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- Amlinger, Lina, et al.
(författare)
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Fluorescent CRISPR Adaptation Reporter for rapid quantification of spacer acquisition
- 2017
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Ingår i: Scientific Reports. - : NATURE PUBLISHING GROUP. - 2045-2322. ; 7
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Tidskriftsartikel (refereegranskat)abstract
- CRISPR-Cas systems are adaptive prokaryotic immune systems protecting against horizontally transferred DNA or RNA such as viruses and other mobile genetic elements. Memory of past invaders is stored as spacers in CRISPR loci in a process called adaptation. Here we developed a novel assay where spacer integration results in fluorescence, enabling detection of memory formation in single cells and quantification of as few as 0.05% cells with expanded CRISPR arrays in a bacterial population. Using this fluorescent CRISPR Adaptation Reporter (f-CAR), we quantified adaptation of the two CRISPR arrays of the type I-E CRISPR-Cas system in Escherichia coli, and confirmed that more integration events are targeted to CRISPR-II than to CRISPR-I. The f-CAR conveniently analyzes and compares many samples, allowing new insights into adaptation. For instance, we show that in an E. coli culture the majority of acquisition events occur in late exponential phase.
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- Amlinger, Lina
(författare)
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The type I-E CRISPR-Cas system : Biology and applications of an adaptive immune system in bacteria
- 2017
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- CRISPR-Cas systems are adaptive immune systems in bacteria and archaea, consisting of a clustered regularly interspaced short palindromic repeats (CRISPR) array and CRISPR associated (Cas) proteins. In this work, the type I-E CRISPR-Cas system of Escherichia coli was studied.CRISPR-Cas immunity is divided into three stages. In the first stage, adaptation, Cas1 and Cas2 store memory of invaders in the CRISPR array as short intervening sequences, called spacers. During the expression stage, the array is transcribed, and subsequently processed into small CRISPR RNAs (crRNA), each consisting of one spacer and one repeat. The crRNAs are bound by the Cascade multi-protein complex. During the interference step, Cascade searches for DNA molecules complementary to the crRNA spacer. When a match is found, the target DNA is degraded by the recruited Cas3 nuclease.Host factors required for integration of new spacers into the CRISPR array were first investigated. Deleting recD, involved in DNA repair, abolished memory formation by reducing the concentration of the Cas1-Cas2 expression plasmid, leading to decreased amounts of Cas1 to levels likely insufficient for spacer integration. Deletion of RecD has an indirect effect on adaptation. To facilitate detection of adaptation, a sensitive fluorescent reporter was developed where an out-of-frame yfp reporter gene is moved into frame when a new spacer is integrated, enabling fluorescent detection of adaptation. Integration can be detected in single cells by a variety of fluorescence-based methods. A second aspect of this thesis aimed at investigating spacer elements affecting target interference. Spacers with predicted secondary structures in the crRNA impaired the ability of the CRISPR-Cas system to prevent transformation of targeted plasmids. Lastly, in absence of Cas3, Cascade was successfully used to inhibit transcription of specific genes by preventing RNA polymerase access to the promoter.The CRISPR-Cas field has seen rapid development since the first demonstration of immunity almost ten years ago. However, much research remains to fully understand these interesting adaptive immune systems and the research presented here increases our understanding of the type I-E CRISPR-Cas system.
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- Bindal, Gargi, et al.
(författare)
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Type I-E CRISPR-Cas System as a Defense System in Saccharomyces cerevisiae
- 2022
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Ingår i: mSphere. - : American Society for Microbiology. - 2379-5042. ; 7:3
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Tidskriftsartikel (refereegranskat)abstract
- Defense against viruses and other mobile genetic elements (MGEs) is important in many organisms. The CRISPR-Cas systems found in bacteria and archaea constitute adaptive immune systems that can acquire the ability to target previously unrecognized MGEs. No CRISPR-Cas system is found to occur naturally in eukaryotic cells, but here, we demonstrate interference by a type I-E CRISPR-Cas system from Escherichia coli introduced in Saccharomyces cerevisiae. The designed CRISPR arrays are expressed and processed properly in S. cerevisiae. Targeted plasmids display reduced transformation efficiency, indicative of DNA cleavage. IMPORTANCE Genetic inactivation of viruses and other MGEs is an important tool with application in both research and therapy. Gene editing using, e.g., Cas9-based systems, can be used to inactivate MGEs in eukaryotes by introducing specific mutations. However, type I-E systems processively degrade the target which allows for inactivation without detailed knowledge of gene function. A reconstituted CRISPR-Cas system in S. cerevisiae can also function as a basic research platform for testing the role of various factors in the interference process. Genetic inactivation of viruses and other MGEs is an important tool with application in both research and therapy. Gene editing using, e.g., Cas9-based systems, can be used to inactivate MGEs in eukaryotes by introducing specific mutations.
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- Brorsson, Sofia, et al.
(författare)
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Increasing Activation of the Gluteus Medius using a New Training Device
- 2012
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Ingår i: Journal of Sport and Health Research. - 1989-6239. ; 4:3, s. 311-320
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Tidskriftsartikel (refereegranskat)abstract
- Objective: The gluteus medius (GM) is a strong abductor and medial rotator of the thigh, and plays an important role in stabilizing the pelvis and controlling the knees during athletic activities.Weakness in the GM can have adverse effects onperformance and increase the risk of lower extremityinjuries. The aim of this study was to validate a newtraining device by comparing the activation of theGM when performing a squat with and without thedevice. Methods: Thirty-two female athletes (mean age 20 ± 3) performed body weight squats on and offthe device, while surface electromyography wasrecorded bilaterally on the GM. Results: All testsubjects were able to perform the squat and toactivate the GM. The activation of the GM was significantly higher when using the new device than when performing squats on the floor (Z=-4.9,P<0.001). Correlation tests between a complete sequence of three squats and one selected repetition revealed that activation was consistent throughout theexercise (right GM: rs=0.93, P<0.001, left GM:rp=0.92, P<0.001). No differences in activation were found between the right and left GM when squatting on the device. Conclusion: The newly developed training device increases muscle activity in the GM during squats. Moreover, the results showed thatsquatting on the device activates the left and rightside of the body equally, and that the GM was activated during the whole hip flexion exercise. This information and the new training device can be usedin training programs to improve stabilization of the pelvis and lower extremities during dynamic exercises.
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