scholarly journals Detection of Spacer Precursors Formed In Vivo During Primed CRISPR Adaptation

2019 ◽  
Author(s):  
Anna A. Shiriaeva ◽  
Ekaterina Savitskaya ◽  
Kirill A. Datsenko ◽  
Irina O. Vvedenskaya ◽  
Iana Fedorova ◽  
...  
Keyword(s):  
High Throughput Sequencing ◽  
Type I ◽  
Dependent Manner ◽  
Dna Fragments ◽  
Crispr Array ◽  
Protospacer Adjacent Motif ◽  
Sequencing Method ◽  
Foreign Dna ◽  
And Function

Type I CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-Cas (CRISPR associated) loci provide prokaryotes with a nucleic-acid-based adaptive immunity against foreign DNA1. Immunity involves “adaptation,” the integration of ~30-bp DNA fragments into the CRISPR array as “spacer” sequences, and “interference,” the targeted degradation of DNA containing a “protospacer” sequence mediated by a complex containing a spacer-derived CRISPR RNA (crRNA)1–4. Specificity for targeting interference to protospacers, but not spacers, occurs through recognition of a 3-bp protospacer adjacent motif (PAM)5 by the crRNA-containing complex6. Interference-driven DNA degradation of protospacer-containing DNA can be coupled with “primed adaptation,” ill which spacers are acquired from DNA surrounding the targeted protospacer in a bidirectional, orientation-dependent manner2,3,7. Here we construct a robust in vivo model for primed adaptation consisting of an Escherichia coli type I-E CRISPR-Cas “self-targeting” locus encoding a crRNA that targets a chromosomal protospacer. We develop a strand-specific, high-throughput-sequencing method for analysis of DNA fragments, “FragSeq,” and use this method to detect short fragments derived from DNA surrounding the targeted protospacer. The detected fragments have sequences matching spacers acquired during primed adaptation, contain ~3- to 4-nt overhangs derived from excision of genomic DNA within a PAM, are generated in a bidirectional, orientation-dependent manner relative to the targeted protospacer, require the functional integrity of machinery for interference and adaptation to accumulate, and function as spacer precursors when exogenously introduced into cells by transformation. DNA fragments with a similar structure accumulate in cells undergoing primed adaptation in a type I-F CRISPR-Cas self-targeting system. We propose the DNA fragments detected in this work are products of universal steps of spacer precursor processing in type I CRISPR-Cas systems.

scholarly journals Detection of spacer precursors formed in vivo during primed CRISPR adaptation

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Anna A. Shiriaeva ◽  
Ekaterina Savitskaya ◽  
Kirill A. Datsenko ◽  
Irina O. Vvedenskaya ◽  
Iana Fedorova ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Nucleic Acid ◽  
High Throughput Sequencing ◽  
Dna Degradation ◽  
Type I ◽  
Dna Fragments ◽  
Crispr Array ◽  
Foreign Dna ◽  
And Function

Abstract Type I CRISPR-Cas loci provide prokaryotes with a nucleic-acid-based adaptive immunity against foreign DNA. Immunity involves adaptation, the integration of ~30-bp DNA fragments, termed prespacers, into the CRISPR array as spacers, and interference, the targeted degradation of DNA containing a protospacer. Interference-driven DNA degradation can be coupled with primed adaptation, in which spacers are acquired from DNA surrounding the targeted protospacer. Here we develop a method for strand-specific, high-throughput sequencing of DNA fragments, FragSeq, and apply this method to identify DNA fragments accumulated in Escherichia coli cells undergoing robust primed adaptation by a type I-E or type I-F CRISPR-Cas system. The detected fragments have sequences matching spacers acquired during primed adaptation and function as spacer precursors when introduced exogenously into cells by transformation. The identified prespacers contain a characteristic asymmetrical structure that we propose is a key determinant of integration into the CRISPR array in an orientation that confers immunity.

