Enzymology seminars

Virginijus Šikšnys traces genome-editing tools to bacterial antiviral defence. Programmable CRISPR-Cas nucleases offer powerful ways to alter DNA, but delivery, unintended effects, immune interactions and insertion efficiency remain challenges. The lecture discusses compact TnpB nucleases and how further study of defence systems may reveal useful enzymatic functions.

Programmed axon death is a widespread and completely preventable mechanism in injury and disease. Mouse and Drosophila studies define a molecular pathway involving activation of SARM1 NA Dase and its prevention by NAD synthesising enzyme NMNAT2 . Loss of axonal NMNAT2 causes its substrate, NMN , to accumulate and activate SARM1 , driving loss of NAD and changes in ATP , ROS and calcium. Animal models caused by genetic mutation, toxins, viruses or metabolic defects can be alleviated by blocking programmed axon death, for example models of CMT1B , chemotherapy-induced peripheral neuropathy (CIPN), rabies and diabetic peripheral neuropathy (DPN). The perinatal lethality of NMNAT2 null mice is completely rescued, restoring a normal, healthy lifespan. Animal models lack the genetic and environmental diversity present in human populations and this is problematic for modelling gene-environment combinations, for example in CIPN and DPN , and identifying rare, pathogenic mutations. Instead, by testing human gene variants in WGS datasets for loss- and gain-of-function, we identified enrichment of rare SARM1 gain-of-function variants in sporadic ALS , despite previous negative findings in SOD1 transgenic mice. We have shown in mice that heterozygous SARM1 loss-of-function is protective from a range of axonal stresses and that naturally-occurring SARM1 loss-of-function alleles are present in human populations. This enables new approaches to identify disorders where blocking SARM1 may be therapeutically useful, and the existence of two dominant negative human variants in healthy adults is some of the best evidence available that drugs blocking SARM1 are likely to be safe. Further loss- and gain-of-function variants in SARM1 and NMNAT2 are being identified and used to extend and strengthen the evidence of association with neurological disorders. We aim to identify diseases, and specific patients, in whom SARM1 -blocking drugs are most likely to be effective.

Cells and microorganisms produce and consume all sorts of chemicals, from nutrients to signalling molecules. The same happens at the nanoscale inside cells themselves, where enzymes catalyse the production and consumption of the chemicals needed for life. In this work, we have found a generic mechanism by which such chemically-active particles, be it cells or enzymes or engineered synthetic colloids, can "sense" each other and ultimately self- organize in a multitude of ways. A peculiarity of these chemical-mediated interactions is that they break action-reaction symmetry : for example, one particle may be repelled from a second particle, which is in turn attracted to the first one, so that it ends up "chasing" it. Such chasing interactions allow for the formation of large clusters of particles that "swim" autonomously. Regarding enzymes, we find that they can spontaneously aggregate into clusters with precisely the right composition, so that the product of one enzyme is passed on, without lack or excess, to the next enzyme in the metabolic cascade.

Emmanuelle Charpentier and Jennifer Doudna explain the discovery and biochemical development of CRISPR-Cas9 as a programmable tool for modifying DNA. The scientific story begins with bacterial defence against invading genetic material and the role of tracrRNA in the CRISPR system. It proceeds through the reconstruction and simplification of the RNA-guided machinery and the demonstration that a chosen guide can direct DNA cleavage to a specified sequence. The lectures connect basic research on microbes and RNA with a method that transformed experimental genetics. They explain the molecular logic of the system and the possibilities it opened for studying gene function, engineering organisms and developing therapeutic approaches.

Gerald Joyce explores attempts to reconstruct features of RNA-based life in the laboratory. Under the RNA world hypothesis, early organisms used RNA to store information and catalyze replication before the emergence of DNA genomes. Directed evolution has produced RNA enzymes that copy diverse templates, synthesize structured and catalytic RNAs, and amplify short templates in an RNA-catalyzed analogue of the polymerase chain reaction. A variant can make DNA from an RNA template, suggesting a possible route between RNA and DNA genomes. Joyce also describes cross-chiral enzymes that synthesize RNA of opposite handedness and can assemble their own enantiomers from fragments. The lecture examines these complementary approaches to replicating functional RNA, their relevance to the origins of life, and potential diagnostic and clinical applications.