Clustered regularly interspaced short palindromic repeats and associated Cas proteins (CRISPR-Cas) are the only known adaptive immune system in prokaryotes. CRISPR-Cas system provides sequence-specific immunity against invasion by foreign genetic elements. It carries out its functions by incorporating a small part of the invading DNA sequence, termed as spacer into the CRISPR array. Although the CRISPR-Cas systems are mainly responsible for adaptive immune functions, their alternative role in the gene regulation, bacterial pathophysiology, virulence, and evolution has started to unravel. In several species, these systems are revealed to regulate the processes beyond adaptive immunity by employing various components of CRISPR-Cas machinery, independently or in combination. The molecular mechanisms entailing the regulatory processes are not clear in most of the instances. In this review, we have discussed some well-known and some recently established noncanonical functions of CRISPR-Cas system and its fast-extending applications in other biological processes.
- Klíčová slova
- CRISPR-Cas, CRISPR-Cas alternative roles, CRISPR-Cas application, CRISPR-Cas in gene regulation, Genome remodeling,
- MeSH
- Archaea MeSH
- Bacteria genetika MeSH
- biologické jevy * MeSH
- CRISPR-Cas systémy * MeSH
- virulence MeSH
- Publikační typ
- časopisecké články MeSH
- přehledy MeSH
Within five years, the CRISPR-Cas system has emerged as the dominating tool for genome engineering, while also changing the speed and efficiency of metabolic engineering in conventional (Saccharomyces cerevisiae and Schizosaccharomyces pombe) and non-conventional (Yarrowia lipolytica, Pichia pastoris syn. Komagataella phaffii, Kluyveromyces lactis, Candida albicans and C. glabrata) yeasts. Especially in S. cerevisiae, an extensive toolbox of advanced CRISPR-related applications has been established, including crisprTFs and gene drives. The comparison of innovative CRISPR-Cas expression strategies in yeasts presented here may also serve as guideline to implement and refine CRISPR-Cas systems for highly efficient genome editing in other eukaryotic organisms.
- Klíčová slova
- CRISPR-Cas, Candida albicans/glabrata, Expression optimization, Metabolic engineering, Pichia pastoris, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Synthetic biology, Yarrowia lipolytica, Yeasts,
- MeSH
- bodová mutace MeSH
- chromozomy hub MeSH
- CRISPR-Cas systémy * MeSH
- editace genu metody MeSH
- geneticky modifikované mikroorganismy MeSH
- klonování DNA MeSH
- kvasinky genetika MeSH
- metabolické inženýrství MeSH
- Pichia genetika MeSH
- regulace genové exprese u hub MeSH
- Saccharomyces cerevisiae genetika MeSH
- technologie gene drive MeSH
- vodící RNA, systémy CRISPR-Cas MeSH
- Yarrowia genetika MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- přehledy MeSH
- Názvy látek
- vodící RNA, systémy CRISPR-Cas MeSH
The discovery of the CRISPR/Cas genome-editing system has revolutionized our understanding of the plant genome. CRISPR/Cas has been used for over a decade to modify plant genomes for the study of specific genes and biosynthetic pathways as well as to speed up breeding in many plant species, including both model and non-model crops. Although the CRISPR/Cas system is very efficient for genome editing, many bottlenecks and challenges slow down further improvement and applications. In this review we discuss the challenges that can occur during tissue culture, transformation, regeneration, and mutant detection. We also review the opportunities provided by new CRISPR platforms and specific applications related to gene regulation, abiotic and biotic stress response improvement, and de novo domestication of plants.
