Adaptation is a landmark property of living cells. Cells have evolved sophisticated regulatory networks to selectively switch their genes on or off depending on the environment. Such regulation allows them to robustly adapt their metabolism and survive in different environments. Failures in metabolic regulation can lead to disease, and regulation can also be exploited by pathogens to survive drug treatments.
While some metabolic responses are encoded in the architecture of a regulatory network, others are the result of evolutionary fine-tuning of the parameters governing molecular interactions. Little is known about the interplay between architecture and parameter fine-tuning, yet this knowledge could allow us to understand metabolic responses in broader contexts, including the emergence of antibiotic resistance, abnormal metabolism in disease, and the detection of weak spots for future therapeutics to disrupt the metabolism of pathogens.
We propose to combine control theory and synthetic biology to single out the genetic mechanisms that control metabolic adaptations. We will integrate theory and experiment to predict, measure and alter the metabolic responses of microbes to environmental shocks. Our focus will be the fatty acid biosynthetic pathway in Escherichia coli, which is a promising antibiotic target and is amenable to genetic manipulations. We will use these genetic manipulations to rewire the native regulation and systematically explore the roles of network architecture and parameters. Together we aim to gain a systemic grasp of the overlapping mechanisms that control metabolic adaptations, establishing the mathematical and experimental groundwork to quantitatively understand other complex regulatory networks found in nature.

The reflection superposition compound eye, found in decapod crustaceans represents a spectacularly complex natural optical device based on biominerals. The eye contains a multilayer reflector in the distal part of the eye to focus light onto the retina and a reflecting tapetum layer which increases light sensitivity. Using an array of physical techniques, including the use of synchrotron radiation we will determine the chemical identity of photonic crystals isolated from the eyes of the crayfish C. quadricarinatus and the prawn M. rosenbergii. We recently determined that magnesium lactate dihydrate, a previously unknown biomineral is present in the reflector material. Using cryo-SEM, we will determine the ultrastructural arrangement of photonic crystals in the distal and tapetal layers under life-like conditions. We will relate the crystal structure, crystal morphology and eye ultrastructural properties to understand better the overall optical function of the eye. In collaboration with Prof. Amir Sagi (Ben-Gurion University), we will study the mechanism of crystal formation by observing changes in the distal reflecting layer in the eye of M. rosenbergii during development from larvae to adult. By studying gene expression during ontogeny a list of 'candidate genes' will be drawn up and their possible role in the biomineralization pathway will be explored by a series of gene silencing experiments. A deeper understanding of the reflector materials in the eye could yield applications in material optics and uncover new paradigms in biologically controlled crystal formation which will have a wide impact in the fields of biomineralization, materials science and pharmacy.

Immune-mediated diseases, including inflammatory bowel disease (IBD), spread rapidly around the globe on the heels of the transition to modern lifestyles. One predominate factor driving this epidemic is the decline in diversity of the human microbiome as a result of our highly hygienic lifestyles. The human gut harbors vast communities of bacteria, protists, helminths and other microbes that have co-evolved with humans over millions of years. Absence of exposure to helminths and altered bacterial communities in the gut (called dysbiosis) are both strong risk factors for immune-mediated disease development. Protists are poorly studied, but likely also important for human health. Humans ancestrally harbored diverse protists communities, and today healthy people are more likely to harbor diverse protists, especially Blastocystis, than those with IBD. The underlying mechanism remains unknown, but likely involves changes in bacterial community composition. Intriguingly, re-introducing helminths or correcting bacterial dysbiosis are both promising disease treatments; animal studies and small trials report successful outcomes in upwards of 60% of cases. However, subsequent unsuccessful clinical trials and reports that helminth therapy can exacerbate pathogenic infections dampen enthusiasm and point to a fundamental gap in our knowledge of how helminths impact the gut ecosystem and how interactions with the other gut microbes such as Blastocystis might affect treatment outcomes. Here, we propose to revisit helminth therapy from an ecological perspective to ask what role the dynamics in the overall gut communities play in the success or failure of these treatments. We will conduct a series of experiments in a rat model system to address two central aims: (i) to characterize the dynamics of the host immune response and the microbiome in the face of disturbance caused by induced colitis and the introduction of the helminth Hymenolepis diminuta and/or Blastocystis ST1, and (ii) disentangle therapeutic impact arising from direct interaction of helminths with host from impacts mediated by shifts in microbiota. We hypothesize that shifting the microbiota to a new stable state may be key to long-term treatment success and believe our complementary expertise uniquely enables us to address this complex research question.

