Although several Cre-regulated CRISPR/Cas platforms exist, a CRISPR/Cas-controlled Cre-system remains a challenge. Here, we present a genetic switch we term SWITCHER based on a floxed wild-type Cre-construct representing a CRISPR-inducible and self-limiting kill switch. By leveraging CRISPR/Cas12a-mediated crRNA-array maturation, we showcase SWITCHER's dual role-not just as a recombinase but as a CRISPR switch, capable of orchestrating distinct Cas12a/crRNA-encoded programs.

• MeSH
• bakteriální proteiny MeSH
• Cas proteiny genetika   metabolismus   MeSH
• CRISPR-Cas systémy * genetika   MeSH
• editace genu * metody   MeSH
• endodeoxyribonukleasy genetika   metabolismus   MeSH
• HEK293 buňky MeSH
• integrasy * genetika   metabolismus   MeSH
• lidé MeSH
• sekvence CRISPR * MeSH
• vodící RNA, systémy CRISPR-Cas genetika   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• bakteriální proteiny MeSH
• Cas proteiny MeSH
• Cas12a protein MeSH Prohlížeč
• Cre recombinase MeSH Prohlížeč
• endodeoxyribonukleasy MeSH
• integrasy * MeSH
• vodící RNA, systémy CRISPR-Cas MeSH

The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system has become the most important tool for targeted genome editing in many plant and animal species over the past decade. The CRISPR/Cas9 technology has also sparked a flood of applications and technical advancements in genome editing in the key cereal crops, including rice, wheat, maize, and barley. Here, we review advanced uses of CRISPR/Cas9 and derived systems in genome editing of cereal crops to enhance a variety of agronomically important features. We also highlight new technological advances for delivering preassembled Cas9-gRNA ribonucleoprotein (RNP)-editing systems, multiplex editing, gain-of-function strategies, the use of artificial intelligence (AI)-based tools, and combining CRISPR with novel speed breeding (SB) and vernalization strategies.

• Klíčová slova
• CRISPR/Cas9, agronomic trait improvement, cereals, multiplex editing, strategies for gain-of-function,
• MeSH
• CRISPR-Cas systémy * genetika   MeSH
• genom rostlinný genetika   MeSH
• jedlá semena * genetika   MeSH
• šlechtění rostlin MeSH
• umělá inteligence MeSH
• vodící RNA, systémy CRISPR-Cas MeSH
• zemědělské plodiny genetika   MeSH
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH
• Názvy látek
• vodící RNA, systémy CRISPR-Cas MeSH

CRISPR/Cas9-mediated genome editing has become an extremely powerful technique used to modify gene expression in many organisms, including parasitic protists. Giardia intestinalis, a protist parasite that infects approximately 280 million people around the world each year, has been eluding the use of CRISPR/Cas9 to generate knockout cell lines due to its tetraploid genome. In this work, we show the ability of the in vitro assembled CRISPR/Cas9 components to successfully edit the genome of G. intestinalis. The cell line that stably expresses Cas9 in both nuclei of G. intestinalis showed effective recombination of the cassette containing the transcription units for the gRNA and the resistance marker. This highly efficient process led to the removal of all gene copies at once for three independent experimental genes, mem, cwp1 and mlf1. The method was also applicable to incomplete disruption of the essential gene, as evidenced by significantly reduced expression of tom40. Finally, testing the efficiency of Cas9-induced recombination revealed that homologous arms as short as 150 bp can be sufficient to establish a complete knockout cell line in G. intestinalis.

