency. However, specifically integrating homologous donor cassettes via HR for replacement studies had proven difficult resulting at most in ∼20% correct integration using CRISPR/Cas9. Here, we demonstrate the CRISPR/Cas9 mediated integration of markerless donor cassettes at efficiencies approaching 100% using a ku70 deletion strain. The Ku70p is involved in NHEJ repair and lack of the protein appears to favor repair via HR near exclusively. While the absolute number of transformants in the Δku70 strain is reduced, virtually all surviving transformants showed correct integration. In the wildtype strain, markerless donor cassette integration was also improved up to 25-fold by placing an autonomously replicating sequence (ARS) on the donor cassette. Alternative strategies for improving donor cassette integration using a Cas9 nickase variant or reducing off targeting associated toxicity using a high fidelity Cas9 variant were so far not successful in our hands in P. pastoris. Furthermore we provide Cas9/gRNA expression plasmids with a Geneticin resistance marker which proved to be versatile tools for marker recycling. The reported CRSIPR-Cas9 tools can be applied for modifying existing production strains and also pave the way for markerless whole genome modification studies in P. pastoris.

• Klíčová slova
• CRISPR/Cas9, KU70, Pichia pastoris, homologous recombination, marker recycling, resistance markers,
• MeSH
• CRISPR-Cas systémy MeSH
• genetické inženýrství MeSH
• genetické markery MeSH
• genový knockout metody   MeSH
• mutace INDEL MeSH
• oprava DNA spojením konců MeSH
• Pichia genetika   růst a vývoj   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• genetické markery MeSH

This work discusses several selected topics of plant genetics and breeding in relation to the 150th anniversary of the seminal work of Gregor Johann Mendel. In 2015, we celebrated the 150th anniversary of the presentation of the seminal work of Gregor Johann Mendel. While Darwin's theory of evolution was based on differential survival and differential reproductive success, Mendel's theory of heredity relies on equality and stability throughout all stages of the life cycle. Darwin's concepts were continuous variation and "soft" heredity; Mendel espoused discontinuous variation and "hard" heredity. Thus, the combination of Mendelian genetics with Darwin's theory of natural selection was the process that resulted in the modern synthesis of evolutionary biology. Although biology, genetics, and genomics have been revolutionized in recent years, modern genetics will forever rely on simple principles founded on pea breeding using seven single gene characters. Purposeful use of mutants to study gene function is one of the essential tools of modern genetics. Today, over 100 plant species genomes have been sequenced. Mapping populations and their use in segregation of molecular markers and marker-trait association to map and isolate genes, were developed on the basis of Mendel's work. Genome-wide or genomic selection is a recent approach for the development of improved breeding lines. The analysis of complex traits has been enhanced by high-throughput phenotyping and developments in statistical and modeling methods for the analysis of phenotypic data. Introgression of novel alleles from landraces and wild relatives widens genetic diversity and improves traits; transgenic methodologies allow for the introduction of novel genes from diverse sources, and gene editing approaches offer possibilities to manipulate gene in a precise manner.

• MeSH
• dějiny 19. století MeSH
• dějiny 20. století MeSH
• dějiny 21. století MeSH
• fenotyp MeSH
• genetická variace MeSH
• geneticky modifikované rostliny genetika   MeSH
• genetika dějiny   MeSH
• genom rostlinný MeSH
• genomika MeSH
• hrách setý genetika   MeSH
• lokus kvantitativního znaku MeSH
• mapování chromozomů MeSH
• selekce (genetika) MeSH
• šlechtění rostlin * MeSH
• Check Tag
• dějiny 19. století MeSH
• dějiny 20. století MeSH
• dějiny 21. století MeSH
• Publikační typ
• biografie MeSH
• časopisecké články MeSH
• historické články MeSH
• přehledy MeSH

• O autorovi
• Mendel, Gregor

Decades of intensive experimental studies of the recognition of DNA sequences by proteins have provided us with a view of a diverse and complicated world in which few to no features are shared between individual DNA-binding protein families. The originally conceived direct readout of DNA residue sequences by amino acid side chains offers very limited capacity for sequence recognition, while the effects of the dynamic properties of the interacting partners remain difficult to quantify and almost impossible to generalise. In this work we investigated the energetic characteristics of all DNA residue-amino acid side chain combinations in the conformations found at the interaction interface in a very large set of protein-DNA complexes by the means of empirical potential-based calculations. General specificity-defining criteria were derived and utilised to look beyond the binding motifs considered in previous studies. Linking energetic favourability to the observed geometrical preferences, our approach reveals several additional amino acid motifs which can distinguish between individual DNA bases. Our results remained valid in environments with various dielectric properties.

• MeSH
• adenin chemie   metabolismus   MeSH
• aminokyselinové motivy * MeSH
• aminokyseliny chemie   metabolismus   MeSH
• cytosin chemie   metabolismus   MeSH
• databáze proteinů MeSH
• DNA vazebné proteiny chemie   genetika   metabolismus   MeSH
• DNA chemie   genetika   metabolismus   MeSH
• guanin chemie   metabolismus   MeSH
• konformace nukleové kyseliny MeSH
• krystalografie rentgenová MeSH
• molekulární modely MeSH
• statistika jako téma metody   MeSH
• terciární struktura proteinů MeSH
• termodynamika MeSH
• thymin chemie   metabolismus   MeSH
• vazba proteinů MeSH
• vazebná místa genetika   MeSH
• výpočetní biologie metody   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• adenin MeSH
• aminokyseliny MeSH
• cytosin MeSH
• DNA vazebné proteiny MeSH
• DNA MeSH
• guanin MeSH
• thymin MeSH