It has generally been assumed that a small number of molecules is sufficient to explain most if not all aspects of early development. This idea has been challenged by our recent discovery that toddler/apela encodes a novel, highly conserved secreted peptide with essential functions during embryogenesis. Genome-wide technologies have allowed us to identify hundreds of other uncharacterized peptides and translated short open reading frames (ORFs) that are expressed during embryogenesis.

These recent findings pose two fundamental questions: (1) What are the functions of these newly discovered small peptides? Small peptides are particularly promising candidates because they are likely to have been missed in prior random mutagenesis screens. (2) What is the role of the expanded universe of translated ORFs? Rather than encoding functional peptides, translation could be neutral, or it could be the translation process itself that serves a function.

The goal of the proposed research is to identify functions for novel peptides and short translated ORFs during early development, using zebrafish as a model system. Aim 1 will investigate the molecular and cellular mechanism of Toddler/Apelin receptor signaling. Aim 2 will investigate the functions of other novel peptides and aim at unraveling regulatory functions for short translated ORFs during developmental transitions.

These studies will identify the roles and mechanisms of novel bioactive peptides in early development. The proposed research has the potential to identify molecules with possible applications in tissue repair and in vitro differentiation, and could uncover novel principles of translational regulation.

Glioblastoma multiforme (GBM), the most prevalent form of primary brain tumors, severely debilitates patients by infiltrating and destroying the brain parenchyma. The molecular mechanisms governing GBM invasion are poorly understood, rendering GBM incurable. Previous work has revealed that 30-50% of GBM tumors overexpress a distinct mesenchymal-like gene signature, which is further enriched in GBM undergoing recurrence, raising the question whether specific mesenchymal genes regulate GBM progression. We propose to tackle this by implementing a three-dimensional (3D) organotypic brain tissue model for the dynamic study of GBM invasion ex vivo. Using glioblastoma neural stem-like (GNS) cell populations, we will perform loss-of-function complemented with gain-of-function genetic screens to identify mesenchymal genes critical for GNS cell invasiveness and GBM tumor progression. To molecularly dissect cell-intrinsic networks in GNS invasion, we will conduct epistasis experiments with pro-invasive genes and downstream pathway effectors. We will also examine cell-extrinsic effects of pro-invasive genes on the neural microenvironment. Potential paracrine feedback by microenvironment-secreted factors, deregulated by invading GNS cells, will be tested in antibody-based blocking experiments and validated using brain tissue from mice genetically inactivated for targeted factors. Finally, the prognostic power of pro-invasive mesenchymal genes in predicting GBM patient outcomes will be assessed. The findings from this proposal, if successful, should provide a molecular handle on GBM invasion by implicating specific mesenchymal genes as key therapeutic targets.

Obesity is a serious medical problem that poses an enormous public health burden. Understanding how the brain integrate and control satiety and hunger signals are vital for unraveling the causes of obesity. Many brain regions are known to regulate feeding behavior including the hippocampus, a brain region usually associated with memory. However, while some studies show a role for the hippocampus to control food intake, the identity of the specific cells that modulate this behavior are not known. I propose to identify and profile the hippocampal cells that are activated during a feeding paradigm in mice. To gain greater insight on how the hippocampus regulate feeding in mice, I will initially focus on the subiculum which was found recently to send projections to the nucleus accumbens (NAc). This will be accomplished using two new profiling technologies that allow the identification of specific molecular markers of neural populations based on changes in their activity (PhosphoTrap) or based on their pattern of projections (RetroTrap). Once these specific cells are identified, I will modulate the activity of these cells using optogenetics, DREADDs (designer receptors exclusively activated by designer drugs) or radiowaves and assay possible effects on feeding behavior in wild-type and leptin-deficient obese (ob/ob) mice. I will also use electrophysiology assays to monitor the effects of activating or inhibiting subiculum cells on the activity of neurons in the nucleus accumbens. This project will generate new tools, provide novel evidence on how the hippocampus controls feeding behavior and potentially identify new targets for genetic manipulation and drug design.

Theory predicts that when a system is close to a catastrophic transition it will take unusually long times to recover from small perturbations. This effect, called critical slowing down, may predict the impending
catastrophe better than other indicators. For example, it indicates when an ecosystem is near to its collapse, while other parameters such as population sizes may only show the collapse when it is already occurring.