• Klíčová slova
• CRISPR/Cas9, Giardia, gene knockout, multiploid,
• MeSH
• CRISPR-Cas systémy * MeSH
• editace genu metody   MeSH
• Giardia lamblia * genetika   MeSH
• lidé MeSH
• tetraploidie MeSH
• vodící RNA, systémy CRISPR-Cas MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• vodící RNA, systémy CRISPR-Cas MeSH

The CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats, CRISPR-associated protein 9) system has become a commonly used tool for genome editing and metabolic engineering. For Komagataella phaffii, commercialized as Pichia pastoris, the CRISPR/Cas9 protocol for genome editing was established in 2016 and since then has been employed to facilitate genetic modifications such as markerless gene disruptions and deletions as well as to enhance the efficiency of homologous recombination.In this chapter, we describe a robust basic protocol for CRISPR-based genome editing, demonstrating near 100% targeting efficiency for gene inactivation via a frameshift mutation. As described in other chapters of this volume, CRISPR/Cas9 technologies for use in P. pastoris have been further optimized for various specific purposes.

• Klíčová slova
• CRISPR/Cas9, Gene knockout, Genome editing, Genome engineering, Guide RNA, Homologous recombination, Komagataella phaffii, Pichia pastoris, Synthetic biology,
• MeSH
• CRISPR-Cas systémy * genetika   MeSH
• editace genu * metody   MeSH
• genom fungální MeSH
• metabolické inženýrství * metody   MeSH
• Pichia * genetika   MeSH
• Saccharomycetales * genetika   MeSH
• vodící RNA, systémy CRISPR-Cas genetika   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• vodící RNA, systémy CRISPR-Cas 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

In the recent years, there was a remarkable advance in research and clinical implementation of the genome editing technologies. The most remarkable was a discovery of the bacterial adaptive immune system called CRISPR and its rapid transformation into a robust and broadly applicable technology that completely revolutionized both basic and applied biomedical research. Implementation of CRISPR makes genome modification easier, faster and significantly cheaper compare to any other currently available technology. It also offers a tremendous potential for desiging novel research approaches and future treatment options for various genetic diseases including multiple myeloma. The hightroughput use of CRISPR in pooled screen formats promises faster identification and validation of valuable drug targets together with revealing high-confidence biomarkers and unknown resistance mechanisms. This can provide clinicians with new diagnostic and prognostic tolls and ultimately allow more accurate patient stratification for personalised treatment with better eficacy. In this review, we summarize current knowledge about the CRISPR technology and focus especially on its impact in exploring gene functions, screening for novel drug targets, diagnostic markers and genes involved in resistance to commonly used drug in the treatment of multiple myeloma. Finally, we also highlight a potential future use of CRISPR in actual clinical practise.Key words: multiple myeloma - CRISPR - therapeutics.

• MeSH
• chemorezistence genetika   MeSH
• cílená molekulární terapie MeSH
• CRISPR-Cas systémy MeSH
• genetická terapie MeSH
• lidé MeSH
• mnohočetný myelom diagnóza   genetika   terapie   MeSH
• nádorové biomarkery genetika   MeSH
• sekvence CRISPR * MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH
• Názvy látek
• nádorové biomarkery MeSH

The CRISPR/Cas9-based targeted genome editing has emerged as a versatile technique, widely employed in plant genome engineering, both to decipher gene function and as an alternative to classical breeding technique for traits improvement in plants. However, to date, no such platform has been developed for hop (Humulus lupulus L.), which is an economically important crop producing valuable secondary metabolites utilized in the brewing and pharmaceutical industries. Here, we present the first report on the successful establishment of efficient CRISPR/Cas9-based genome editing using the visible endogenous marker gene phytoene desaturase (PDS) involved in carotenoid biosynthesis to demonstrate successful genome editing in hop. Agrobacterium tumefaciens-mediated transformation of in vitro generated internodal explants was used for the stable integration of constructs expressing plant codon-optimized Cas9 and a pair of co-expressed guide RNAs to target the distinct genomic sites of the PDS gene of hop. Analysis of RNA-guided genome-editing events, including mutant lines screening and homozygosity assessment using the T7 endonuclease assay showed that 33.3% of transformed plants were successfully edited at the target site, displaying albino and mosaic regenerants. Intriguingly, the detected mutations were ranges of deletions (16 bp to 39 bp) which led to disruption of the exon-intron boundary, few base substitutions, and a 1 bp insertion at 3 bp upstream of the PAM region of the target site. The decrease in chlorophyll a/b, and carotenoid content in the mutant lines further confirmed the functional disruption of the HlPDS gene. Taken together, our results demonstrate that the CRISPR/Cas9 system can precisely edit the targeted genome sequences, which may revolutionize our way to overcome some of the obstacles that have plagued the traits improvement in hop.

