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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IntroductionPreimplantation Genetic Diagnosis (PGD) was developed in the 1990s and has been used since to diagnose and discard embryos with genetic conditions or chromosomal abnormalities. CRISPR-Cas9 was discovered in 2012 and has been used in research, but has not become clinical practice on humans yet. CRISPR-Cas9 could potentially be applied to treat and prevent genetic disorders.AimThe aim was to investigate the ethical dilemmas of each method through a set of research questions. The ethics of applying PGD according to Swedish guidelines and applying CRISPR-Cas9 on humans was investigated.MethodologyThis was not a systematic literature review. Instead, articles have been selected based on their explanation of each method and uniqueness or volume of ethical arguments surrounding each method, that is of relevance for the discussed issues.ResultsArguments in favour of PGD addressed among other things the somatic and psychological health of future children and parents along with the economical benefits. Arguments against PGD addressed different dilemmas of discarding an embryo and thereby a future individual. Arguments against CRISPR-Cas9 addressed technical limitations, our limited knowledge of genetics and more. Arguments in favour addressed benefits in clinical medicine and research.ConclusionsPGD according to Swedish guidelines was found to be ethically acceptable, since its restrictive use that have not given room for ethically dubious applications. CRISPR-Cas9 was found not to be safe enough for human applications at this moment due to technical limitations. If these were to be solved, caution and restraint must be urged.

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T cells modified for expression of Chimeric Antigen Receptors (CARs) were the first genemodified cell products approved for use in cancer immunotherapy. CAR-T cells engineered with gammaretroviral or lentiviral vectors (RVs/LVs) targeting B-cell lymphomas and leukemias have shown excellent clinical efficacy and no malignant transformation due to insertional mutagenesis to date. Large-scale production of RVs/ LVs under good-manufacturing practices for CAR-T cell manufacturing has soared in recent years. However, manufacturing of RVs/LVs remains complex and costly, representing a logistical bottleneck for CAR-T cell production. Emerging gene-editing technologies are fostering a new paradigm in synthetic biology for the engineering and production of CAR-T cells. Firstly, the generation of the modular reagents utilized for gene editing with the CRISPR-Cas systems can be scaled-up with high precision under good manufacturing practices, are interchangeable and can be more sustainable in the long-run through the lower material costs. Secondly, gene editing exploits the precise insertion of CARs into defined genomic loci and allows combinatorial gene knock-ins and knock-outs with exciting and dynamic perspectives for T cell engineering to improve their therapeutic efficacy. Thirdly, allogeneic edited CAR-effector cells could eventually become available as “off-the-shelf” products. This review addresses important points to consider regarding the status quo, pending needs and perspectives for the forthright evolution from the viral towards gene editing developments for CAR-T cells.

The last decades of research in cardiovascular prevention have been characterized by successful bench-to-bedside developments for the treatment of low-density lipoprotein (LDL) hypercholesterolemia. Recent examples include the inhibition of proprotein convertase subtilisin/kexin type 9 (PCSK9) with monoclonal antibodies, small interfering RNA and antisense RNA drugs. The cumulative effects of LDL cholesterol on atherosclerosis make early, potent, and long-term reductions in LDL cholesterol desirable—ideally without the need of regular intake or application of medication and importantly, without side effects. Current reports show durable LDL cholesterol reductions in primates following one single treatment with PCSK9 gene or base editors. Use of the CRISPR/Cas system enables precise genome editing down to single-nucleotide changes. Provided safety and documentation of a reduction in cardiovascular events, this novel technique has the potential to fundamentally change our current concepts of cardiovascular prevention. In this review, the application of the CRISPR/Cas system is explained and the current state of in vivo approaches of PCSK9 editing is presented.