We propose to use critical slowing down to predict the death of an animal. The theory is powerful enough to address this question but has never been applied to individual animals, which involve new challenges such as inter-individual variability, restricted access to the state variables and highly interconnected and heterogeneous subsystems.

We will test our predictions in the nematode Caenorhabditis elegans, which is experimentally tractable yet at the same time is a complex multicellular organism with distinct organs and systems. We will focus on osmoregulation, as perturbations are easy to apply, the effects of osmotic shocks are easy to measure, and a sufficiently severe osmotic shock will induce death. We predict that quantitative measurements of osmoregulation in individual animals will reveal an increase in time to recover from osmotic shocks as the animal approaches death, regardless of whether the death is caused by osmotic stress or other reasons, such as heat stress or even aging. We will try to show not only that the effect exists on average across replicates, but also that it has diagnostic power on individual animals, identifying those individuals less capable of withstanding a given condition.

Understanding tissue morphogenesis and function is of fundamental importance in developmental biology. Resolving the underlying mechanisms relies on tracking dynamic cellular behavior in toto and requires new microscopes able to image the developing embryo over a range of spatiotemporal domains. Selective plane illumination microscopy (SPIM) represents the state of the art in time-lapse imaging. However, optical penetration is fundamentally limited in turbid media and multi-view imaging unnecessarily exposes the sample and yields large datasets that are often sparse in information content. Typically, the ballistic regime defines the superficial region available to conventional optical imaging. This limitation will be overcome via a range of imaging modalities available in parallel. Self-healing beams and multi-photon excitation with adaptive wavefront correction will allow even deep tissues to be imaged without loss of contrast. New transgenic lines will be developed for multi-color infrared imaging of cardiac morphogenesis to produce 4D biophysical models of the heart. Quantitative analysis performed in real time will allow highly selective imaging of evolving regions of interest thus minimizing sample exposure and maximizing sample throughput. This smart SPIM will be used to map and image not only vascularization but also earlier developmental events such as germ layer coordination during gastrulation with isotropic resolution. I envisage these strategies will provide the basis for the next generation of smart SPIM and allow real-time system level study of zebrafish embryogenesis.

Cell migration is a fundamental processes of life, yet also one of the molecularly most complex ones. The majority of mechanistic insight of cell migration stems from observations on coverslips. While efforts are underway to observe cell migration in entire organisms, from fruit flies to mice, the molecular detail and statistical power that can be achieved are limited. Instead, I propose here to use ex vivo, but physiologically relevant samples of human cell migration that are amenable to systematic and quantitative high-resolution imaging. I hypothesize that some modes of migration in 3D share mechanical similarities with 2D migration, whereas other modes will exhibit completely novel mechanisms. To test this, I will first identify a finite set of migration modes as a function of the mechanical properties of the external cellular matrix and correlate them with the dynamics of actomyosin organization, cell cortex contractility, and adhesion patterns. To measure actomyosin dynamics I will adapt an imaging modality known as fluorescent speckle microscopy to high-resolution light-sheet imaging. I will be trained by, and in constant collaboration with, colleagues in the Danuser lab at UT Southwestern who are experts in fluorescent probe design and optical engineering. I will contribute to this team with my expertise in image signal processing and computer vision. Using speckle microscopy we will then determine the dynamic interactions between actin filaments and myosin motors, map out spatial organization of micro-strains in the cortex, and relate these parameters to the formation of cell adhesions, cell protrusions and retractions, and the efficiency of whole cell migration.

Satellite cells (SCs) are stem cells required for growth, maintenance, and regeneration of skeletal muscle. In response to molecular triggers from injuries or disease, subsets of SCs activate and have the ability to self renew, proliferate as myoblasts, or undergo myogenic differentiation to regenerate damaged tissue. Central to the specification and function of SCs is the PAX3 transcription factor, which activates factors that mediate migration of muscle progenitor cells during development. In adult SCs however, PAX3 expression is down-regulated in all but a few muscle groups, such as the diaphragm muscle. Pax3 expression is controlled via alternative polyadenylation (APA) of its 3'untranslated region (3'UTR), which alters the ability of microRNA-206 (miR-206) to bind and knock-down Pax3 transcripts. Recently, it was discovered that the poly-A binding protein nuclear 1 (PABPN1) regulates the length of 3’UTRs in human cells by suppressing APA. Moreover, mutations in PABPN1 are linked to the late onset myopathy, oculopharyngeal muscular dystrophy (OPMD). Here, we hypothesize that PABPN1 mediates Pax3 3’UTR alternative polyadenylation. Transient expression of PAX3 is associated with SC activation and corresponds with a proliferative state of myoblasts. Thus, PABPN1 may also regulate SC function. I will first assess the role of PABPN1 in Pax3 regulation and satellite cell proliferation ex vivo. I will then determine the binding profile of PABPN1 to Pax3 pre-mRNA in satellite cells from different muscle groups. Finally, I will investigate the affect of PABPN1 knockout or mutation in mouse models of satellite cell activation.

