Bleris, Leonidas

Permanent URI for this collectionhttps://hdl.handle.net/10735.1/2891

Leonidas Bleris is interested in the intersection of synthetic and systems biology, mathematics and control theory. He is the head of the Synthetic & Systems Biology Research Lab. Learn more about Dr. Bleris's research on his Research Explorer page

Browse

Now showing 1 - 9 of 9

Mapping the Operational Landscape of MicroRNAs in Synthetic Gene Circuits.
(Nature Partner Journals, 2018-10-22) Quarton, Tyler; Ehrhardt, Kristina; Lee, James; Kannan, Srijaa; Li, Yi; Ma, Lan; Bleris, Leonidas; Quarton, Tyler; Ehrhardt, Kristina; Lee, James; Kannan, Srijaa; Li, Yi; Ma, Lan; Bleris, Leonidas
MicroRNAs are a class of short, noncoding RNAs that are ubiquitous modulators of gene expression, with roles in development, homeostasis, and disease. Engineered microRNAs are now frequently used as regulatory modules in synthetic biology. Moreover, synthetic gene circuits equipped with engineered microRNA targets with perfect complementarity to endogenous microRNAs establish an interface with the endogenous milieu at the single-cell level. The function of engineered microRNAs and sensor systems is typically optimized through extensive trial-and-error. Here, using a combination of synthetic biology experimentation in human embryonic kidney cells and quantitative analysis, we investigate the relationship between input genetic template abundance, microRNA concentration, and output under microRNA control. We provide a framework that employs the complete operational landscape of a synthetic gene circuit and enables the stepwise development of mathematical models. We derive a phenomenological model that recapitulates experimentally observed nonlinearities and contains features that provide insight into the microRNA function at various abundances. Our work facilitates the characterization and engineering of multi-component genetic circuits and specifically points to new insights on the operation of microRNAs as mediators of endogenous information and regulators of gene expression in synthetic biology.
Exploiting the CRISPR/Cas9 PAM Constraint for Single-Nucleotide Resolution Interventions
(2016-01-20) Li, Yi; Mendiratta, Saurabh; Ehrhardt, Kristina; Kashyap, Neha; White, Michael A.; Bleris, Leonidas; 0000 0001 2535 9739 (Bleris, L)
CRISPR/Cas9 is an enabling RNA-guided technology for genome targeting and engineering. An acute DNA binding constraint of the Cas9 protein is the Protospacer Adjacent Motif (PAM). Here we demonstrate that the PAM requirement can be exploited to specifically target single-nucleotide heterozygous mutations while exerting no aberrant effects on the wildtype alleles. Specifically, we target the heterozygous G13A activating mutation of KRAS in colorectal cancer cells and we show reversal of drug resistance to a MEK small-molecule inhibitor. Our study introduces a new paradigm in genome editing and therapeutic targeting via the use of gRNA to guide Cas9 to a desired protospacer adjacent motif.
Mir-192-Mediated Positive Feedback Loop Controls the Robustness of Stress-Induced P53 Oscillations in Breast Cancer Cells
(Public Library of Science) Moore, Richard; Ooi, Hsu Kiang; Kang, Taek; Bleris, Leonidas; Ma, Lan; 55515673900 (Ma, L); Moore, Richard; Ooi, Hsu Kiang; Kang, Taek; Bleris, Leonidas; Ma, Lan
The p53 tumor suppressor protein plays a critical role in cellular stress and cancer prevention. A number of post-transcriptional regulators, termed microRNAs, are closely connected with the p53-mediated cellular networks. While the molecular interactions among p53 and microRNAs have emerged, a systems-level understanding of the regulatory mechanism and the role of microRNAs-forming feedback loops with the p53 core remains elusive. Here we have identified from literature that there exist three classes of microRNA-mediated feedback loops revolving around p53, all with the nature of positive feedback coincidentally. To explore the relationship between the cellular performance of p53 with the microRNA feedback pathways, we developed a mathematical model of the core p53-MDM2 module coupled with three microRNA-mediated positive feedback loops involving miR-192, miR-34a, and miR-29a. Simulations and bifurcation analysis in relationship to extrinsic noise reproduce the oscillatory behavior of p53 under DNA damage in single cells, and notably show that specific