Graduate Students Mini Symposium IX-2024

01:15 PM - Timon Lindeboom (CF Schindler)
Using synthetic yeast chromosomes to map and probe large scale-deletions in high-throughput

As biology is transitioning from an observing science to a science focused on engineering DNA, high-throughput approaches to probing DNA become more relevant. Here, I showcase a qPCR-based pipeline for screening genetic modifications in synthetic yeast through use of ‘Loxtags’. Furthermore, I showcase a use of this pipeline. We first integrate a counter selectable marker into the synthetic genome in a random manner. We then remove this marker using a variety of different endonucleases creating a library of many different mutations. We then easily screen these mutants in high-throughput for low cost. Finally, mutants are phenotyped using lab-automation.

01:45 PM - Selina Rust (RG Randau)
Functional characterization of Pseudomonas oleovorans Type IV-A1 CRISPR-Cas activity in vivo

CRISPR-Cas systems are commonly used in genome editing. While many different types of these systems are known, Type IV-A is one of the least understood. We revealed that the Type IV-A1 CRISPR-Cas system of P. oleovorans is not able to cleave its target DNA, but represses gene expression by blocking transcription. We further examined native CRISPR interference activity and established a recombinant system in Escherichia coli BL21-AI. Our results show that Type IV-A1 is able to target plasmid and genomic DNA, while gene downregulation can be accomplished by targeting regions up to 3 kb upstream of a gene of interest.

02:15 PM - Nathalie Klein (RG Randau)
Structural basis and engineering of Type IV-A CRISPR-Cas system activity

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated (Cas) proteins provide adaptive immunity against invading nucleic acids in prokaryotes. The CRISPR interference mechanism generally relies on sequence-specific target binding and its nucleolytic degradation. However, type IV-A CRISPR-Cas systems use a nuclease-independent method to regulate gene expression and combat invaders. We examined the cryo-EM structures of two type IV-A complexes to understand their mechanism, including DNA recognition and recruitment of the CasDinG effector helicase. Additionally, we engineered CasDinG into a helicase/nuclease and obtained large genomic deletions. Our studies provide a detailed view of type IV-A mediated DNA interference and highlight the potential of type IV-A CRISPR-Cas systems for gene repression studies.