In order to maintain the integrity of the proteome, eukaryotic cells have evolved a complex array of spatial protein quality control (PQC) mechanisms that recognize misfolded proteins and triage them for refolding, degradation or--if these processes fail--sequestration. Soluble misfolded proteins in yeast are rapidly sequestered in dynamic punctate inclusions called Q-bodies; these coalesce into larger structures, where the misfolded proteins are degraded by the ubiquitin-proteasome system or targeted to a juxtanuclear quality control compartment. By contrast, a separate pathway--involving permanent sequestration into a peripheral insoluble protein deposit--seems to exist for proteins that cannot be degraded, such as disease-linked amyloid proteins. To date, studies have described spatial PQC using conventional optical microscopy, thereby providing limited information as to the ultrastructural basis of this system. I will use correlative light-and-electron microscopy and super-resolution fluorescent microscopy to pinpoint the location of fluorescently-tagged misfolded proteins at various stages of spatial PQC with respect to the subcellular architecture in mammalian cells. Combining this approach with cutting-edge proteomic tools that allow analysis of specific PQC compartments, together with various genetic, biochemical and cell biological methods of modulating protein function, will provide unprecedented mechanistic detail into the role of various PQC machineries in these pathways. My integrated approach should considerably advance our understanding of misfolded protein triage in mammalian cells, both under physiological conditions and in response to cellular stresses.

Our ability to identify disease-associated genomic alterations by next generation sequencing and to genetically model those alterations using novel genome editing tools will revolutionize biomedical research. Studying phenotypic extremes and the role of rare variants in human disease such as cancer will provide us with key insights into basic biology and might even unravel novel therapeutic strategies. Cancer sequencing projects are currently uncovering hundreds of genes associated with cancer, which show a classic long tail distribution revealing the enormous heterogeneity not only between different cancers but even within a tumor itself. Mining this information for therapies and personalized cancer medicine is predicated on weeding out ‘bystander’ mutation and identifying ‘driver’ mutations and molecular pathways responsible for tumor initiation, progression and metastasis. However, the complex nature of cancer involving multiple genes, several cell types and stromal cues necessitates the use of model organisms to faithfully recapitulate physiological and pathological processes, which has impeded a systematic and comprehensive characterization of rare variants and the search for synthetic lethal interactions. I propose to develop direct in vivo genome editing technologies to specifically modify and assess function of hundreds of putative cancer genes within intact organs of a living mouse in a mosaic fashion. By integrating human cancer genomics and mouse modeling with in vivo screening, I hope to (1) rapidly discover and functionally characterize potent but low penetrance cancer driver mutations and to (2) elucidate novel cancer vulnerabilities.

Traits can be inherited in a non-Mendelian fashion by a variety of mechanisms including transgenerational epigenetic inheritance. Previous research has shown that mutation of a C. elegans histone H3 lysine 4 dimethyl (H3K4me2) demethylase, SPR-5, causes progressive sterility and extended longevity (Katz DJ et al Cell 2009, Greer EL et al Cell Reports 2014, Greer EL et al unpublished). Previous work has shown that the H3K4me2 mark and a novel DNA modification increase in successive generations of worms lacking spr-5. However, how this non-genetic information is transmitted from one generation to the next is still unknown. The goal of this proposal is to identify the mode and mechanism of epigenetic transgenerational inheritance that leads to progressive generational sterility in C. elegans with mutated spr-5. I hypothesize that dysregulation of SPR-5 causes an increase in H3K4me2 which is “remembered” from generation to generation by an epigenetic memory of DNA methylation. My goal is to identify whether H3K4me2 or this DNA methylation event are transmitted from one generation to the next in instances of transgenerational epigenetic inheritance and to identify regulators of DNA methylation. Understanding how epigenetic inheritance is dysregulated in spr-5 mutant worms will enhance our understanding of how epigenetic signals are propagated from generation to generation. In the long term, I will determine whether these moieties are conserved and whether they play a conserved role in regulating transgenerational epigenetic processes in mammals.

