On CRISPR-controlled Proteases

This dissertation provides an experimental and conceptual characterization of Craspase, a CRISPR-controlled protease. The array of functionalities inherent to Craspase — including precise protein cleavage, guided RNA recognition, and self-regulatory capabilities — highlights the complexity that can emerge from the endless bacterium-virus...

Cliquepolitik: Multimodal online discourse coalitions on CRISPR-Cas genome editing technology

The influence of visualizations on decision-making about controversial policy issues is increasingly recognized in the political and policy sciences. In this paper, we explore how combinations of visuals and text on Twitter (now X) lead to the formation of networks of actors sharing similar textual and visual framings about a policy issue in...

Expanding the genome editing toolbox of Saccharomyces cerevisiae with the endonuclease ErCas12a

ErCas12a is a class 2 type V CRISPR-Cas nuclease isolated from Eubacterium rectale with attractive fundamental characteristics, such as RNA self-processing capability, and lacks reach-through royalties typical for Cas nucleases. This study aims to develop a ErCas12a-mediated genome editing tool applicable in the model yeast Saccharomyces...

Approaches for bacteriophage genome engineering

In recent years, bacteriophage research has been boosted by a rising interest in using phage therapy to treat antibiotic-resistant bacterial infections. In addition, there is a desire to use phages and their unique proteins for specific biocontrol applications and diagnostics. However, the ability to manipulate phage genomes to understand and...

Single cell variability of CRISPR-Cas interference and adaptation

While CRISPR-Cas defence mechanisms have been studied on a population level, their temporal dynamics and variability in individual cells have remained unknown. Using a microfluidic device, time-lapse microscopy and mathematical modelling, we studied invader clearance in Escherichia coli across multiple generations. We observed that CRISPR...

A CRISPR-associated transposase presents null cargo integration efficiency when targeting a transcriptionally highly active region

CRISPR-Cas effectors (e.g. Cas9) have been widely used to perform genetic knock-outs. Performing knock-ins however, remains challenging due to the inefficiency of the endogenous pathway cells use to integrate a donor genetic cargo into its genome (homology directed repair) when compared to other repair pathways like non-homologous end joining....

Genome-wide Off-target Cleavage Prediction of CRISPR/Cas Nucleases

Predicting off-target cleavage sites for CRISPR/Cas nucleases on the human genome based on biophysical modelling and high-throughput biochemical profiling.

Genome analysis and engineering of industrial lager brewing yeasts

Lager beer, also referred to as Pilsner, is the most popular alcoholic beverage in the world, with an annual consumption of almost 200 billion litres per year. To make lager beer, brewer’s wort is fermented with the yeast <i style="mso-bidi-font-style:normal"...

Multiplex genome editing of microorganisms using CRISPR-Cas

Microbial production of chemical compounds often requires highly engineered microbial cell factories. During the last years, CRISPR-Cas nucleases have been repurposed as powerful tools for genome editing. Here, we briefly review the most frequently used CRISPR-Cas tools and describe some of their applications. We describe the progress made...

Keeping crispr in check: diverse mechanisms of phage-encoded anti-crisprs

CRISPR-Cas represents the only adaptive immune system of prokaryotes known to date. These immune systems are widespread among bacteria and archaea, and provide protection against invasion of mobile genetic elements, such as bacteriophages and plasmids. As a result of the arms-race between phages and their prokaryotic hosts, phages have...

Conserved motifs in the CRISPR leader sequence control spacer acquisition levels in Type I-D CRISPR-Cas systems

Integrating short DNA fragments at the correct leader-repeat junction is key to successful CRISPR-Cas memory formation. The Cas1-2 proteins are responsible to carry out this process. However, the CRISPR adaptation process additionally requires a DNA element adjacent to the CRISPR array, called leader, to facilitate efficient localization of...

Cleavage and unbinding times of CRISPR associated proteins

In this thesis the CRISPR-Cas9 mechanism, a promising mechanism for geneediting, is considered. Closed form expressions are derived for the probability and time to cleave or unbind for the associated Cas9 protein. The mechanism can be modelled mathematically by a birth and death process, therefore the expressions could be derived using Markov...

Cas4 Facilitates PAM-Compatible Spacer Selection during CRISPR Adaptation

CRISPR-Cas systems adapt their immunological memory against their invaders by integrating short DNA fragments into clustered regularly interspaced short palindromic repeat (CRISPR) loci. While Cas1 and Cas2 make up the core machinery of the CRISPR integration process, various class I and II CRISPR-Cas systems encode Cas4 proteins for which...

Microbial Warfare: Illuminating CRISPR adaptive immunity using single-molecule fluorescence

Bacteria and archaea are constantly threatened by a large array of viruses and other genetic elements. Driven by evolution, these organisms have acquired a wide arsenal of defense mechanisms that allow the host organism to fight off the invaders. Among these defense mechanisms is an adaptive and inheritable that is conveyed through Clustered...

Cas3-Derived Target DNA Degradation Fragments Fuel Primed CRISPR Adaptation

Prokaryotes use a mechanism called priming to update their CRISPR immunological memory to rapidly counter revisiting, mutated viruses, and plasmids. Here we have determined how new spacers are produced and selected for integration into the CRISPR array during priming. We show that Cas3 couples CRISPR interference to adaptation by producing...