Browsing by Author "Wang, Kaiyuan"

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    Compact engineered human mechanosensitive transactivation modules enable potent and versatile synthetic transcriptional control
    (Springer Nature, 2023) Mahata, Barun; Cabrera, Alan; Brenner, Daniel A.; Guerra-Resendez, Rosa Selenia; Li, Jing; Goell, Jacob; Wang, Kaiyuan; Guo, Yannie; Escobar, Mario; Parthasarathy, Abinand Krishna; Szadowski, Hailey; Bedford, Guy; Reed, Daniel R.; Kim, Sunghwan; Hilton, Isaac B.
    Engineered transactivation domains (TADs) combined with programmable DNA binding platforms have revolutionized synthetic transcriptional control. Despite recent progress in programmable CRISPR–Cas-based transactivation (CRISPRa) technologies, the TADs used in these systems often contain poorly tolerated elements and/or are prohibitively large for many applications. Here, we defined and optimized minimal TADs built from human mechanosensitive transcription factors. We used these components to construct potent and compact multipartite transactivation modules (MSN, NMS and eN3x9) and to build the CRISPR–dCas9 recruited enhanced activation module (CRISPR-DREAM) platform. We found that CRISPR-DREAM was specific and robust across mammalian cell types, and efficiently stimulated transcription from diverse regulatory loci. We also showed that MSN and NMS were portable across Type I, II and V CRISPR systems, transcription activator-like effectors and zinc finger proteins. Further, as proofs of concept, we used dCas9-NMS to efficiently reprogram human fibroblasts into induced pluripotent stem cells and demonstrated that mechanosensitive transcription factor TADs are efficacious and well tolerated in therapeutically important primary human cell types. Finally, we leveraged the compact and potent features of these engineered TADs to build dual and all-in-one CRISPRa AAV systems. Altogether, these compact human TADs, fusion modules and delivery architectures should be valuable for synthetic transcriptional control in biomedical applications.
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    CRISPR-based Transcriptional Activators for Dissecting Human Gene-Regulatory Mechanisms
    (2022-10-13) Wang, Kaiyuan; Hilton, Isaac B
    Nuclease-inactivated CRISPR/Cas-based (dCas-based) systems have emerged as powerful technologies to reshape the human epigenome and gene expression synthetically. Despite the increasing adoption of these platforms, their relative potencies lack optimization, and their mechanistic differences are incompletely characterized. Here we expanded the dCas9-based activator toolbox to incorporate human CBP and GCN5 and tested their efficacy as transcriptional activators. For the optimization of the dCas9-toolbox, we showed that affinity-purification-mass spectrometry could be applied to identify the antagonistic gene for these dCas9-based tools in the cellular context, which enables us to improve dCas9-based transcriptional activation by targeted antagonist neutralization through either small-molecule inhibition or RNA interference. Eventually, we systematically compared the most widely adopted dCas9-based transcriptional activators to investigate the interconnections between different aspects of transcription regulation, including eRNA transcription, mRNA transcription, histone acetylation and chromatin spatial rearrangement. We used dCas9-based activators to demonstrate that an intrinsic transcriptional and epigenetic reciprocity can exist between human enhancers and promoters and that enhancer-mediated tracking and engagement of a downstream promoter can be synthetically driven by targeting dCas9-based transcriptional activators to an enhancer. Collectively, our study increases the expanse of the dCas9-activator toolbox, creates new systematic approaches for their functional improvement, and unravels the potential of the dCas9-based activator toolbox to provide new insights into the enhancer-mediated control of human gene expression.
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    Programmable human histone phosphorylation and gene activation using a CRISPR/Cas9-based chromatin kinase
    (Springer Nature, 2021) Li, Jing; Mahata, Barun; Escobar, Mario; Goell, Jacob; Wang, Kaiyuan; Khemka, Pranav; Hilton, Isaac B.; Bioengineering; Biosciences
    Histone phosphorylation is a ubiquitous post-translational modification that allows eukaryotic cells to rapidly respond to environmental stimuli. Despite correlative evidence linking histone phosphorylation to changes in gene expression, establishing the causal role of this key epigenomic modification at diverse loci within native chromatin has been hampered by a lack of technologies enabling robust, locus-specific deposition of endogenous histone phosphorylation. To address this technological gap, here we build a programmable chromatin kinase, called dCas9-dMSK1, by directly fusing nuclease-null CRISPR/Cas9 to a hyperactive, truncated variant of the human MSK1 histone kinase. Targeting dCas9-dMSK1 to human promoters results in increased target histone phosphorylation and gene activation and demonstrates that hyperphosphorylation of histone H3 serine 28 (H3S28ph) in particular plays a causal role in the transactivation of human promoters. In addition, we uncover mediators of resistance to the BRAF V600E inhibitor PLX-4720 in human melanoma cells using genome-scale screening with dCas9-dMSK1. Collectively, our findings enable a facile way to reshape human chromatin using CRISPR/Cas9-based epigenome editing and further define the causal link between histone phosphorylation and human gene activation.
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    Systematic comparison of CRISPR-based transcriptional activators uncovers gene-regulatory features of enhancer–promoter interactions
    (Oxford University Press, 2022) Wang, Kaiyuan; Escobar, Mario; Li, Jing; Mahata, Barun; Goell, Jacob; Shah, Spencer; Cluck, Madeleine; Hilton, Isaac B; Bioengineering; Biosciences
    Nuclease-inactivated CRISPR/Cas-based (dCas-based) systems have emerged as powerful technologies to synthetically reshape the human epigenome and gene expression. Despite the increasing adoption of these platforms, their relative potencies and mechanistic differences are incompletely characterized, particularly at human enhancer–promoter pairs. Here, we systematically compared the most widely adopted dCas9-based transcriptional activators, as well as an activator consisting of dCas9 fused to the catalytic core of the human CBP protein, at human enhancer–promoter pairs. We find that these platforms display variable relative expression levels in different human cell types and that their transactivation efficacies vary based upon the effector domain, effector recruitment architecture, targeted locus and cell type. We also show that each dCas9-based activator can induce the production of enhancer RNAs (eRNAs) and that this eRNA induction is positively correlated with downstream mRNA expression from a cognate promoter. Additionally, we use dCas9-based activators to demonstrate that an intrinsic transcriptional and epigenetic reciprocity can exist between human enhancers and promoters and that enhancer-mediated tracking and engagement of a downstream promoter can be synthetically driven by targeting dCas9-based transcriptional activators to an enhancer. Collectively, our study provides new insights into the enhancer-mediated control of human gene expression and the use of dCas9-based activators.