Genome Editing and Systems Genetics
We develop CRISPR-based technologies for high-throughput precision genome editing to study how genome sequence variation modulates cellular processes to ultimately shape the phenotypic diversity that we observe in natural populations. We also apply these technologies to explore natural and synthetic sequence variation for generating superior strains for industrial applications. The yeast S. cerevisiae, which is a model for many basic processes in humans and other biological systems and an industrially important microbe, is our main model system.
VIB Group Leader from May 2021
- Postdoc at European Molecular Biology Laboratory in Heidelberg, Germany, 2015 - 2020
- PhD student at ETH Zürich, Switzerland, 2010 - 2015
Genomics is the study of all the genes of an organism (the genome), including interactions of those genes with each other and with the organism’s environment.
Next-generation, or high-throughput, sequencing is a set of technologies that allows sequencing large runs of DNA or RNA faster and cheaper than ever before. This generates large amounts of biologically relevant data, waiting to be explored.
Systems biology studies biological systems taking into account the interactions of key elements such as DNA, RNA, proteins, and cells with respect to one another (eg as in a system).
Yeasts are a group of single-celled eukaryotic microorganisms. They are part of the fungus kingdom and there are at least 1500 species of them. Several of them are important for the production of foods and beverages due to their fermentation abilities. Some of them can be pathogenic.
Through advances in sequencing technologies we are now able to generate detailed maps of sequence variation across entire genomes. However, our knowledge on the functional consequences of the majority of these variants is lagging behind, even for well-characterized organisms such as the baker’s yeast S. cerevisiae. Methods for precise and accurate manipulation of genomes enable assessing the function of genes and gene variants directly, analyzing their contribution to phenotypes of interest, and engineering cells with desired characteristics. RNA guided programmable DNA nucleases of CRISPR systems, in particular the type II CRISPR/Cas9 system from Streptococcus pyogenes, have been widely adopted for efficient and precise genome manipulation.
The Cas9 nuclease can be directed to cut any genomic location using a short guide RNA (gRNA) with homology to the target region if a protospacer-adjacent motif (PAM) is next to the target sequence. Following gRNA-directed, Cas9-mediated cutting of the genome, donor DNA templates encoding desired mutations flanked by homologies to the target site are used in homologous recombination (HR)-mediated repair of the cut. Array-based oligonucleotide synthesis allows fast and affordable creation of guide-donor libraries directed against thousands of targets. In our research we develop highly multiplexed CRISPR/Cas9 based methods to systematically perturb genomes, and are broadly interested in applying our tools to study how natural sequence variation shapes phenotypic complexity and to generate, screen, and identify sequence variants with advantageous properties for industrial and medical applications.
We also develop new, more scalable and more affordable (functional) genomics technologies, often combined with leveraging homemade enzymes, for genomic and functional characterization of our edited strains. We mainly work with the yeast S. cerevisiae, which is a model for many basic processes in humans and other biological systems and an industrially important microbe.
High-throughput CRISPR-based precision genome editing
We develop CRISPR technologies for massively parallel precision genome editing and direct functional screening of sequence variation in microbes. This builds on our multiplexed variant engineering and barcode-based phenotyping platform MAGESTIC (Multiplexed Accurate Genome Editing with Short, Trackable, Integrated Cellular barcodes), which uses pools of array-synthesized oligos encoding a gRNA and a donor DNA to introduce a designed variant by HR (Roy, Smith, Vonesch et al. 2018). Importantly, variant strains are tagged by DNA barcodes integrated at a dedicated genomic locus, allowing to efficiently track mutations in cell populations during functional screens, and read out variant identities via sequencing of the barcode locus. We will develop versions of MAGESTIC that will allow us to explore more complex genetic interactions, and that can be more widely deployed across the genome, other strains and microbes.
Scalable measurement of molecular responses to genetic perturbations
We have established sensitive protocols to measure small variations in fitness of cells carrying defined sequence variants via competitive growth followed by barcode sequencing. To understand how these variants lead to changes in fitness, and to more broadly assess the roles of sequence variants that may not lead to a noticeable fitness change we will develop reporter systems that we benchmark for quantitative readout of dynamic molecular responses and pathway activities. To gain a more global overview of the molecular impact of defined genetic perturbations we will simultaneously measure the dynamics of core sets of genes by adapting a recently developed strategy for multiplexed detection of SARS-CoV-2 (Vonesch et al. 2020a).
Applying precision editing to systematically study and exploit natural and synthetic sequence variation
We apply our tools to improve our understanding of how traits are controlled by variation at individual nucleotides, and how these variants impact on molecular and genetic networks to shape phenotypic complexity. We are particularly interested in variants that alter dynamic molecular response parameters upon exposure of cells to stresses, drugs and other challenging environments. We are also interested in better understanding how diverse genetic contexts alter the functional consequences of promising variants and exploiting these insights in translational applications.
The unexplored sequence diversity in natural populations is a large reservoir of untapped potential for biotechnology, synthetic biology and medicine. Using our tools we can efficiently design, engineer and screen large libraries of natural and synthetic sequence variation for advantageous properties for industrial and medical applications. Combined with our simplified protocol for microbial genome sequencing (Vonesch et al. 2020b) for fast and inexpensive strain validation we have the capability to generate designer biological systems with unprecedented speed, precision and efficiency.
Vonesch SC#, Bredikhin D, Dobrev N, Villacorta L, Kleinendorst R, Cacace E, Flock J, Frank M, Jung F, Kornienko J, Mitosch K, Osuna-Lopez M, Zimmermann J, Göttig S, Hamprecht A, Kräusslich HG, Knop M, Typas A, Steinmetz LM#, Benes V#, Remans K#, Krebs AR# (2020a). McQ – an open-source multiplexed SARS-CoV-2 quantification platform. medRXiv.
(# corresponding authors)
Vonesch SC#, Li S, Szu Tu C, Hennig BP, Dobrev N, Steinmetz LM# (2020b). Fast and inexpensive whole genome sequencing library preparation from intact yeast cells. G3 jkaa009 (accepted). bioRxiv
(# corresponding authors)
Roy KR*, Smith JD*, Vonesch SC*, Lin G, Szu Tu C, Lederer AR, Chu A, Suresh S, Nguyen M, Horecka J, Tripathi A, Burnett WT, Morgan MA, Schulz J, Orsley KM, Wei W, Aiyar RS, Davis RW, Bankaitis VA, Haber JE, Salit ML, St.Onge RP, Steinmetz LM (2018). Multiplexed precision genome editing with trackable genomic barcodes in yeast. Nature Biotechnology 36, 512–520. (* equal contribution)