One question standing upfront in the developmental biology field is how the developmental transition from unipotency to totipotency takes place post fertilization. It is believed, that totipotency results from extensive epigenetic reprogramming of the germline state into an embryonic state. My host lab and others have recently highlighted that both the highly asymmetric mature gametes contributes to this developmental reprogramming and hence the zygotic genome activation (ZGA). Genes specifically expressed at the onset of the ZGA, factors controlling their activation and the parental contribution of such regulatory factors remain unknown. To achieve this, we propose to perform a computational analyses combined with in-vivo functional characterization. Our preliminary analysis on published data sets, by filtering and measuring the intronic transcripts in early embryos, have already gave us a list of early transcribed genes and the transcription factors (TFs) potentially determining the regulatory states of the cells. Furthermore, I propose to investigate the influence of germline inheritance of chromatin states on ZGA, by generating expression data from genetically hybrid embryos enabling the distinction between parental genomes. This will provide an unprecedented insight into the parental contribution of selected ZGA regulators. Subsequently, I will perform a functional characterization of these potential ZGA regulators by applying loss- and gain-of- function studies in early embryos. I will extend my studies and use CRISPR/Cas system to generate transgenic mouse for further in-depth studies.

Extracellular vesicles (EVs) are found in all body fluids and play an important role in cell-cell communication by being able to fuse with target cells while carrying and transferring various signaling molecules. They are also known to be involved in several types of disease. Several studies suggest that the mechanical properties of particles play a major role in their cellular uptake. However, a systematic investigation of this effect is still lacking. Hence, I propose to test the hypothesis that mechanical properties of EVs affect the efficiency and kinetics of their uptake by cells. To this end, I will prepare synthetic lipid vesicles (SVs) with varying stiffness by incorporating a crosslinked internal polymeric shell and characterize their mechanical strength. Next, I will measure the forces involved in the adhesion and possibly internalization of the vesicles by cultured cells. I will do this using advanced nanomanipulation techniques such as Atomic Force Microscopy (AFM) and Acoustic Force Spectroscopy (AFS), combined with fluorescence imaging. I will test whether lipid mixing and content mixing between the vesicle bilayer and the cell membrane take place by double fluorescent labeling of the aqueous core and the lipid membrane. A better understanding of the link between SVs mechanical properties and uptake rate and mechanism will provide insight into fundamental biological processes mediated by vesicles, e.g the release of neurotransmitters in synapses, viral infections etc. In addition, my findings will facilitate the design of efficient synthetic exosomes for future therapeutic solutions.

During the last decades, signaling pathways responsible for the initiation of gastrulation in mammalian embryos have been identified. However, the physical rules governing the tissue spatial patterning and the extensive morphogenetic movements occurring during that process are still elusive. We have recently shown using micro-patterned adhesive substrates, that geometric confinement is sufficient to trigger self-organized patterning in hESCs and that this intrinsic tendency of ESCs to make patterns can be harnessed to provides a quantitative assay to study early developmental patterning and differentiation. By taking advantage the micro-fabrication and the micro-fluidics toolboxes, we propose here to study how the physical (stiffness of the substrate, size and shape of the tissue) and chemical (spatio-temporal profile of differentiating signals) properties of the cells micro-environment are affecting the final pattern, in order to shed light on the complex morphogenetic processes occurring during mammalian gastrulation.

Shelterin, a complex of proteins that recognizes repetitive DNA sequences present in the telomeres, efficiently acts as a suppressor of chromosomal fusions and in turn promote cellular genomic integrity. In addition to telomeres, regions of highly repetitive DNA sequences – often involving non-B DNA secondary structural conformations – can be found throughout the genome. These regions are often involved in various genomic rearrangements involving insertion, deletion or inter-chromosomal recombination. The mechanism by which such deleterious events occur is still poorly understood. We hypothesize that in addition to its estabilished role at telomeres, shelterin binds non-telomeric repetitive elements and blocks recombination initiating from these sites. The proposed project aims to analyze the distribution of key shelterin components, such as TRF2, in the repetitive-rich regions genome-wide as well as its specific accumulation at DNA damaged sites. Moreover, specialized and aberrant recombination events, which occur at the repetitive DNA regions in vivo will be assessed by utilizing TRF2-ablated mouse model systems. Finally, to understand the role of shelterin complex at the recombinogenic-prone sites, we will compare the TRF2-associated protein complex in telomere and other repetitive regions that are susceptible to genomic rearrangements. These approaches will enable us to learn ways in which genome stability is preserved. The completion of the project will provide a deeper understanding of diseases associated with trinucleotide repeat instability, as well as various lymphoma-derived cancers.

