Emine Kaya

Bio: Emine Kaya is an academic researcher from Ludwig Maximilian University of Munich. The author has contributed to research in topics: Amino acid & Click chemistry. The author has an hindex of 8, co-authored 9 publications receiving 1288 citations. Previous affiliations of Emine Kaya include Protein Sciences & University of California, Berkeley.

Structures of Cas9 Endonucleases Reveal RNA- Mediated Conformational Activation

Abstract: Type II CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated) systems use an RNA-guided DNA endonuclease, Cas9, to generate double-strand breaks in invasive DNA during an adaptive bacterial immune response. Cas9 has been harnessed as a powerful tool for genome editing and gene regulation in many eukaryotic organisms. We report 2.6 and 2.2 angstrom resolution crystal structures of two major Cas9 enzyme subtypes, revealing the structural core shared by all Cas9 family members. The architectures of Cas9 enzymes define nucleic acid binding clefts, and single-particle electron microscopy reconstructions show that the two structural lobes harboring these clefts undergo guide RNA-induced reorientation to form a central channel where DNA substrates are bound. The observation that extensive structural rearrangements occur before target DNA duplex binding implicates guide RNA loading as a key step in Cas9 activation.

A genetically encoded norbornene amino acid for the mild and selective modification of proteins in a copper-free click reaction.

Abstract: Methods for the site-specific chemical modification of proteins are currently of immense importance for the synthesis of protein–hybrid compounds for pharmaceutical and diagnostic purposes. Most of the methods rely on the reaction of free protein thiols with maleimides or the reaction of lysine side chains with activated esters. These methods provide only limited specificity, which is prompting researchers to develop alternative strategies that involve the incorporation of special unnatural amino acid into proteins to enable site-specific bioorthogonal functionalization. Among the developed methods, the Cu-catalyzed reaction of a protein containing an alkyne amino acid with azides stands out as the most thoroughly investigated technology. 6] However, the need for Cu salts, which may harm the protein structure, limits the technology. This fuels current interest to develop copperfree coupling reactions that are compatible with fragile protein structures. Here we show that these requirments can be met with a specially encoded norbornene amino acid which reacts selectively with nitrile imines. In order to insert a norbornene amino acid into a protein we used the amber suppression technique based on the pyrrolysyl tRNA/pyrrolysyl-tRNA synthetase (tRNA/ PylRS) pair from Methanosarcina mazei. The main task of the project was to evolve the pyrrolysine synthetase so that it accepts the synthetic norbornene amino acid 1 (Scheme 1 a) for loading onto the pyrrolysyl-tRNA. For the study we synthesized the norbornene-containing Pyl analogue 1 in seven steps from readily available starting materials (see the Supporting Information). To test to what extent 1 is accepted by wild-type (wt) PylRS, we used E. coli cells encoding the full tRNA/PylRS pair and a modified yellow fluorescent protein (YFP) containing one in-frame TAG stop codon. In this system the full-length and hence fluorescent YFP can only be produced when the corresponding Pyl analogue is accepted by the PylRS and successfully loaded onto the tRNA for subsequent incorporation into the protein at the amber stop codon site. The so-prepared E. coli cells were grown in a medium containing 5 mm 1. In addition to the wild-type PylRS we also tested a PylRS mutant (Y384F) previously used by Yanagisawa and co-workers. These initial experiments provided a just faint fluorescence when the mutant PylRS(Y384F) was used. No flurescence and hence no full-length YFP was generated in the presence of wild-type PylRS. In order to increase the PylRS activity we evolved the protein using iterative saturation mutagenesis (ISM) developed by Reetz and co-workers. Based on the co-crystal structure of PylRS in complex with adenylated pyrrolysine (PDB 2Q7H) we selected five residues in the substrate-binding pocket of wt-PylRS for the experiments. After transformation of the plasmid-based PylRS library into E. coli, single colonies were grown in liquid cultures supplemented with 1. The incorporation was monitored by means of the YFP fluorescence intensity of the cells. The most efficient PylRS variants were then sequenced and used in the next round of the saturation mutagenesis. In Scheme 1. a) Structure of norbornene Pyl analogue 1 and b) schematic representation of the click reactions. Top: A nitrile imine is generated by base-promoted HCl elimination from the hydrazonoyl chloride and then used in a cycloaddition reaction with the norbornene. Middle: Alternatively the nitrile imine is generated from a tetrazole in a photochemical reaction. Bottom: The norbornene modification can also react with tetrazines in a reversed-electron-demand Diels–Alder reaction. (Protein representations generated from PDB 3IN5.)

RNA-dependent RNA targeting by CRISPR-Cas9

Abstract: Double-stranded DNA (dsDNA) binding and cleavage by Cas9 is a hallmark of type II CRISPR-Cas bacterial adaptive immunity. All known Cas9 enzymes are thought to recognize DNA exclusively as a natural substrate, providing protection against DNA phage and plasmids. Here, we show that Cas9 enzymes from both subtypes II-A and II-C can recognize and cleave single-stranded RNA (ssRNA) by an RNA-guided mechanism that is independent of a protospacer-adjacent motif (PAM) sequence in the target RNA. RNA-guided RNA cleavage is programmable and site-specific, and we find that this activity can be exploited to reduce infection by single-stranded RNA phage in vivo. We also demonstrate that Cas9 can direct PAM-independent repression of gene expression in bacteria. These results indicate that a subset of Cas9 enzymes have the ability to act on both DNA and RNA target sequences, and suggest the potential for use in programmable RNA targeting applications.