• Klíčová slova
• CRISPR/Cas9, Genome editing, Hop, Phytoene desaturase, Transformation and T7E1 assay,
• MeSH
• Agrobacterium tumefaciens MeSH
• chlorofyl a MeSH
• chlorofyl MeSH
• CRISPR-Cas systémy * MeSH
• editace genu MeSH
• geneticky modifikované rostliny genetika   MeSH
• genom rostlinný genetika   MeSH
• Humulus enzymologie   genetika   MeSH
• mutageneze MeSH
• oxidoreduktasy genetika   MeSH
• vodící RNA, systémy CRISPR-Cas genetika   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• chlorofyl a MeSH
• chlorofyl MeSH
• chlorophyll b MeSH Prohlížeč
• oxidoreduktasy MeSH
• phytoene dehydrogenase MeSH Prohlížeč
• vodící RNA, systémy CRISPR-Cas 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 a FANCD1 gene 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). FANCD1 function 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 primary FANCD1 cells in a precise manner. These proof of principle studies support expanded application of gene editing for FA.

• Klíčová slova
• CRISPR/Cas9, Fanconi anemia, Fanconi anemia D1, fibroblasts, gene editing, poly adenosine diphosphate-ribose polymerase inhibitors,
• 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
• Názvy látek
• BRCA2 protein, human MeSH Prohlížeč
• protein BRCA2 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.

• Klíčová slova
• CRISPR/Cas9, agricultural production, genome editing, industrial applications, livestock, therapeutics,
• 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

BACKGROUND: Many prokaryotic genomes comprise Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRs) offering defense against foreign nucleic acids. These immune systems are conditioned by the production of small CRISPR-derived RNAs matured from long RNA precursors. This often requires a Csy4 endoribonuclease cleaving the RNA 3'-end. METHODS: We report extended explicit solvent molecular dynamic (MD) simulations of Csy4/RNA complex in precursor and product states, based on X-ray structures of product and inactivated precursor (55 simulations; ~3.7μs in total). RESULTS: The simulations identify double-protonated His29 and deprotonated terminal phosphate as the likely dominant protonation states consistent with the product structure. We revealed potential substates consistent with Ser148 and His29 acting as the general base and acid, respectively. The Ser148 could be straightforwardly deprotonated through solvent and could without further structural rearrangements deprotonate the nucleophile, contrasting similar studies investigating the general base role of nucleobases in ribozymes. We could not locate geometries consistent with His29 acting as general base. However, we caution that the X-ray structures do not always capture the catalytically active geometries and then the reactive structures may be unreachable by the simulation technique. CONCLUSIONS: We identified potential catalytic arrangement of the Csy4/RNA complex but we also report limitations of the simulation technique. Even for the dominant protonation state we could not achieve full agreement between the simulations and the structural data. GENERAL SIGNIFICANCE: Potential catalytic arrangement of the Csy4/RNA complex is found. Further, we provide unique insights into limitations of simulations of protein/RNA complexes, namely, the influence of the starting experimental structures and force field limitations. This article is part of a Special Issue entitled Recent developments of molecular dynamics.

• Klíčová slova
• Cas6 superfamily, Endoribonuclease, Force field, Molecular dynamic simulation, Protein/RNA complex, RNA cleavage,
• MeSH
• Cas proteiny chemie   metabolismus   MeSH
• časové faktory MeSH
• CRISPR-Cas systémy * MeSH
• endoribonukleasy chemie   metabolismus   MeSH
• katalytická doména MeSH
• krystalografie rentgenová MeSH
• sekvence CRISPR * MeSH
• simulace molekulární dynamiky * MeSH
• vazba proteinů MeSH
• vazebná místa MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• Cas proteiny MeSH
• endoribonukleasy MeSH