Many cortical areas cooperate to produce visually-guided behaviors but the way this cooperation happens is not well understood. Specifically, I will address the competing hypotheses of whether cortical areas interact in a “serial” versus a “distributed” fashion; that is, whether each area performs a distinct processing step in turn or whether some steps are accomplished jointly across areas. To distinguish between these hypotheses, I propose to use novel microelectrodes to record simultaneously from six key cortical areas in the pathway from sensation to action: primary and anteromedial visual cortex; posterior parietal cortex; cingulate cortex; primary and secondary motor cortex. Recordings will be made while awake, head-fixed mice perform a task in which they use their forepaws to turn a wheel left or right to indicate the location of the higher-contrast of two visual stimuli presented on a screen. I will analyze the responses of populations of neurons recorded from each area to determine whether and when the following pieces of information can be decoded on single trials: the onset and contrast of each visual stimulus; the integrated evidence available to the animal to solve the task, i.e. the difference in contrast between the two stimuli; the choice of the animal; the timing of the onset and the duration of the wheel-turn response. The temporal evolution of these signals on single trials will reveal whether there is a serial processing chain versus a computation distributed across cortical areas, thus answering a fundamental question about the way cortical areas cooperate to perform visually-guided behaviors.

Stem cell niches, composed of cells or cells together with extracellular matrix, produce secreted or cell surface factors, such as Notch, Wnt, FGF, and EGF, to control stem cell renewal, maintenance, or survival through the activation of various signaling pathways. Deciphering the relationships between these pathways is critical to our understanding of the niche function. The Drosophila adult midgut has emerged as an attractive system to study adult stem cells. Although many conserved signaling pathways regulating Drosophila intestinal stem cells (ISCs) have been characterized, major gaps in our understanding are (1) whether their activation are sequential or simultaneous, and (2) how stem cells integrate these signals to change the program of gene expression during regeneration.

I propose to characterize how stem cells are regulated during regeneration. In Aim 1, Dynamic fluorescent Pathway (DFP) reporters will be used to visualize where and when signaling pathways are activated. DFP reporters will provide quantitative read-outs of pathway activities and help dissect the logic of signaling events. In Aim 2, I will use the Targeted DamID (TADA) method to profile the transcriptional changes following the activation of signaling pathways to decipher their crosstalk and integration. In Aim 3, I will perform a genome-wide RNAi screen to identify transcription factors modulating stem cell proliferation. Together, these studies will provide an unprecedented systems-level understanding of the signaling pathways involved in stem cell-niche interactions during the regenerative process.

Over 100 post-transcriptional RNA modifications are known with N6-methyladenosine (m6A) being the most frequent. m6A has been reported to play a role in many human biological processes (e.g. obesity and cancer), but the detailed mechanisms remain unknown. Detailed study of the function of m6A requires the site-specific introduction of m6A into specific RNAs. However, this appears difficult using endogenous m6A modification enzymes because only a few such enzymes modify a wide range of RNAs. Here, I propose a methodology for introducing site-specific m6A modification in vivo by engineering of the C/D small nucleolar ribonucleoprotein (snoRNP). The C/D snoRNP catalyzes the 2’-O methylation of a substrate RNA and consists of four proteins and a C/D snoRNA, which recognizes the substrate RNA by base pairing. Therefore, the substrate specificity of C/D snoRNP can be easily changed by altering the C/D snoRNA sequence. In this research, I will engineer the catalytic protein of C/D snoRNP to catalyze m6A modification using rational design and directed evolution. First, the catalytic core of C/D snoRNP will be redesigned based on the structure of known m6A modification enzymes. Then, the engineered C/D snoRNP will be randomly mutated to construct a library to perform directed evolution. After directed evolution, the activity and substrate specificity of the engineered C/D snoRNP will be determined. Finally m6A modifications of target RNAs will be altered to assess the biological response. A similar approach could also be applied to other RNA modifications and this technique has the potential to be a fundamental tool to control and study various RNA modifications.