The archaeal cofactor F0 is a light-harvesting antenna chromophore in eukaryotes

Abstract: Archae possess unique biochemical systems quite distinct from the pathways present in eukaryotes and eubacteria. 7,8-Dimethyl-8-hydroxy-5deazaflavin (F(0)) and F(420) are unique deazaflavin-containing coenzyme and methanogenic signature molecules, essential for a variety of biochemical transformations associated with methane biosynthesis and light-dependent DNA repair. The deazaflavin cofactor system functions during methane biosynthesis as a low-potential hydrid shuttle F(420)/F(420)H(2). In DNA photolyase repair proteins, the deazaflavin cofactor is in the deprotonated state active as a light-collecting energy transfer pigment. As such, it converts blue sunlight into energy used by the proteins to drive an essential repair process. Analysis of a eukaryotic (6-4) DNA photolyase from Drosophila melanogaster revealed a binding pocket, which tightly binds F(0). Residues in the pocket activate the cofactor by deprotonation so that light absorption and energy transfer are switched on. The crystal structure of F(0) in complex with the D. melanogaster protein shows the atomic details of F(0) binding and activation, allowing characterization of the residues involved in F(0) activation. The results show that the F(0)/F(420) coenzyme system, so far believed to be strictly limited to the archael kingdom of life, is far more widespread than anticipated. Analysis of a D. melanogaster extract and of a DNA photolyase from the primitive eukaryote Ostreococcus tauri provided direct proof for the presence of the F(0) cofactor also in higher eukaryotes.

Synthesis of Threefold Glycosylated Proteins using Click Chemistry and Genetically Encoded Unnatural Amino Acids

Abstract: Eukaryotic proteins and in particular cell-surface proteins are frequently glycosylated, which has fueled the interest of chemist to develop new methods for the synthesis of glycosylated proteins. Protein glycosylation is essential for the proper function of the respective proteins and in the case of glycosylated protein therapeutics, such as erythropoietin, the activity of the protein strongly depends on glycosylation patterns. The synthesis and production of glycosylated proteins to either elucidate their biochemical function or to bring a new therapeutic to the market is a formidable challenge. The current most widely used methods for the production of glycosylated proteins involve over-expression of the protein in particular host systems, such as mammalian or plant cell cultures, moss cultures or tobacco plants, to name a few, that are able to make either partial or even fully glycosylated proteins. Alternatively, solid-phase synthesis of glycosylated peptides and their insertion into the protein in question by native or expressed chemical ligation has emerged as a powerful chemical strategy. We thought that one possibility to produce glycosylated proteins would be to cotranslationally insert unnatural alkyne or alkene amino acids into proteins. These amino acids enable site-specific protein glycosylation by Cu-free or Cu-catalyzed click chemistry methods, for example, with azide modified sugar moieties as reaction partners for the alkynes. The linkage between the protein and the sugar units would be an artificial triazole instead of the naturally occurring O,Oor O,N-acetals (sugar connection via Ser/Thr and Asn, respectively). However, this rather small change of the linker unit is thought to keep the biochemical properties of the glycosylated protein unchanged. The problem associated with a strategy like this is that in the literature mainly methods for the incorporation of a single artificial amino acid into a protein by wellestablished techniques are reported, while glycosylated proteins often possess multiple glycosylation sites. For in vitro incorporation of two different amino acids see refs. [40]–[41] . Here we report the in vivo incorporation of up to three artificial alkyne or alkene amino acids into the yellow fluorescent protein (YFP) and the subsequent glycosylation of these sites with different sugar azides using the Huisgen–Meldal–Sharpless click chemistry method, which was used in our group for the synthesis of multiple sugar modified oligonucleotides. Incorporation of unnatural, mostly aromatic amino acids can be efficiently achieved through the amber suppression technique with the help of a specially evolved, orthogonal tyrosyl-tRNA synthetase/tRNACUA pair. [43] Alternatively, the amber suppression with different Methanosarcina MS pyrrolysyl-tRNA synthetase/tRNACUA pair was reported. [44–52] This particular method was used by Yokoyama et al. and Chin et al. to insert one click site into proteins. 50] Here we show that the system Methanosarcina mazei (Mm) MS pyrrolysyl-tRNA synthetase/tRNACUA pair has the potential to read through more than just one amber stop codon present in the messenger RNA to achieve insertion of up to three artificial alkyne or alkene amino acids. We experimented with various protected Lys derivatives and found that the propargyl-protected Lys derivative 1, also used by Chin, and the commercially available allocLys derivative 2 are efficient substrates for the pyrrolysyl-tRNA synthetase. 53] In order to incorporate them into a protein and to set up an assay that would report efficient stop-codon suppression we transformed E. coli with a vector containing the genes for the M. mazei pyrrolysyl-tRNA synthetase, the M. mazei tRNACUA, a C-terminal StrepII-tag modified YFP with initially one later three amber TAG stop codons at positions 27, 114, and 132 as well as an ampicilline resistance gene. We added the alkyne amino acid 1 and the alkene amino acid 2 to the medium containing the transformed growing E. coli cells. As depicted in Figure 1, the E. coli cells containing the eYFP (Figure 1 A) are strongly fluorescent showing that the YFP protein is efficiently produced by the bacteria. E. coli cells containing the YFP protein with one TAG stop codon in the absence of the alkyne or alkene amino acids 1 or 2 are not fluorescent (Figure 1 B); this shows that in this case the full-length protein cannot be produced. Figure 1 C and D show E. coli cells containing the YFP protein with one and three TAG stop codons, respectively, in the presence of the alkyne amino acid 1. Figure 1 E and F are the corresponding pictures in the presence of the alkene amino acid 2. Here fluorescent cells are clearly detected showing that the stop codons are suppressed in the presence of the artificial amino acids in the medium. The fluorescence in Figure 1 D is clearly weaker; this indicates that although suppression of three stop codons is possible, the total suppression efficiency decreases. In order to quantify the protein yield, we isolated the full-length mutant YFP proteins with the C-terminal StrepII-tag from E. coli cultures (1 L) using a two-step purification procedure (Strep-tag affinity chromatography and anion exchange chromatography). The yield was determined to be about 0.3 mg for the YFP proteins comprising one alkyne or alkene amino acid (1 mut). For comparison we also quantified [a] E. Kaya, K. Gutsmiedl, Dr. M. Vrabel, Dr. M. M ller, P. Thumbs, Prof. Dr. T. Carell Center for Integrated Protein Science (CiPS) Department of Chemistry and Biochemistry, LMU Munich Butenandtstrasse 5–13, 81377 Munich (Germany) Fax: (+ 49) 89-2180-77756 E-mail : thomas.carell@cup.uni-muenchen.de [] These authors contributed equally to this work. Supporting information for this article is available on the WWW under http ://dx.doi.org/10.1002/cbic.200900625.