microRNA abrogation can disrupt the wild-type cellular phenotype when the ubiquitous cell-to-cell variability is taken into account. To assess these in silico results we conducted microRNA-perturbation experiments in MCF7 breast cancer cells. Time-lapse microscopy of cell-population behavior in response to DNA double-strand breaks, together with image classification of single-cell phenotypes across a population, confirmed that the cellular p53 oscillations are compromised after miR-192 perturbations, matching well with the model predictions. Our study via modeling in combination with quantitative experiments provides new evidence on the role of microRNA-mediated positive feedback loops in conferring robustness to the system performance of stress-induced response of p53.;
Discriminating Direct and Indirect Connectivities in Biological Networks
(National Academy of Sciences) Kang, Taek; Moore, Richard; Li, Yi; Sontag, Eduardo; Bleris, Leonidas; 0000 0001 2535 9739 (Bleris, L); Kang, Taek; Moore, Richard; Li, Yi; Bleris, Leonidas
Reverse engineering of biological pathways involves an iterative process between experiments, data processing, and theoretical analysis. Despite concurrent advances in quality and quantity of data as well as computing resources and algorithms, difficulties in deciphering direct and indirect network connections are prevalent. Here, we adopt the notions of abstraction, emulation, benchmarking, and validation in the context of discovering features specific to this family of connectivities. After subjecting benchmark synthetic circuits to perturbations, we inferred the network connections using a combination of nonparametric single-cell data resampling and modular response analysis. Intriguingly, we discovered that recovered weights of specific network edges undergo divergent shifts under differential perturbations, and that the particular behavior is markedly different between topologies. Our results point to a conceptual advance for reverse engineering beyond weight inference. Investigating topological changes under differential perturbations may address the longstanding problem of discriminating direct and indirect connectivities in biological networks.;
CRISPR-Based Self-Cleaving Mechanism for Controllable Gene Delivery in Human Cells
(Oxford University Press, 2014-12-18) Moore, Richard; Spinhirne, Alec; Lai, Michael J.; Preisser, Samantha; Li, Yi; Kang, Taek; Bleris, Leonidas; 0000 0001 2535 9739 (Bleris, L); 2012076942‏ (Bleris, L)
Controllable gene delivery via vector-based systems remains a formidable challenge in mammalian synthetic biology and a desirable asset in gene therapy applications. Here, we introduce a methodology to control the copies and residence time of a gene product delivered in host human cells but also selectively disrupt fragments of the delivery vehicle. A crucial element of the proposed system is the CRISPR protein Cas9. Upon delivery, Cas9 guided by a custom RNA sequence cleaves the delivery vector at strategically placed targets thereby inactivating a co-expressed gene of interest. Importantly, using experiments in human embryonic kidney cells, we show that specific parameters of the system can be adjusted to fine-tune the delivery properties. We envision future applications in complex synthetic biology architectures, gene therapy and trace-free delivery.;
Assembly and Validation of Versatile Transcription Activator-Like Effector Libraries
(Nature Publishing Group, 2014-05-06) Li, Yi; Ehrhardt, Kristina; Zhang, Michael Q.; Bleris, Leonidas; 0000 0001 2535 9739 (Bleris, L); 0000 0001 1707 1372 (Zhang, MQ); 2012076942‏ (Bleris, L); 99086074‏ ‎(Zhang, MQ); Zhang, Michael Q.
The ability to perturb individual genes in genome-wide experiments has been instrumental in unraveling cellular and disease properties. Here we introduce, describe the assembly, and demonstrate the use of comprehensive and versatile transcription activator-like effector (TALE) libraries. As a proof of principle, we built an 11-mer library that covers all possible combinations of the nucleotides that determine the TALE-DNA binding specificity. We demonstrate the versatility of the methodology by constructing a constraint library, customized to bind to a known p53 motif. To verify the functionality in assays, we applied the 11-mer library in yeast-one-hybrid screens to discover TALEs that activate human SCN9A and miR-34b respectively. Additionally, we performed a genome-wide screen using the complete 11-mer library to confirm known genes that confer cycloheximide resistance in yeast. Considering the highly modular nature of TALEs and the versatility and ease of constructing these libraries we envision broad implications for high-throughput genomic assays. ;
