CSB Research
Permanent URI for this collectionhttps://hdl.handle.net/10735.1/3686
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Browsing CSB Research by Author "0000 0001 2535 9739 (Bleris, L)"
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Item 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. ;Item 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.;Item 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.Item 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.;Item 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.;