scholarly journals Avoidance of Trinucleotide Corresponding to Consensus Protospacer Adjacent Motif Controls the Efficiency of Prespacer Selection during Primed Adaptation

mBio ◽  
2018 ◽  
Vol 9 (6) ◽  
Author(s):  
Olga Musharova ◽  
Danylo Vyhovskyi ◽  
Sofia Medvedeva ◽  
Jelena Guzina ◽  
Yulia Zhitnyuk ◽  
...  
Keyword(s):  
Selection Process ◽  
Natural Populations ◽  
Type I ◽  
Dna Fragments ◽  
Dna Arrays ◽  
Content Type ◽  
Factors Affecting ◽  
Crispr Array ◽  
Protospacer Adjacent Motif ◽  
Foreign Dna

ABSTRACT CRISPR DNA arrays of unique spacers separated by identical repeats ensure prokaryotic immunity through specific targeting of foreign nucleic acids complementary to spacers. New spacers are acquired into a CRISPR array in a process of CRISPR adaptation. Selection of foreign DNA fragments to be integrated into CRISPR arrays relies on PAM (protospacer adjacent motif) recognition, as only those spacers will be functional against invaders. However, acquisition of different PAM-associated spacers proceeds with markedly different efficiency from the same DNA. Here, we used a combination of bioinformatics and experimental approaches to understand factors affecting the efficiency of acquisition of spacers by the Escherichia coli type I-E CRISPR-Cas system, for which two modes of CRISPR adaptation have been described: naive and primed. We found that during primed adaptation, efficiency of spacer acquisition is strongly negatively affected by the presence of an AAG trinucleotide—a consensus PAM—within the sequence being selected. No such trend is observed during naive adaptation. The results are consistent with a unidirectional spacer selection process during primed adaptation and provide a specific signature for identification of spacers acquired through primed adaptation in natural populations. IMPORTANCE Adaptive immunity of prokaryotes depends on acquisition of foreign DNA fragments into CRISPR arrays as spacers followed by destruction of foreign DNA by CRISPR interference machinery. Different fragments are acquired into CRISPR arrays with widely different efficiencies, but the factors responsible are not known. We analyzed the frequency of spacers acquired during primed adaptation in an E. coli CRISPR array and found that AAG motif was depleted from highly acquired spacers. AAG is also a consensus protospacer adjacent motif (PAM) that must be present upstream from the target of the CRISPR spacer for its efficient destruction by the interference machinery. These results are important because they provide new information on the mechanism of primed spacer acquisition. They add to other previous evidence in the field that pointed out to a “directionality” in the capture of new spacers. Our data strongly suggest that the recognition of an AAG PAM by the interference machinery components prior to spacer capture occludes downstream AAG sequences, thus preventing their recognition by the adaptation machinery.

scholarly journals Extracellular Vesicles promote host immunity during an M. tuberculosis infection through RNA Sensing

2018 ◽  
Author(s):  
Yong Cheng ◽  
Jeffery S. Schorey
Keyword(s):  
Extracellular Vesicles ◽  
Type I ◽  
Dependent Manner ◽  
Drug Resistant ◽  
Interferon Production ◽  
Bacterial Killing ◽  
Novel Approach ◽  
And Function ◽  
Dependent Pathway

AbstractExtracellular vesicles (EVs) have been shown to carry microbial components and function in the host defense against infections. In this study, we demonstrate that Mycobacterium tuberculosis (M.tb) RNA is delivered into macrophage-derived EVs through an M.tb SecA2-dependent pathway, and that EVs released from M.tb-infected macrophages stimulate a host RIG-I/MAVS/TBK1/IRF3 RNA sensing pathway, leading to type I interferon production in recipient cells. These EVs also promote, in a RIG-I/MAVS-dependent manner, the maturation of M.tb-containing phagosomes through a noncanonical LC3 modification, leading to increased bacterial killing. Moreover, treatment of M.tb-infected macrophages or mice with a combination of moxifloxacin and EVs, isolated from M.tb-infected macrophages, significantly lowered bacterial burden relative to either treatment alone. We propose that EVs, which are preferentially removed by macrophages in vivo, may be developed in combination with effective antibiotics as a novel approach to treat drug-resistant TB.

