Browsing by Author "Goell, Jacob"

Now showing 1 - 4 of 4
  • Thumbnail Image
    Item
    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.
  • Thumbnail Image
    ItemEmbargo
    Engineering chromatin modifiers as biological discovery tools and epigenome editors
    (2024-04-19) Goell, Jacob; Hilton, Isaac
    Epigenetics is the study of heritable traits driven by alterations in the genome that occur on top of the DNA sequences encoding our genome. These features include histone and DNA modifications, chromatin architecture, and transcription factors that enable the emergence of cell types and a diversity of traits that are responsive to external stimuli. The study of the epigenome has thus been important for understanding basic biology and human disease. Until the emergence of CRISPR/Cas systems for programmable gene/epigenome editing, the field of epigenetics relied heavily on observational studies and genome-wide correlations to reach conclusions on the role epigenetic features play. The rapid adoption of CRISPR-based tools allowed for the targeted modification of DNA and deposition of precise epigenetic modifications through the fusion of chromatin-modifying factors to a catalytically inactivated version of a CRISPR system. While promising in its use for both basic and translational applications, the field still requires improved tools and a better understanding of their function to address inherent toxicity and off-targeting concerns, especially with respect to histone acylation, a modification linked to gene activation. Using the widely adopted epigenome editing effector domain and histone acyltransferase, p300, I address these concerns by (1) engineering mutations into p300 to alter its acylation deposition profile and cytotoxicity and (2) applying multi-omics and functional genomics methods to characterize these effectors for off-targets and benchmark their utility in a non-coding enhancer mapping screen.
  • Thumbnail Image
    Item
    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.
    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.
  • Thumbnail Image
    Item
    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
    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.