CRISPR technologies have revolutionized genome editing in medicine, agriculture, biotechnology, and life-sciences research with their ability to generate virtually any DNA edit at any genomic site in any organism. However, editing frequencies can be restrictively low, requiring extensive screening to identify cells harboring desired edits. What remains elusive is a way to greatly boost the frequency of editing. While characterizing CRISPR-Cas systems, bacterial defense systems and the source of CRISPR technologies, we discovered a new mechanism that could kill unedited cells yet spare edited cells, regardless of the type of gene edit. This versatile and sequence-specific approach can be described as programmable counter-selection, as undesired cells are targeted for removal, and those cells can be targeted in a programmable way. If proven, this capability could radically boost the effective editing frequencies by removing unedited cells regardless of the underlying edit, providing much-needed relief to the burden of screening imposed on diverse genome-editing applications. Here, we propose to perform proof-of-concept experiments demonstrating this capability in human cells while exploring which editing applications would best benefit from this capability. The associated tasks build on my extensive work at the interface of CRISPR biology and technologies and leverages his numerous academic and industrial contacts. I ultimately aim to translate a novel biological insight from my group’s ERC Consolidator project into an innovative foundational technology, with a clear path toward its broad use in genome editing.

Antimicrobial resistance is a global health threat, urgently calling for the development of novel anti-infective strategies. We will adopt a multidisciplinary approach to design, synthesize and optimize derivatives targeting ECF transporters as antibacterial agents. The ECF transporters are transmembrane proteins involved in the uptake of vitamins predominantly in Gram-positive pathogens (e.g., Streptococcus pneumoniae, Enterococcus faecalis, E. faecium, Staphylococcus aureus). Their inhibition prevents the uptake of vitamins from the environment, leading to starvation followed by cell death. Due to their critical role in the homeostasis of vitamins in bacteria as well as their absence in humans, ECF transporters are considered a promising novel antimicrobial target. We will thoroughly profile our inhibitors for their in vitro on-target and antibacterial activities. We will, on the one hand, further validate the cellular target through advanced target engagement studies and attempt to achieve a co-crystal structure using cryo-electron microscopy. Furthermore, evaluation of the inhibitors for their in vitro ADME-T properties and in vivo pharmacokinetic profiles will set the stage for in vivo infection models in mice. The extensive data that we will gather will enable us to nominate the most promising compound(s) for further multiparameter optimization en route to the nomination of a preclinical candidate. Ultimately, this opens access to novel anti-infectives with an unprecedented mode of action.

Bacterial infections are now a global threat demanding novel treatments due to the appearance of resistances against antibiotics at a high pace. The ESKAPE pathogens are those with highest importance in the EU and chronic infections due to biofilm formation are a particular task. Noninvasive pathogen-specific imaging of the infected tissue is not clinically available. Its successful implementation will enable the choice of appropriate therapy and boost efficacy. Furthermore, Gram-negative bacteria have a highly protective cellular envelope as an important resistance mechanism for drugs acting intracellularly, resulting in an alarmingly empty drug-pipeline. To overcome this gap, I will establish Lectin-directed Theranostics targeting pathogens via their extracellular carbohydrate-binding proteins at the site of infection for specific imaging and treatment. This will be implemented for the highly resistant ESKAPE pathogen Pseudomonas aeruginosa through 3 different work packages. WP1 Sweet Imaging: Design & conjugation of lectin-directed ligands to imaging probes, Optimization of ligand/linker, in vivo proof-of-concept imaging study. WP2 Sweet Targeting: Delivery of antibiotics to the infection through covalent linking of lectindirecting groups. Employing different antibiotics, assessment of bactericidal potency and targeting efficiency. Manufacturing of nano-carriers with surface exposed lectin-directed ligands, noncovalent charging with antibiotics. In vitro and in vivo targeting. WP3 Sweet SMART Targeting: Conjugates as SMART drugs: specific release of anti-biofilm lectin inhibitor and drug cargo upon contact with pathogen, development of linkers cleavable by pathogenic enzymes. SWEETBULLETS will establish fundamentally novel lectin-directed theranostics to fight these deleterious infections and provide relief to nosocomially infected and cystic fibrosis patients. It is rapidly extendable towards other ESKAPE pathogens, e.g. Klebsiella spp..