Hierarchical binding of DNA fragments derived from scaffold-attached regions: correlation of properties in vitro and function in vivo

Biochemistry ◽  
1990 ◽  
Vol 29 (32) ◽  
pp. 7475-7485 ◽  
Author(s):  
Christian Mielke ◽  
Yoshinori Kohwi ◽  
Terumi Kohwi-Shigematsu ◽  
Juergen Bode
Keyword(s):  
Dna Fragments ◽  
And Function

Isoform-specific expression and function of neuregulin

Development ◽  
1997 ◽  
Vol 124 (18) ◽  
pp. 3575-3586 ◽  
Author(s):  
D. Meyer ◽  
T. Yamaai ◽  
A. Garratt ◽  
E. Riethmacher-Sonnenberg ◽  
D. Kane ◽  
...  
Keyword(s):  
Biological Effects ◽  
Type I ◽  
Mouse Development ◽  
Specific Expression ◽  
Independent Functions ◽  
Cell Growth And Differentiation ◽  
Cranial Ganglia ◽  
And Function

Neuregulin (also known as NDF, heregulin, ARIA, GGF or SMDF), induces cell growth and differentiation. Biological effects of neuregulin are mediated by members of the erbB family of tyrosine kinase receptors. Three major neuregulin isoforms are produced from the gene, which differ substantially in sequence and in overall structure. Here we use in situ hybridization with isoform-specific probes to illustrate the spatially distinct patterns of expression of the isoforms during mouse development. Ablation of the neuregulin gene in the mouse has demonstrated multiple and independent functions of this factor in development of both the nervous system and the heart. We show here that targeted mutations that affect different isoforms result in distinct phenotypes, demonstrating that isoforms can take over specific functions in vivo. Type I neuregulin is required for generation of neural crest-derived neurons in cranial ganglia and for trabeculation of the heart ventricle, whereas type III neuregulin plays an important role in the early development of Schwann cells. The complexity of neuregulin functions in development is therefore due to independent roles played by distinct isoforms.

Orchestrated biomechanical, structural, and biochemical stimuli for engineering anisotropic meniscus

2019 ◽  
Vol 11 (487) ◽  
pp. eaao0750 ◽  
Author(s):  
Zheng-Zheng Zhang ◽  
You-Rong Chen ◽  
Shao-Jie Wang ◽  
Feng Zhao ◽  
Xiao-Gang Wang ◽  
...  
Keyword(s):  
Rabbit Model ◽  
Proper Function ◽  
Type I ◽  
Specific Expression ◽  
Tissue Organization ◽  
Meniscus Tissue ◽  
Biomimetic Scaffold ◽  
And Function

Reconstruction of the anisotropic structure and proper function of the knee meniscus remains an important challenge to overcome, because the complexity of the zonal tissue organization in the meniscus has important roles in load bearing and shock absorption. Current tissue engineering solutions for meniscus reconstruction have failed to achieve and maintain the proper function in vivo because they have generated homogeneous tissues, leading to long-term joint degeneration. To address this challenge, we applied biomechanical and biochemical stimuli to mesenchymal stem cells seeded into a biomimetic scaffold to induce spatial regulation of fibrochondrocyte differentiation, resulting in physiological anisotropy in the engineered meniscus. Using a customized dynamic tension-compression loading system in conjunction with two growth factors, we induced zonal, layer-specific expression of type I and type II collagens with similar structure and function to those present in the native meniscus tissue. Engineered meniscus demonstrated long-term chondroprotection of the knee joint in a rabbit model. This study simultaneously applied biomechanical, biochemical, and structural cues to achieve anisotropic reconstruction of the meniscus, demonstrating the utility of anisotropic engineered meniscus for long-term knee chondroprotection in vivo.