“Bioinformatics” 특집을 내면서

Abstract: BIOE 402. Medical Technology Assessment. 2 or 3 hours. Bioentrepreneur course. Assessment of medical technology in the context of commercialization. Objectives, competition, market share, funding, pricing, manufacturing, growth, and intellectual property; many issues unique to biomedical products. Course Information: 2 undergraduate hours. 3 graduate hours. Prerequisite(s): Junior standing or above and consent of the instructor.

The new frontier of genome engineering with CRISPR-Cas9

Abstract: The advent of facile genome engineering using the bacterial RNA-guided CRISPR-Cas9 system in animals and plants is transforming biology. We review the history of CRISPR (clustered regularly interspaced palindromic repeat) biology from its initial discovery through the elucidation of the CRISPR-Cas9 enzyme mechanism, which has set the stage for remarkable developments using this technology to modify, regulate, or mark genomic loci in a wide variety of cells and organisms from all three domains of life. These results highlight a new era in which genomic manipulation is no longer a bottleneck to experiments, paving the way toward fundamental discoveries in biology, with applications in all branches of biotechnology, as well as strategies for human therapeutics.

Development and Applications of CRISPR-Cas9 for Genome Engineering

Abstract: Recent advances in genome engineering technologies based on the CRISPR-associated RNA-guided endonuclease Cas9 are enabling the systematic interrogation of mammalian genome function. Analogous to the search function in modern word processors, Cas9 can be guided to specific locations within complex genomes by a short RNA search string. Using this system, DNA sequences within the endogenous genome and their functional outputs are now easily edited or modulated in virtually any organism of choice. Cas9-mediated genetic perturbation is simple and scalable, empowering researchers to elucidate the functional organization of the genome at the systems level and establish causal linkages between genetic variations and biological phenotypes. In this Review, we describe the development and applications of Cas9 for a variety of research or translational applications while highlighting challenges as well as future directions. Derived from a remarkable microbial defense system, Cas9 is driving innovative applications from basic biology to biotechnology and medicine.

Optimized sgRNA design to maximize activity and minimize off-target effects of CRISPR-Cas9

Abstract: CRISPR-Cas9-based genetic screens are a powerful new tool in biology. By simply altering the sequence of the single-guide RNA (sgRNA), one can reprogram Cas9 to target different sites in the genome with relative ease, but the on-target activity and off-target effects of individual sgRNAs can vary widely. Here, we use recently devised sgRNA design rules to create human and mouse genome-wide libraries, perform positive and negative selection screens and observe that the use of these rules produced improved results. Additionally, we profile the off-target activity of thousands of sgRNAs and develop a metric to predict off-target sites. We incorporate these findings from large-scale, empirical data to improve our computational design rules and create optimized sgRNA libraries that maximize on-target activity and minimize off-target effects to enable more effective and efficient genetic screens and genome engineering.