- Klíčová slova
- CA18111, CRISPR applications, CRISPR platforms, PlantEd, gene regulation, mutant detection, plant regeneration,
- MeSH
- CRISPR-Cas systémy * genetika MeSH
- editace genu * MeSH
- geneticky modifikované rostliny genetika MeSH
- genom rostlinný genetika MeSH
- šlechtění rostlin MeSH
- zemědělské plodiny genetika MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- přehledy 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
CRISPR-Cas pathways provide prokaryotes with acquired "immunity" against foreign genetic elements, including phages and plasmids. Although many of the proteins associated with CRISPR-Cas mechanisms are characterized, some requisite enzymes remain elusive. Genetic studies have implicated host DNA polymerases in some CRISPR-Cas systems but CRISPR-specific replicases have not yet been discovered. We have identified and characterised a family of CRISPR-Associated Primase-Polymerases (CAPPs) in a range of prokaryotes that are operonically associated with Cas1 and Cas2. CAPPs belong to the Primase-Polymerase (Prim-Pol) superfamily of replicases that operate in various DNA repair and replication pathways that maintain genome stability. Here, we characterise the DNA synthesis activities of bacterial CAPP homologues from Type IIIA and IIIB CRISPR-Cas systems and establish that they possess a range of replicase activities including DNA priming, polymerisation and strand-displacement. We demonstrate that CAPPs operonically-associated partners, Cas1 and Cas2, form a complex that possesses spacer integration activity. We show that CAPPs physically associate with the Cas proteins to form bespoke CRISPR-Cas complexes. Finally, we propose how CAPPs activities, in conjunction with their partners, may function to undertake key roles in CRISPR-Cas adaptation.
- MeSH
- Bacteria enzymologie genetika MeSH
- Bacteroidetes enzymologie genetika MeSH
- bakteriální proteiny genetika metabolismus MeSH
- Cas proteiny metabolismus MeSH
- CRISPR-Cas systémy * MeSH
- dimerizace MeSH
- DNA primery biosyntéza MeSH
- DNA-dependentní DNA-polymerasy genetika metabolismus MeSH
- DNA-primasa genetika metabolismus MeSH
- Escherichia coli metabolismus MeSH
- exprese genu MeSH
- fylogeneze MeSH
- mutace MeSH
- prokaryotické buňky metabolismus MeSH
- rekombinantní proteiny MeSH
- ribonukleotidy metabolismus MeSH
- výpočetní biologie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- bakteriální proteiny MeSH
- Cas proteiny MeSH
- DNA primery MeSH
- DNA-dependentní DNA-polymerasy MeSH
- DNA-primasa MeSH
- rekombinantní proteiny MeSH
- ribonukleotidy MeSH
CAS is a docking protein downstream of the proto-oncogene Src with a role in invasion and metastasis of cancer cells. The CAS SH3 domain is indispensable for CAS-mediated signaling, but structural aspects of CAS SH3 ligand binding and regulation are not well understood. Here, we identified the consensus CAS SH3 binding motif and structurally characterized the CAS SH3 domain in complex with ligand. We revealed the requirement for an uncommon centrally localized lysine residue at position +2 of CAS SH3 ligands and two rather dissimilar optional anchoring residues, leucine and arginine, at position +5. We further expanded the knowledge of CAS SH3 ligand binding regulation by manipulating tyrosine 12 phosphorylation and confirmed the negative role of this phosphorylation on CAS SH3 ligand binding. Finally, by exploiting the newly identified binding requirements of the CAS SH3 domain, we predicted and experimentally verified two novel CAS SH3 binding partners, DOK7 and GLIS2.
- MeSH
- aminokyseliny metabolismus MeSH
- fosforylace fyziologie MeSH
- lidé MeSH
- ligandy MeSH
- protoonkogen Mas MeSH
- sekvence aminokyselin MeSH
- signální transdukce fyziologie MeSH
- src homologní domény fyziologie MeSH
- substrátový protein asociovaný s Crk metabolismus MeSH
- vazba proteinů fyziologie MeSH
- vazebná místa fyziologie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- aminokyseliny MeSH
- ligandy MeSH
- MAS1 protein, human MeSH Prohlížeč
- protoonkogen Mas MeSH
- substrátový protein asociovaný s Crk MeSH
CAS is a docking protein, which was shown to act as a mechanosensor in focal adhesions. The unique assembly of structural domains in CAS is important for its function as a mechanosensor. The tension within focal adhesions is transmitted to a stretchable substrate domain of CAS by focal adhesion-targeting of SH3 and CCH domain of CAS, which anchor the CAS protein in focal adhesions. Mechanistic models of the stretching biosensor propose equal roles for both anchoring domains. Using deletion mutants and domain replacements, we have analyzed the relative importance of the focal adhesion anchoring domains on CAS localization and dynamics in focal adhesions as well as on CAS-mediated mechanotransduction. We confirmed the predicted prerequisite of the focal adhesion targeting for CAS-dependent mechanosensing and unraveled the critical importance of CAS SH3 domain in mechanosensing. We further show that CAS localizes to the force transduction layer of focal adhesions and that mechanical stress stabilizes CAS in focal adhesions.