scholarly journals Processing and integration of functionally oriented prespacers in the Escherichia coli CRISPR system depends on bacterial host exonucleases

2019 ◽  
Vol 295 (11) ◽  
pp. 3403-3414 ◽  
Author(s):  
Anita Ramachandran ◽  
Lesley Summerville ◽  
Brian A. Learn ◽  
Lily DeBell ◽  
Scott Bailey
Keyword(s):  
Escherichia Coli ◽  
Type I ◽  
Bacterial Host ◽  
E Coli ◽  
Correct Orientation ◽  
Protospacer Adjacent Motif ◽  
Functional Immunity ◽  
Functional Orientation ◽  
Biochemical Approach ◽  
Foreign Dna

CRISPR-Cas systems provide bacteria with adaptive immunity against viruses. During spacer adaptation, the Cas1-Cas2 complex selects fragments of foreign DNA, called prespacers, and integrates them into CRISPR arrays in an orientation that provides functional immunity. Cas4 is involved in both the trimming of prespacers and the cleavage of protospacer adjacent motif (PAM) in several type I CRISPR-Cas systems, but how the prespacers are processed in systems lacking Cas4, such as the type I-E and I-F systems, is not understood. In Escherichia coli, which has a type I-E system, Cas1-Cas2 preferentially selects prespacers with 3′ overhangs via specific recognition of a PAM, but how these prespacers are integrated in a functional orientation in the absence of Cas4 is not known. Using a biochemical approach with purified proteins, as well as integration, prespacer protection, sequencing, and quantitative PCR assays, we show here that the bacterial 3′–5′ exonucleases DnaQ and ExoT can trim long 3′ overhangs of prespacers and promote integration in the correct orientation. We found that trimming by these exonucleases results in an asymmetric intermediate, because Cas1-Cas2 protects the PAM sequence, which helps to define spacer orientation. Our findings implicate the E. coli host 3′–5′ exonucleases DnaQ and ExoT in spacer adaptation and reveal a mechanism by which spacer orientation is defined in E. coli.

scholarly journals Isopsoralen Enhanced Osteogenesis by Targeting AhR/ERα

Molecules ◽  
2018 ◽  
Vol 23 (10) ◽  
pp. 2600 ◽  
Author(s):  
Luna Ge ◽  
Yazhou Cui ◽  
Kai Cheng ◽  
Jinxiang Han
Keyword(s):  
Osteoblast Differentiation ◽  
Estrogen Receptor Alpha ◽  
Biological Effects ◽  
Hormone Deficiency ◽  
In Vivo Studies ◽  
Osteogenic Potential ◽  
Type I ◽  
Dependent Manner

Isopsoralen (IPRN), one of the main effective ingredients in Psoralea corylifolia Linn, has a variety of biological effects, including antiosteoporotic effects. In vivo studies show that IPRN can increase bone strength and trabecular bone microstructure in a sex hormone deficiency-induced osteoporosis model. However, the mechanism underlying this osteogenic potential has not been investigated in detail. In the present study, we investigated the molecular mechanism of IPRN-induced osteogenesis in MC3T3-E1 cells. Isopsoralen promoted osteoblast differentiation and mineralization, increased calcium nodule levels and alkaline phosphatase (ALP) activity and upregulated osteoblast markers, including ALP, runt-related transcription factor 2 (RUNX2), and collagen type I alpha 1 chain (COL1A1). Furthermore, IPRN limited the nucleocytoplasmic shuttling of aryl hydrocarbon receptor (AhR) by directly binding to AhR. The AhR target gene cytochrome P450 family 1 subfamily A member 1 (CYP1A1) was also inhibited in vitro and in vivo. This effect was inhibited by the AhR agonists indole-3-carbinol (I3C) and 3-methylcholanthrene (3MC). Moreover, IPRN also increased estrogen receptor alpha (ERα) expression in an AhR-dependent manner. Taken together, these results suggest that IPRN acts as an AhR antagonist and promotes osteoblast differentiation via the AhR/ERα axis.

