Engineered anti-CRISPR proteins for optogenetic control of CRISPR-Cas9

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DNA-binding proteins physically interact with the DNA and directly affect genomic functions. The eukaryotic genome is compacted into chromatin, limiting the DNA access to nuclear factors. In this Ph.D. thesis, I explored the dynamic mechanisms, that allow ...

Many challenges are faced in the conversion of biomass into advanced biofuels, one of which is finding the correct organism for the job. The filamentous fungus Aspergillus niger has been chosen as a biocatalyst for cellulose, hemicellulose, and lignin degr ...

Type I CRISPR-Cas systems typically rely on a two-step process to degrade DNA. First, an RNA-guided complex named Cascade identifies the complementary DNA target. The helicase-nuclease fusion enzyme Cas3 is then recruited in trans for processive DNA degrad ...

Host attachment is often a critical step in the onset of pathogenesis. To attach to host cells, bacteria have evolved a range of adhesins that bind to specific receptors. Some of these adhesins have been thoroughly characterized using biochemical technique ...

The sheer size of the protein sequence space is massive: a protein of 100 residues can have 20^100 possible sequence combinations; and knowing that this exceeds the number of atoms in the universe, the chance of randomly discovering a stable new sequence w ...

Optogenetic control of CRISPR-Cas9 systems has significantly improved our ability to perform genome perturbations in living cells with high precision in time and space. As new Cas orthologues with advantageous properties are rapidly being discovered and en ...

Background The advent of the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 technology marked the beginning of a new era in the field of molecular biology, allowing the efficient and precise creation of targeted mutations in the ge ...

The advancements in genome editing techniques over the past years have rekindled interest in rational metabolic engineering strategies. While Metabolic Control Analysis (MCA) is a well-established method for quantifying the effects of metabolic engineering ...

Single-molecule measurements provide detailed mechanistic insights into molecular processes, for example in genome regulation where DNA access is controlled by nucleosomes and the chromatin machinery. However, real-time single-molecule observations of nucl ...

Anti-CRISPR (Acr) proteins are powerful tools to control CRISPR-Cas technologies. However, the available Acr repertoire is limited to naturally occurring variants. Here, we applied structure-based design on AcrIIC1, a broad-spectrum CRISPR-Cas9 inhibitor, ...

The Krüppel-associated box zinc finger protein (KRAB-ZFP) family diversified in mammals. The majority of human KRAB-ZFPs bind transposable elements (TEs), however, since most TEs are inactive in humans it is unclear whether KRAB-ZFPs emerged to suppress TE ...

Background: The discovery of the CRISPR-Cas9-based gene editing method has opened unprecedented new potential for biological and medical engineering, sparking a growing public debate on both the potential and dangers of CRISPR applications. Given the speed ...

Since most dominant human mutations are single nucleotide substitutions(1,2), we explored gene editing strategies to disrupt dominant mutations efficiently and selectively without affecting wild-type alleles. However, single nucleotide discrimination can b ...

Aspergillus niger and other filamentous fungi are widely used in industry, but efficient genetic engineering of these hosts remains nascent. For example, while molecular genetic tools have been developed, including CRISPR/Cas9, facile genome engineering of ...

Gene delivery using vector or viral-based methods is often limited by technical and safety barriers. A promising alternative that circumvents these shortcomings is the direct delivery of proteins into cells. Here we introduce a non-viral, ligand-mediated p ...

Hearing Loss (HL) is the most prevalent sensorineural disorder, affecting 360 million people worldwide. As genetic causes lead to 50% of pre-lingual deafness, gene therapy is considered as a potential therapeutic strategy. Mutations in the TMC1 gene are li ...

Plasmodium falciparum is the main cause of disease and death from malaria. P. falciparum virulence resides in the ability of infected erythrocytes (IEs) to sequester in various tissues through the interaction between members of the polymorphic P. falciparu ...

Inherited retinal dystrophies (IRDs) are a large and heterogeneous group of degenerative diseases caused by mutations in various genes. Given the favorable anatomical and immunological characteristics of the eye, gene therapy holds great potential for thei ...

Recent progress in protein engineering, empowered by the rapidly evolving fields of synthetic biology and DNA synthesis, has enabled the generation of various custom-designed proteins. Their thorough analysis has led to applications in areas including medi ...

The controlled delivery of antibodies by immunoisolated bioimplants containing genetically engineered cells is an attractive and safe approach for chronic treatments. To reach therapeutic antibody levels there is a need to generate renewable cell lines, wh ...

Engineered anti-CRISPR proteins for optogenetic control of CRISPR-Cas9

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Development of multiplexed single-molecule imaging techniques to study chromatin invasion dynamics of chromatin effector proteins

An optimized high throughput platform, from genetic transformation to fermentation, to screen for secreted laccases in Aspergillus niger

Allosteric control of type I-A CRISPR-Cas3 complexes and establishment as effective nucleic acid detection and human genome editing tools

Engineering synthetic adhesins for biophysical investigation and rewiring of host-microbe interactions

Towards automating de novo protein design for novel functionalities: controlling protein folds and protein-protein interactions

Optogenetic control of Neisseria meningitidis Cas9 genome editing using an engineered, light-switchable anti-CRISPR protein

A ribonucleoprotein transfection strategy for CRISPR/Cas9-mediated gene editing and single cell cloning in rainbow trout cells

Constraint-based metabolic control analysis for rational strain engineering

Multiplexed Single-Molecule Experiments Reveal Nucleosome Invasion Dynamics of the Cas9 Genome Editor

Computational design of anti-CRISPR proteins with improved inhibition potency

KRAB-zinc finger protein gene expansion in response to active retrotransposons in the murine lineage

Assessing Public Opinion on CRISPR-Cas9: Combining Crowdsourcing and Deep Learning

Allele-specific gene editing prevents deafness in a model of dominant progressive hearing loss

A new approach to Cas9-based genome editing in Aspergillus niger that is precise, efficient and selectable

A ligand-based system for receptor-specific delivery of proteins

Delivery of CRISPR/Cas9 using AAV-PHP.B in the inner ear leads to allele-specific inactivation of the mutated Tmc1 allele and protects auditory function in Beethoven mice

Phosphorylation of the VAR2CSA extracellular region is associated with enhanced adhesive properties to the placental receptor CSA

Gene Editing Preserves Visual Functions in a Mouse Model of Retinal Degeneration

Development of microfluidic tools and methods for the biochemical characterization of scFv and Cas9 affinity reagents and their applications in molecular detection

Genetic engineering of cell lines using lentiviral vectors to achieve antibody secretion following encapsulated implantation