Secondary structure dynamics of biopolymers play a vital role in many of the complex processes within a cell. However, due to the substantial number of atoms in the involved biopolymers along with the multitude of interactions that occur between the molecules, understanding these processes in detail is challenging and often involves computationally demanding simulations. In this thesis, the secondary structure dynamics of three different biopolymer systems were modeled using a single approach, which is based on intuitive principles that facilitate the interpretation. To this end, the kinetic behavior of each system was experimentally determined, and described by simplified reaction schemes, which were then connected to Markov chain models encompassing all principal secondary structural conformations. Firstly, we investigated the toehold-mediated strand displacement reaction, which is widely applied in nanotechnology to create DNA-based nano-devices and biochemical reaction networks. Our model correctly described the impact of base pair mismatches on the kinetics of these reactions, as measured by bulk fluorescence experiments. Additionally, it revealed that incumbent dissociation, base pair fraying, and internal loop formation are important processes during strand displacement. Furthermore, we established two dissipative elements to enhance temporal control over toehold-mediated strand displacement reactions. The first element allowed a reversible and repeatable incumbent strand release, whereas the second element provided the possibility to start the displacement reaction after a programmable temporal delay. Secondly, we studied the target recognition by the CRISPR-Cas effector complex Cascade, a highly promising protein for applications in genome engineering. Our model successfully reproduced all aspects of the torque- and mismatch-dependent R-loop formation time by Cascade obtained by single-molecule torque and bulk fluorescence measurements. Furthermore, we demonstrated that the seed effect observed for Cascade results from DNA supercoiling, rather than a structural property of the protein complex. Lastly, we explored the folding/unfolding of α-helices, which plays a critical role in the folding and function of proteins. Our model accurately described α-helix unfolding kinetics obtained by fast triplet-triplet energy transfer. Moreover, we showed that the complex α-helix unfolding does not follow a simple Einstein-type diffusion but is a combination of the sub-diffusive boundary diffusion and the rather peptide-length-independent coil nucleation. The presented models enabled access to the diverse timescales of the characterized processes, which are generally difficult to access experimentally, despite utilizing just a single approach. In particular, we obtained: tens of microseconds for the branch migration step time of the toehold-mediated strand displacement, hundreds of microseconds for the R-loop formation steps by Cascade, and tens of nanoseconds for folding or unfolding of an α-helix by a single residue. Given the simplicity and accessibility of the established models, we are confident that they will become useful tools for researchers to analyze the dynamics of biomolecules, and anticipate that similar modeling approaches can be applied to other biopolymer systems, being well-described by probabilistic models. / Die Sekundärstrukturdynamik von Biopolymeren spielt eine entscheidende Rolle bei vielen komplexen Prozessen innerhalb einer Zelle. Aufgrund der beträchtlichen Anzahl von Atomen in den beteiligten Biopolymeren und der Vielzahl an Wechselwirkungen zwischen den Molekülen ist es jedoch eine Herausforderung diese Prozesse im Detail zu verstehen, und erfordert oft rechenintensive Simulationen. In dieser Arbeit wurde die Sekundärstrukturdynamik von drei verschiedenen Biopolymersystemen mit einem einzigen Ansatz modelliert, welcher auf intuitiven Prinzipien beruht und somit eine erleichterte Interpretation der Ergebnisse ermöglicht. Hierzu wurde das kinetische Verhalten jedes Systems experimentell bestimmt und durch vereinfachte Reaktionsschemata beschrieben. Diese wurden anschließend mit Markov-Kettenmodellen verknüpft, welche alle wichtigen Konformationen der Sekundärstruktur abbilden. Als erstes System untersuchten wir die DNA Strangaustauschreaktion, welche in der Nanotechnologie häufig zur Herstellung von DNA-basierten Nanomaschinen und biochemischen Reaktionsnetzwerken eingesetzt wird. Unser Modell beschrieb die durch Ensemble-Fluoreszenz-Experimente gemessenen Auswirkungen von Basenfehlpaarungen auf die Kinetik dieser Reaktionen korrekt. Des Weiteren zeigte sich, dass die vorzeitige Strangablösung, das Ausfransen von Basenpaaren und die Bildung interner Schleifen wichtige Prozesse während des Strangaustausches sind. Darüber hinaus konnten wir zwei dissipative Elemente etablieren, um die zeitliche Kontrolle über die Strangaustauschreaktionen zu verbessern. Das erste Element ermöglicht eine reversible und wiederholbare Strangablösung, während das zweite Element die Möglichkeit bietet die Strangaustauschreaktionen nach einer programmierbaren zeitlichen Verzögerung zu starten. Zweitens untersuchten wir den Zielerkennungsprozess durch den CRISPR-Cas Komplex Cascade, ein vielversprechendes Protein für Anwendungen in der Genomtechnologie. Unser Modell reproduzierte erfolgreich alle Aspekte der torsions- und fehlpaarungs-abhängigen R-Schleifenbildung durch Cascade, welche durch Einzelmolekül-Torsions- und Ensemble-Fluoreszenz-Messungen ermittelt wurden. Zusätzlich konnten wir nachweisen, dass der für Cascade beobachtete „seed“-Effekt auf DNA-Verdrehung und nicht auf eine strukturelle Eigenschaft des Proteinkomplexes zurückzuführen ist. Schließlich untersuchten wir die Faltung/Entfaltung von α-Helices, welche eine entscheidende Rolle bei der Faltung und Funktion von Proteinen spielen. Unser Modell beschrieb die durch schnelle Triplett-Triplett-Energietransfer Experimente ermittelte α-Helix-Entfaltungskinetik exakt. Darüber hinaus konnten wir zeigen, dass die komplexe α-Helix-Entfaltung nicht einer einfachen Diffusion vom Einstein-Typ folgt, sondern eine Kombination aus subdiffusiver Grenzdiffusion und der eher peptidlängenunabhängigen Coil-Nukleation ist. Obwohl nur ein einziger Ansatz verwendet wurde, ermöglichten die vorgestellten Modelle den Zugang zu den vielschichtigen Zeitskalen der charakterisierten Prozesse, welche im Allgemeinen experimentell schwer zugänglich sind. Insbesondere konnten die folgenden zeitlichen Bereiche bestimmt werden: Dutzende von Mikrosekunden für die Schrittzeit der Strangaustauschreaktion, Hunderte von Mikrosekunden für die Schritte der R-Schleifenbildung durch Cascade, und Dutzende von Nanosekunden für die Faltung oder Entfaltung einer α-Helix um ein einzelnes Segment. Angesichts der Simplizität und Zugänglichkeit der etablierten Modelle sind wir zuversichtlich, dass sie zu nützlichen Werkzeugen für Forscher werden, um die Dynamik von Biomolekülen zu analysieren. Zusätzlich gehen wir davon aus, dass ähnliche Modellierungsansätze auf andere Biopolymersysteme angewendet werden können, sofern sie gut durch probabilistische Modelle beschrieben werden.