Synthetic Mammalian Transgene Negative Autoregulation
(2013-06-04) Shimoga, Vinay; White, Jacob T.; Li, Yi; Sontag, Eduardo; Bleris, Leonidas; 0000 0001 2535 9739 (Bleris, L); 2012076942‏ (Bleris, L)
Biological networks contain overrepresented small-scale topologies, typically called motifs. A frequently appearing motif is the transcriptional negative-feedback loop, where a gene product represses its own transcription. Here, using synthetic circuits stably integrated in human kidney cells, we study the effect of negative-feedback regulation on cell-wide (extrinsic) and gene-specific (intrinsic) sources of uncertainty. We develop a theoretical approach to extract the two noise components from experiments and show that negative feedback results in significant total noise reduction by reducing extrinsic noise while marginally increasing intrinsic noise. We compare the results to simple negative regulation, where a constitutively transcribed transcription factor represses a reporter protein. We observe that the control architecture also reduces the extrinsic noise but results in substantially higher intrinsic fluctuations. We conclude that negative feedback is the most efficient way to mitigate the effects of extrinsic fluctuations by a sole regulatory wiring.;
Transcripts for Combined Synthetic MicroRNA and Gene Delivery
(2013-06-26) Kashyap, Neha; Pham, Bich; Xie, Zhen; Bleris, Leonidas; 0000 0001 2535 9739 (Bleris, L)
MicroRNAs (miRNAs) are a class of short noncoding RNAs which are endogenously expressed in many organisms and regulate gene expression by binding to messenger RNA (mRNA). MicroRNAs are either produced from their independent transcription units in intergenic regions or lie in intragenic regions. Intragenic miRNAs and their host mRNAs are produced from the same transcript by the microprocessor and the spliceosome protein complex respectively. The details and exact timing of the processing events have implications for downstream RNA interference (RNAi) efficiency and mRNA stability. Here we engineer and study in mammalian cells a range of synthetic intragenic miRNAs co-expressed with their host genes. Furthermore, we study transcripts which carry the target of the miRNA, thereby emulating a common regulation mechanism. We perform fluorescence microscopy and flow cytometry to characterize the engineered transcripts and investigate the properties of the underlying biological processes. Our results shed additional light on miRNA and pre-mRNA processing but importantly provide insight into engineering transcripts customized for combined delivery and use in synthetic gene circuits.;
Transcription Activator-Like Effector Hybrids for Conditional Control and Rewiring of Chromosomal Transgene Expression
Li, Yi; Moore, Richard; Guinn, Michael; Bleris, Leonidas; 0000 0001 2535 9739 (Bleris, L); 2012076942‏ (Bleris, L); Erik Jonsson School of Engineering and Computer Science. Center for Systems Biology.
The ability to conditionally rewire pathways in human cells holds great therapeutic potential. Transcription activator-like effectors (TALEs) are a class of naturally occurring specific DNA binding proteins that can be used to introduce targeted genome modifications or control gene expression. Here we present TALE hybrids engineered to respond to endogenous signals and capable of controlling transgenes by applying a predetermined and tunable action at the single-cell level. Specifically, we first demonstrate that combinations of TALEs can be used to modulate the expression of stably integrated genes in kidney cells. We then introduce a general purpose two-hybrid approach that can be customized to regulate the function of any TALE either using effector molecules or a heterodimerization reaction. Finally, we demonstrate the successful interface of TALEs to specific endogenous signals, namely hypoxia signaling and microRNAs, essentially closing the loop between cellular information and chromosomal transgene expression. © 2012 Macmillan Publishers Limited. All rights reserved.

Browse

Recent Submissions