scholarly journals Induction of type I interferons and interferon-inducible Mx genes during respiratory syncytial virus infection and reinfection in cotton rats

2008 ◽  
Vol 89 (1) ◽  
pp. 261-270 ◽  
Author(s):  
Lioubov M. Pletneva ◽  
Otto Haller ◽  
David D. Porter ◽  
Gregory A. Prince ◽  
Jorge C. G. Blanco
Keyword(s):  
Gene Expression ◽  
Respiratory Syncytial Virus ◽  
Virus Replication ◽  
Surrogate Marker ◽  
Type I Interferons ◽  
Type I ◽  
Dependent Manner ◽  
Type I Ifn ◽  
Syncytial Virus

Respiratory syncytial virus (RSV) is the primary cause of bronchiolitis in young children. In general, RSV is considered to be a poor inducer of type I (alpha/beta) interferons (IFNs). Measurement of active type I IFN production during infection in vivo is demanding, as multiple IFN subtypes with overlapping activities are produced. In contrast, Mx gene expression, which is tightly regulated by type I IFN expression, is easily determined. This study therefore measured Mx expression as a reliable surrogate marker of type I IFN activity during RSV infection in vivo in a cotton rat model. It was shown that expression of Mx genes was dramatically augmented in the lungs of infected animals in a dose- and virus strain-dependent manner. The expression of Mx genes in the lungs was paralleled by their induction in the nose and spleen, although in spleen no simultaneous virus gene expression was detected. Reinfection of RSV-immune animals leads to abortive virus replication in the lungs. Thus, type I IFN and Mx gene expression was triggered in reinfected animals, even though virus could not be isolated from their lungs. Furthermore, it was demonstrated that immunity to RSV wanes with time. Virus replication and Mx gene expression became more prominent with increasing intervals between primary infection and reinfection. These results highlight the role of type I IFN in modulation of the immune response to RSV.

scholarly journals Hydroxyl radical induced structural changes of collagen

2007 ◽  
Vol 21 (2) ◽  
pp. 91-103 ◽  
Author(s):  
Helan Xiao ◽  
Guoping Cai ◽  
Mingyao Liu
Keyword(s):  
Free Radical ◽  
Hydroxyl Radicals ◽  
Structural Changes ◽  
Type I Collagen ◽  
Free Radical Scavenger ◽  
Radical Scavenger ◽  
Type I ◽  
Dependent Manner ◽  
First Time

Extracellular matrix (ECM) plays an important role in cell differentiation, growth, migration and apoptosis. Collagen is the most abundant protein familyin vivo, but its function has still not been clearly defined yet. Reactive oxygen species (ROS) have a central role in oxidative cell stress. Electron spin resonance (ESR) spectroscopy indicates that type I collagen could uniquely scavenge hydroxyl radicals in dose- and time-dependent manner; whereas BSA and gelatin (a denatured collagen) have no such an effect. However, the mechanism by which type I collagen scavenges hydroxyl radicals is different from that of GSH, a well-known free radical scavenger. Using a new method, two-dimensional FTIR correlation analysis, for the first time, we show that the order of functional group changes of type I collagen in this process is amide I earlier than amide II than amide III than –CH– thanν(C=O). The results indicates that the structure of the main chain of collagen changed first, followed by more residue groupν(C=O) exposed to hydroxyl radicals. The reaction with the carbonyl group in collagen causes the hydroxyl free radicals to be scavenged. Therefore, ECM can effectively scavenge ROS under normal physiological conditions. When the proteins of ECM were denatured in the same way as gelatin, they lost their function as a free radical scavenger. All of these results provide new insight into therapy or prevention of oxidative stress, apoptosis and ageing.

Sign in / Sign up

Export Citation Format

Share Document