RESEARCH
POST-TRANSCRIPTIONAL GENE EXPRESSION REGULATION
Post-transcriptional gene expression regulation pathways have emerged as critical components underlying the diversification and spatiotemporal control of the proteome and transcriptome. Key regulatory factors include RNA-binding proteins (RBPs), non-coding RNAs and RNA modifications (Lapinaite et al. Nature, 2013; Graziadei et al. RNA, 2016) that function either individually or cooperatively to influence gene expression via alteration of RNA metabolism.
We are interested in understanding how RNA modifications, and RNA-protein and RNA-microRNA interactions contribute to gene expression regulation and impact gene expression in health and disease. This will serve as a foundation for developement of novel therapeutics.
PROKARYOTIC DEFENSE SYSTEMS
Microbes are under constant attack by harmful viruses and mobile genetic elements. Thus, they evolved a variety of defense mechanisms such as restriction-modification (RM), toxin-antitoxin (TA), CRISPR-Cas, and prokaryotic Argonaute (pAgo) based systems to defend against these threats. Each of these defense systems are extremely diverse and the molecular mechanisms of most of them are still unknown.
We are interested in understanding the molecular mechanisms of nucleic acid guided systems (pAgo and CRISPR-Cas) via the investigation of their structure-activity relationships using state-of-art structural biology, biochemistry and systems biology approaches. The acquired knowledge will be harnessed in the development of exciting new genome, transcriptome, and epitranscriptome modulating technologies with important biotechnological and biomedical applications (Kriukiene et al. Nature Communications, 2012; Lapinaite et al. PNAS, 2018; Richter et al. Nature Biotechnology, 2020; Lapinaite & Knott et al. Science, 2020).
PRECISION GENOME EDITING
CRISPR–Cas9 genome editing is based on the site specific double stranded break of the genomic DNA performed by the Cas9-guide RNA complex and relies on the subsequent repair by the cell’s DNA-repair machinery. This process yields random and heterogeneous changes in the genomic DNA, unless a template is used. Programmable CRISPR-Cas based DNA base editors (Cas9 and deaminase fusions) are able to replace one nucleotide with another (A-to-G or C-to-U) by avoiding double stranded break and yield efficient precision genome editing necessary especially for the therapeutic applications. Recently, we have obtained the first cryo-EM structure of an Adenine Base Editor (ABE8e) in a DNA bound state, which in combination with biochemical assays unveil the molecular mechanism of DNA base editing (Richter et al. Nature Biotechnology, 2020; Lapinaite & Knott et al. Science, 2020). ABE8e behaves as two beads on a string: Cas9-gRNA locates a target site, while the deaminase domain catalyzes the deamination reaction. However, the activity of the deaminase domain is Cas9 independent – it is able to deaminate any single stranded DNA. ABE8e has high probability to perform unintended deamination. Thus, we are interested in designing DNA base editors with enhanced specificity where the catalytic domain is an integral part of Cas9 and is turned on only when Cas9 is bound to the correct target.