Browsing by Author "Lee, Sangsin"

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    Acoustically targeted measurement of transgene expression in the brain
    (AAAS, 2024) Seo, Joon Pyung; Trippett, James S.; Huang, Zhimin; Lee, Sangsin; Nouraein, Shirin; Wang, Ryan Z.; Szablowski, Jerzy O.; Applied Physics Program;Systems, Synthetic, and Physical Biology Program;Rice Neuroengineering Initiative
    Gene expression is a critical component of brain physiology, but monitoring this expression in the living brain represents a major challenge. Here, we introduce a new paradigm called recovery of markers through insonation (REMIS) for noninvasive measurement of gene expression in the brain with cell type, spatial, and temporal specificity. Our approach relies on engineered protein markers that are produced in neurons but exit into the brain’s interstitium. When ultrasound is applied to targeted brain regions, it opens the blood-brain barrier and releases these markers into the bloodstream. Once in blood, the markers can be readily detected using biochemical techniques. REMIS can noninvasively confirm gene delivery and measure endogenous signaling in specific brain sites through a simple insonation and a subsequent blood test. REMIS is reliable and demonstrated consistent improvement in recovery of markers from the brain into the blood. Overall, this work establishes a noninvasive, spatially specific method of monitoring gene delivery and endogenous signaling in the brain.
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    Engineered serum markers for non-invasive monitoring of gene expression in the brain
    (2024-08-05) Lee, Sangsin; Szablowski, Jerzy
    Gene expression provides the physiological underpinning for myriad biological processes, including those in the brain. However, monitoring the brain gene expression has been challenging due to the brain’s natural, confined architecture which restricts access to its thick, delicate tissue. Consequently, most existing approaches directly measure RNA abundance or transcriptional changes in an invasive manner, such as by histology or implanted optical devices, the result of which is irreversible physical damage to the brain. The aim of this thesis is to develop a new paradigm of molecular technology capable of measuring brain gene expression non-invasively, while ensuring high sensitivity, scalability, and precise spatial, temporal, and cellular resolution. As a first step towards this goal, we engineered a new class of synthetic, serum-based markers, called Released Markers of Activity (RMAs), that allows for non-invasive monitoring of gene expression with a simple blood test. Genetic tagging of specific genes of interest in the brain with RMA results in the release of RMA reporters from the brain into the bloodstream in response to the level of molecular activity of the gene. Once RMAs are deposited inside the blood, the blood samples can be analyzed using compatible biochemical techniques. Our initial readout method quantifies released RMAs in the blood by measuring luciferase activity, which provided high sensitivity with detectability down to approximately twelve labeled neurons in the mouse brain. Further, genetic tagging of RMAs to the neuronal activity-related gene Fos enabled the discrimination of brain activity through blood analysis. Because RMAs function as protein markers, this paradigm delivers greater scalability for measuring multiple genes or even enabling high-throughput gene analysis of the brain. The next devised strategy which combines the methods of nanopore protein sequencing and a machine learning-based classifier measures differentially barcoded RMAs for non-invasive, parallel readout of multiple genes in the brain. Taken together, our technology presents a novel and radical new reporter system for safe, repeatable, and multiplexed measurement of gene expression in an intact brain.
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    Single C-to-T substitution using engineered APOBEC3G-nCas9 base editors with minimum genome- and transcriptome-wide off-target effects
    (American Association for the Advancement of Science, 2020) Lee, Sangsin; Ding, Ning; Sun, Yidi; Yuan, Tanglong; Li, Jing; Yuan, Qichen; Liu, Lizhong; Yang, Jie; Wang, Qian; Kolomeisky, Anatoly B.; Hilton, Isaac B.; Zuo, Erwei; Gao, Xue; Center for Theoretical and Biological Physics
    Cytosine base editors (CBEs) enable efficient cytidine-to-thymidine (C-to-T) substitutions at targeted loci without double-stranded breaks. However, current CBEs edit all Cs within their activity windows, generating undesired bystander mutations. In the most challenging circumstance, when a bystander C is adjacent to the targeted C, existing base editors fail to discriminate them and edit both Cs. To improve the precision of CBE, we identified and engineered the human APOBEC3G (A3G) deaminase; when fused to the Cas9 nickase, the resulting A3G-BEs exhibit selective editing of the second C in the 5′-CC-3′ motif in human cells. Our A3G-BEs could install a single disease-associated C-to-T substitution with high precision. The percentage of perfectly modified alleles is more than 6000-fold for disease correction and more than 600-fold for disease modeling compared with BE4max. On the basis of the two-cell embryo injection method and RNA sequencing analysis, our A3G-BEs showed minimum genome- and transcriptome-wide off-target effects, achieving high targeting fidelity.