- MeSH
- buněčná adheze MeSH
- buněčný převod mechanických signálů * MeSH
- fibroblasty cytologie metabolismus MeSH
- fokální adheze metabolismus MeSH
- mechanický stres MeSH
- mutantní proteiny chemie MeSH
- myši MeSH
- proteinové domény MeSH
- rekombinantní fúzní proteiny metabolismus MeSH
- signální transdukce MeSH
- stabilita proteinů MeSH
- substrátový protein asociovaný s Crk chemie metabolismus MeSH
- vztahy mezi strukturou a aktivitou MeSH
- zelené fluorescenční proteiny metabolismus MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- Bcar1 protein, mouse MeSH Prohlížeč
- mutantní proteiny MeSH
- rekombinantní fúzní proteiny MeSH
- substrátový protein asociovaný s Crk MeSH
- zelené fluorescenční proteiny MeSH
Focal adhesions are cellular structures through which both mechanical forces and regulatory signals are transmitted. Two focal adhesion-associated proteins, Crk-associated substrate (CAS) and vinculin, were both independently shown to be crucial for the ability of cells to transmit mechanical forces and to regulate cytoskeletal tension. Here, we identify a novel, direct binding interaction between CAS and vinculin. This interaction is mediated by the CAS SRC homology 3 domain and a proline-rich sequence in the hinge region of vinculin. We show that CAS localization in focal adhesions is partially dependent on vinculin, and that CAS-vinculin coupling is required for stretch-induced activation of CAS at the Y410 phosphorylation site. Moreover, CAS-vinculin binding significantly affects the dynamics of CAS and vinculin within focal adhesions as well as the size of focal adhesions. Finally, disruption of CAS binding to vinculin reduces cell stiffness and traction force generation. Taken together, these findings strongly implicate a crucial role of CAS-vinculin interaction in mechanosensing and focal adhesion dynamics.
- MeSH
- aminokyselinové motivy MeSH
- biomechanika MeSH
- buněčná adheze MeSH
- buněčné linie MeSH
- fibroblasty cytologie metabolismus MeSH
- fokální adheze metabolismus ultrastruktura MeSH
- fokální adhezní tyrosinkinasy metabolismus MeSH
- fosforylace MeSH
- mapy interakcí proteinů MeSH
- myši MeSH
- peptidy chemie metabolismus MeSH
- src homologní domény MeSH
- substrátový protein asociovaný s Crk analýza metabolismus MeSH
- vazba proteinů MeSH
- vinkulin analýza metabolismus MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- fokální adhezní tyrosinkinasy MeSH
- peptidy MeSH
- polyproline MeSH Prohlížeč
- substrátový protein asociovaný s Crk MeSH
- vinkulin MeSH
Ca and S K-edge spectra of CaS are calculated by the full-potential Green's function multiple-scattering method, by the FLAPW method and by the finite-difference method. All three techniques lead to similar spectra. Some differences remain close to the edge, both when comparing different calculations with each other and when comparing the calculations with earlier experimental data. Here it is found that using the full potential does not lead to significant improvement over the atomic spheres approximation and that the effect of the core hole can be limited to the photoabsorbing atom alone. Doping CaS with Eu will not affect the Ca and S K-edge XANES of CaS significantly but may give rise to a pre-edge structure not present for clean CaS.
- Klíčová slova
- CaS, XANES, core hole, full potential,
- Publikační typ
- časopisecké články MeSH
- MeSH
- duševní procesy fyziologie MeSH
- elementární částice MeSH
- lidé MeSH
- mozek fyziologie MeSH
- myšlení fyziologie MeSH
- vnímání času fyziologie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- anglický abstrakt MeSH
- časopisecké články MeSH