Clustered regularly interspaced short palindromic repeats (CRISPRs) and associated (Cas) proteins form the basis of a prokaryotic adaptive immune system. Acquired sections of viral DNA are stored within the host genome as ‘spacers' flanked by ‘repeat' sequences. The CRISPR arrays are transcribed and processed to release mature CRISPR RNAs (crRNAs) – containing a single, intact spacer sequence – that are used by effector complexes to base-pair with matching hostile genetic elements and silence future infections. crRNA-biogenesis is thus an essential step within the defence pathway. Within Type I and III systems, the primary processing of the CRISPR transcript at repeat sites is performed almost exclusively by the CRISPR-specific riboendonuclease, Cas6. This thesis seeks to probe the catalytic mechanism of a Cas6 enzyme from the crenarchaeon Sulfolobus solfataricus (sso). Despite analogous generation of crRNA, ssoCas6 paralogues differ from previously characterised Cas6 examples in their lack of a canonical active site histidine residue. The work here builds on recent crystallographic evidence that the ssoCas6-1 paralogue unexpectedly adopts a dimeric conformation (PDB 3ZFV, 4ILR), to show that not only is the ssoCas6-1 dimer stable in solution but that this atypical arrangement is important to the activity of this particular enzyme. Furthermore, the ssoCas6-1 paralogue is shown to be the first in this family of endonucleases to employ multiple-turnover kinetics. The widespread diversity in Cas6 catalytic mechanisms reflects the plastic nature of the Cas6 active site and rapid co-evolution with substrate repeat sequences. The CRISPR/Cas environment within S. solfataricus is highly complex, containing three co-existing system types (Type I-A, III-A, III-B), five Cas6 paralogues and two families of CRISPR loci (AB and CD) that differ by repeat sequence. By probing the activity of an additional ssoCas6 paralogue (ssoCas6-3), which reveals different substrate specificities to those of ssoCas6-1, evidence emerges for functional coupling between ssoCas6 paralogues and downstream effector complexes, sufficient to regulate crRNA uptake and possibly even complex assembly.

Clustered regularly interspaced short palindromic repeats (CRISPRs) act to prevent viral infection and horizontal gene transfer in prokaryotes. The genomic CRISPR array contains short sequences (“spacers”) that are derived from foreign genetic elements. The CRISPR array is transcribed and processed into CRISPR RNAs (crRNAs) used in the sequence-specific degradation of foreign nucleic acids. This process is called interference and is mediated by CRISPR-associated (Cas) proteins. This thesis has focused on the structural and functional characterisation of four Cas proteins from the CRISPR/Cas system of Sulfolobus solfataricus. The crystal structure of Cmr7 (Sso1725), a Sulfolobales-specific subunit of the ssRNA-degrading CMR complex, allowed for the identification of a putative protein-binding site, though no specific function could be ascribed to the protein. Cas6 (Sso1437) is the enzyme responsible for crRNA maturation and the characterisation of this protein allowed for the molecular rationalisation of its atypical RNA cleavage mechanism. Csa5 and Cas8a2 are subunits of the aCascade complex that targets dsDNA. Csa5 (Sso1398) was shown to have a putative role in R-loop stabilisation during interference while the role of Cas8a2 (Sso1401) was not determined. The structures of these two proteins were used to define relationships between the subunits of interference complexes from various CRISPR/Cas systems. A second aspect of this work has been the expression and purification of eukaryotic ion channels for structural studies. The acid sensing ion channel (ASIC) and FMRFamide-gated sodium channel (FaNaC) are gated ion channels with unknown mechanisms of channel activation. These ion channels must be expressed in eukaryotic systems and so human embryonic kidney (HEK) cells and baculovirus-insect cell expression systems were developed to express ASIC and FaNaC constructs. The expression and purification protocols have been optimised to allow for the preparation of soluble protein that will in future be used for crystallography and electron paramagnetic resonance (EPR) studies.