13:15 – 14:15 CRISPR-Cas9: a game changer in genome engineering
Professor Dr. Emmanuelle Charpentier, Helmholtz Center for Infection Research, Braunschweig
The RNA-programmable CRISPR-Cas9 system has recently emerged as a transformative technology in biological sciences, allowing rapid and efficient targeted genome editing, chromosomal marking and gene regulation in a large variety of cells and organisms. In this system, the endonuclease Cas9 or catalytically inactive Cas9 variants are programmed with single guide RNAs (sgRNAs) to target site-specifically any DNA sequence of interest given the presence of a short sequence (Protospacer Adjacent Motif, PAM) juxtaposed to the complementary region between the sgRNA and target DNA. The system is efficient, versatile and easily programmable.
Originally, CRISPR-Cas is an RNA-mediated adaptive immune system that protects bacteria and archaea from invading mobile genetic elements (phages, plasmids). Short crRNA (CRISPR RNA) molecules containing unique genome-targeting spacers commonly guide Cas protein(s) to invading cognate nucleic acids to affect their maintenance. CRISPR-Cas has been classified into three main types and further subtypes. CRISPR-Cas9 originates from the type II CRISPR-Cas system that has evolved unique molecular mechanisms for maturation of crRNAs and targeting of invading DNA, which my laboratory has identified in the human pathogen Streptococcus pyogenes. During the step of crRNA biogenesis, a unique CRISPR-associated RNA, tracrRNA, base pairs with the repeats of precursor-crRNA to form anti-repeat-repeat dual-RNAs that are cleaved by RNase III in the presence of Cas9 (formerly Csn1), generating mature tracrRNA and intermediate forms of crRNAs. Following a second maturation event, the mature dual-tracrRNA-crRNAs guide the endonuclease Cas9 to cleave cognate target DNA and thereby affect the maintenance of invading genomes. We have shown that the endonuclease Cas9 can be programmed with sgRNAs mimicking the natural dual-tracrRNA-crRNAs to target site-specifically any DNA sequence of interest. I will discuss the biological roles of CRISPR-Cas9, the mechanisms involved, the evolution of type II CRISPR-Cas components in bacteria and the applications of CRISPR-Cas9 as a novel genome engineering technology.
Microbial Rhodopsins are widely used in these days as optogenetic tools in neuro and cell biology. We were able to show that rhodopsins from the unicellar alga Chlamydomonas reinhardtii with the 7 transmembrane helix motif act as light-gated ion channels, which we named channelrhodopsins (ChR1,ChR2). Together with the light driven Cl- pump Halorhodopsin ChR2 is used for the non-invasive manipulation of excitable cells and living animals by light with high temporal resolution and more important with extremely high spatial resolution The basic functional and structural description of this unusual class of ion channels is given (electrophysiology, noise analysis, flash photolysis and 2D crystallography). New tools and their application with a biomedical perspective in the cochlea and the restoration of vision are presented.
18:00 – 19:00 Connectomics: the dense reconstruction of neuronal networks
Dr. Moritz Helmstädter, Max Planck Institute for Brain Research, Frankfurt am Main
The mapping of neuronal connectivity is one of the main challenges in neuroscience. Only with the knowledge of wiring diagrams is it possible to understand the computational capacities of neuronal networks, both in the sensory periphery, and especially in the mammalian cerebral cortex. Our methods for dense circuit mapping are based on 3-dimensional electron microscopy (EM) imaging of tissue, which allows imaging nerve tissue at nanometer-scale resolution across substantial volumes (typically hundreds of micrometers per spatial dimension) using Serial Block-Face Scanning Electron Microscopy (SBEM). The most time-consuming aspect of circuit mapping, however, is image analysis; analysis time far exceeds the time needed to acquire the data. Therefore, we developed methods to make circuit reconstruction feasible by increasing analysis speed and accuracy, using a combination of crowd sourcing and machine learning. We have applied these methods to circuits in the mouse retina, mapping the complete connectivity graph between almost a thousand neurons, and we are currently improving these methods for the application to neuronal circuits in the neocortex using automated image analysis, together with online science games.